Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Calcium regulation of calcium transport by sarcoplasmic reticulum Gilchrist, James Stuart Charles 1990

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
[if-you-see-this-DO-NOT-CLICK]
UBC_1991_A1 G54.pdf [ 14.06MB ]
Metadata
JSON: 1.0076804.json
JSON-LD: 1.0076804+ld.json
RDF/XML (Pretty): 1.0076804.xml
RDF/JSON: 1.0076804+rdf.json
Turtle: 1.0076804+rdf-turtle.txt
N-Triples: 1.0076804+rdf-ntriples.txt
Original Record: 1.0076804 +original-record.json
Full Text
1.0076804.txt
Citation
1.0076804.ris

Full Text

CALCIUM REGULATION OF CALCIUM TRANSPORT BY SARCOPLASMIC RETICULUM By  James Stuart Charles Gilchrist B.Sc.(Hons.), Liverpool Polytechnic, 1981 M.Sc. University of Alberta, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Interdisciplinary Studies  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June 1991 ©James Stuart Charles Gilchrist, 1990  In  presenting  this thesis  in partial fulfilment of  the  requirements  for  an  advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. copying  of this thesis for scholarly  department  or  by  his  or  her  I further agree that permission for  purposes  may be granted by the head of  representatives.  It  is  understood  that  publication of this thesis for financial gain shall not be allowed without permission.  Department of  Lwbl'&Pljrtfi&.l  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  extensive  ^Toblt*?  copying  my or  my written  ii  ABSTRACT  The sarcoplasmic reticulum (SR) of skeletal muscle is an intracellular membraneous network that, through the cyclical release and re-uptake of C a  2 +  into and from, respectively, the cytoplasmic space, regulates myofilament shortening and, therefore, muscle contraction. SR derived from the terminal cisternae (HSR) demonstrates the property of Ca -induced C a 2+  attainment of a threshold intralumenal C a extralumenal C a  2 +  ligand gated C a  2 +  2 +  2 +  release. Upon  load, application of a small pulse of  stimulates the release of a pool of intralumenal C a permeable pore of the C a  2 +  2 +  via the  release channel/ryanodine  receptor complex. It was hypothesised that intralumenal C a  2 +  regulates the  opening of the release channel. HSR  vesicles were purified from skeletal and cardiac muscle by a novel  technique.  Structural characterisation of these membranes demonstrated an  enrichment of harvested fractions in the C a intralumenal C a  2 +  release channel and the  2 +  binding protein, calsequestrin.  In radiometric studies,  skeletal HSR vesicles were shown to bind ryanodine with high capacity at both low and high affinity sites, with 2 fold stimulation of C a polyorganic cation C a  2 +  2 +  accumulation by the  channel blocker, ruthenium red. HSR vesicles passively  loaded C a . Passive loading of HSR vesicles with C a 2+  linearly dependent upon the concentration of C a  2 +  This suggested the presence of 2 intralumenal C a  2 +  2 +  was found to be non-  within the loading medium. binding sites with different  affinities for C a . A spectroscopic dual-wavelength assay of C a 2 +  2 +  release was  developed that took advantage of peculiar spectral properties of the metallochromic  sensitive dye Antipyrylazo III. In the presence of mM  MgATP and mM Mg2+ the initial fast phase of HSR C a  2 +  was well resolved.  Evidence was presented that initial rapid uptake was associated with high  affinity binding to an intralumenal  compartment.  C a -induced C a 2 +  z +  release was shown to occur with a threshold loading of intralumenal C a . The 2 +  intralumenal C a  2 +  threshold for Ca -induced C a 2+  the presence of ryanodine. Ryanodine induced C a  2 +  2 +  release was decreased in  release was also dependent  upon the amount of intralumenal C a . Ryanodine was also shown to inhibit 2 +  sustained Ca -induced C a 2+  Ca  2 +  release by apparent inhibition of the binding of  to intralumenal sites.  2 +  transitions of the C a  2 +  channel and calsequestrin were interdependent.  Purified mM and mM C a cleaved the C a  2 +  These results suggested that junctional state  2 +  activated neutral protease isoforms selectively  channel into 410 and 150kDa peptides with limited proteolysis.  This was demonstrated in both HSR vesicles and the purified C a  2 +  release  channel. A novel 88kDa protein was also shown to be fragmented by both CANP isoforms. The identity of this prominent HSR associated protein remains obscure. 4^Ca  2+  CANP fragmentation of HSR protein elevated passive and active loading in vesicles. This indicated that selective structural modification  of the cytoplasmic portion of the release channel modified the comformational states of a intralumenal C a spectroscopic  2 +  binding compartment in HSR vesicles.  In  studies, CANP proteolysis of HSR proteins increased the  sensitivity to C a  2 +  and ryanodine-induced C a  required intralumenal C a  2 +  2 +  release through decreases in the  threshold for release. These functional alterations  coincided with apparent single site cleavage of the release channel. Further proteolysis of the initial 410 and 150kDa peptides was without further significant effect upon function. Based upon the hypothesis that primary sequences rich in proline (P), glutamate (E), aspartate (D), serine (S) and threonine (T) (PEST regions) are recognition sites for CANP binding to substrates, a search for PEST regions within the C a  2 +  channel was undertaken. It was tentatively proposed that two  iv  PEST regions near the N-terminal of the Ca  release channel may represent  z  sites close to the CANP cleavage site. The results of this work were discussed in relation to a possible role of C a 2 +  induced C a  2 +  release in regulating the patterning of C a  The frequency and amplitude of cytosolic C a  2 +  2 +  cytosolic transients.  transients appear to be important  in regulating protein expression. The requirement of intralumenal Ca -induced 2+  Ca  2 +  release may be a means by which the cyclical uptake and release of C a  2 +  during muscle relaxation and contraction can be coordinated. This coordination may define the patterning of cytosolic C a to Ca -induced C a 2+  2 +  2 +  transients. The increased sensitivity  release by HSR after CANP treatment may represent a  means by which the patterning of cytosolic C a  2 +  transients can be altered to  effect changes in protein synthesis.  Signature of thesis co-supervisor  Signature of thesis co-supervisor  ACKNOWLEDGEMENTS I cannot begin to describe the gratitude that I have for my principle advisor and long time friend, Dr Angelo Belcastro. It is his support, imagination, and keen insights which have contributed immeasurably to my thinking and my work. I shall miss him and his wonderful family. I thank you.  I would like to extend my deepest thanks to Dr Sidney Katz for his support, his trust, his guidance, and his friendship. It has been a thoroughly rewarding experience working with you. Many thanks.  I wish to thank the members of my supervisory committee, Dr Angelo Belcastro, Dr Sidney Katz, Dr Peter Hochachka, and Dr David Godin.  Thank you for  having listened to me. Thankyou all for your support.  I would like to acknowledge the friendship and lively informative exchanges with Mr Bruce Allen and Dr Kevin Wang. I learned much from these two talented people. It has been an honour working with you.  I would like to acknowledge the skilled technical assistance of Mr Anthony Borel with the ryanodine purification. In addition, I would like to thank Ms Cheryl Machan for skilled assistance with many aspects of this work.  I would like to thank all members of Dr Katz's laboratory for their friendship.  Many thanks to Dr T Kuo for the generous gift of the CANP antibodies.  vi  I wish to thank all members of the Faculty of Pharmaceutical Sciences and staff and members of the School of Physical Education and Recreation at the University of British Columbia.  Finally, I wish to thank my wonderful wife, Jo-Anne, for having supported me throughout all the trials and tribulations of these graduate years. Thankyou for the typing of this manuscript. Thankyou for being a friend.  DEDICATION  To Jo-Anne, you have always been there for To my family, especially my parents.  TABLE OF CONTENTS CONTENT  Page  ABSTRACT  ii  ACKNOWLEDGMENTS  v  DEDICATION  vii  TABLE OF CONTENTS  viii  LIST OF FIGURES  xiv  LIST OF TABLES  xvii  LIST OF ABBREVIATIONS  xviii  INTRODUCTION  1  I.  Role of calcium in living organisms  1  II.  Control of calcium movements  7  1. Receptor mediated control  7  a. Phosphorylation effects b. Myoinositol triphosphate effects 2. Compartment mediated control of calcium movements a. Mitochondria b. Plasma membrane  8 11 12 12 13  (i) . Calcium ATPase (ii) . Sodium-calcium exchange (iii) . Calcium channels  13 13 15  c. Endoplasmic reticulum and related structures d. Sarcoplasmic reticulum  16  III. Sarcoplasmic reticulum structure and function  17 17  1. Historical overview  17  2. Morphology of SR  18  3. Protein composition of SR  21  a. Calcium-ATPase b. Ryanodine receptor  22 25  c. Calsequestrin d. Calreticulin e. 53kDa Glycoprotein f. Sarcalumenin g. 55kDa Thyroid hormone binding protein 4. SR calcium uptake a. Calcium pump regulation (i) . Calcium (ii) . Magnesium (iii) . ATP 5. SR calcium release a. Overview b. Triggering mechanism of SR calcium release (i) . Charge movement triggered calcium release (ii) . Calcium-induced calcium release c. Modification of calcium release (i) . Nucleotides (ii) . Extravesicular calcium (iii) . Drugs (iv) . Magnesium (v) . Anaesthetics (vi) . Organic poly cations (vii) . Protons (viii) . Reactive group modification (ix) . Myoinositol triphosphate (x) . Calmodulin (xi) . Intralumenal calcium (xii) . Proteases IV. Calcium Activated Protease 1. Regulation of CANP a. Phospholipid b. Calpastatin 2. Role of CANP V. Objectives of this study. MATERIALS AND METHODS I. Materials. II. Methods 1. Isolation of HSR membranes.  2. Ryanodine receptor purification. 3. Purification of CANP. 4. Protein determination 5. Assay of CANP proteolytic activity 6. Antipyrylazo III purification 7. Double-beam spectroscopy 8. Calculation of calcium:dye (CaD2) dissociation constants 9. Spectroscopic determination of HSR calcium transport 10. Determination of calcium stimulated ATPase activity 11. Determination of inorganic phosphate 12. Calcium release from passively loaded vesicles 13. Assay of calcium transport in CANP treated HSR a. Spectroscopic analysis b. Radiometric analysis 14. [^HjRyanodine purification 15. [^HjRyanodine binding 16. SDS-polyacrylamide gel electrophoresis 17. Staining of proteins resolved by SDS-PAGE a. Coomassie blue b. Stains-All 18. CANP proteolysis of HSR resolved by SDS-PAGE 19. Detergent solubilisation of HSR membranes for immunolocalisation of membrane associated CANP  xi  20. Immunostaining of HSR membranes  82  21. Calculation of free ion concentrations  83  22. Data analysis  83  RESULTS I. Protein purification  84  1. Purification of HSR membranes  84  2. Ryanodine receptor purification  93  3. Purification of CANP  93  II. Functional characterisation of HSR membranes  98  1. C a dependent activation of membrane bound Ca " "-ATPase activity  1107  2. Passive C a  113  2 +  2  2 +  1  loading and C a  3. Spectroscopy of HSR C a  2 +  2 +  release  transport  116  a. Ca :APIII difference spectral characteristics  116  2+  (i) . M g effects upon CaD :APIII (ii) . Mg.ATP effects upon divalent catiomAPIII 2 +  2  b. Spectroscopic resolution of Ca2+ uptake and release  120 120  (i) . Wavelength pair selection (ii) . M g effects upon HSR C a uptake and release 2 +  c. Intralumenal C a HSR C a release  2 +  116 120  dependence of  2 +  123 123 129  2 +  (i). Ca -induced C a release (iiL Repetitive triggering of Ca -induced Ca " release 2+  2 +  24  4. Filtration studies of Ca2+ release from actively loaded vesicles a. Pi accumulation during active C a transport b. C a  2 +  release  129 132 135  2 +  135 138  xii  5. Ryanodine effects upon C a and C a release  2 +  uptake  138  2 +  a. C a  2 +  uptake  138  b. Intralumenal C a threshold for Ca -induced C a release  143  c. Intralumenal C a threshold for ryanodine-induced C a release  149  2 +  2+  2 +  2 +  2 +  III. CANP effects upon HSR structure and function  149  1. CANP proteolysis of HSR proteins  149  a. Endogenous CANP effects  149  b. Exogenous CANP effects  152  2. Immunolocalisation of CANP to HSR membranes  162  3. CANP effects upon HSR function  169  a. Passive Ca -loading and Ca -induced C a release  169  b. ATP dependent C a  accumulation  173  c. Spectroscopic studies of CANP effects upon HSR C a transport  173  2+  2+  2 +  4 5  2 +  2 +  (i) . C a  2 +  (ii) . C a  2 +  uptake release  4. CANP effects upon [^H]Ryanodine binding  173 173 183  DISCUSSION I.  HSR structural characterisation  188  II. HSR functional characterisation  190  III. CANP characterisation  192  IV. CANP effects upon HSR structure  194  V.  178  HSR calcium release  VI. CANP effects upon HSR calcium release VII Contribution of this work to the existing literature  213 218  VIII Conclusions  226  BIBLIOGRAPHY  LIST OF FIGURES Figures  Page  1.  The EF-hand or calmodulin fold.  5  2.  Schematic representation of signal cascades.  9  3.  Structural diagram of the SR Ca -ATPase.  23  4.  Gating of the calcium release channel.  49  5.  Purification of HSR membranes from rabbit skeletal muscle.  86  6.  Stains-All staining of skeletal and cardiac HSR proteins resolved by SDS-PAGE.  89  7.  SDS-PAGE comparison of skeletal and cardiac HSR.  91  8.  Calmodulin (CaM)-agarose affinity chromatography of CHAPS solubilised HSR protein.  94  9.  SDS-PAGE resolution of CaM-agarose affinity purified HSR proteins.  96  10.  DEAE-sepharose CL-4B anion exchange chromatography of ammonium sulphate precipitated u- and mCANP from rabbit skeletal muscle homogenates.  99  11.  Phenyl-sepharose CL-4B chromatography of DEAE separated |i- and mCANP.  101  12.  Omega-hexylamine-agarose chromatography of phenyl-sepharose CL-4B isolated \iand mCANP.  103  13.  Gel permeation (Ultrogel AcA 34) chromatography of co-hexylamine-agarose isolated CANP.  105  14.  Purification scheme of CANP purification followed by SDS-PAGE.  107  15.  Calcium dependent stimulation of HSR  111  2+  ATPase activity. Effects of varying extralumenal calcium upon HSR calcium loading and calcium-induced calcium release. Double beam spectroscopy of AP IILdivalent cation AA spectra. Effect of Mg.ATP upon AP IILdivalent cation difference spectra. Effect of wavelength pair upon spectroscopic resolution of initial HSR calcium uptake. Effect of elevated magnesium upon HSR calcium uptake. Intralumenal calcium requirement for calcium -induced calcium release. Effects of creatine phosphate concentration upon HSR calcium uptake and release. Calcium and inorganic phosphate (Pi) retention by HSR vesicles undergoing ATP dependent calcium accumulation. Effect of elevated (20mM) creatine phosphate (CP) upon calcium-induced calcium release from actively loaded vesicles. Ryanodine effects upon HSR calcium uptake. Effect of ryanodine upon calcium stimulation of calcium release. Effect of intralumenal calcium load upon ryanodine induced calcium release. Endogenous CANP effects upon HSR protein structure. Calcium and protease dependence of U.CANP mediated proteolysis of HSR proteins. Calcium and protease dependence of mCANP mediated proteolysis of HSR proteins. Comparison of the calcium dependence of CANP mediated proteolysis of (A) casein and (B) the skeletal muscle ryanodine receptor. Comparison of the effects of u.CANP upon the CHAPS  XVI  solubilised and vesicular 550kDa protein. 33.  CANP proteolysis of cardiac HSR proteins.  163  34.  Immunostaining of SR microsomal protein reactive to CANP polyclonal antisera.  165  35.  Immunostaining of CHAPS solubilised protein reactive to CANP polyclonal antisera.  167  36.  Calcium loading and C a induced C a and mCANP treated HSR membranes.  170  37.  Effect ofjiCANP and mCANP upon assive C a loading and release y HSR vesicles.  2 +  P  4 5  2 +  release  174  2 +  38.  Effect of CANP upon ATP dependent C a uptake by HSR vesicles.  39.  Effect of CANP mediated proteolysis of HSR upon calcium uptake.  179  40.  Effects of CANP upon intralumenal calcium dependence of calcium induced calcium release.  181  41.  Effect of u,CANP and mCANP upon [ H]ryanodine binding to HSR membranes.  185  42.  Scatchard Analysis of [ H]ryanodine binding to HSR membranes.  187  4 5  2 +  3  3  176  xvii  LIST OF TABLES Table  Page  1.  Subfamilies of Calcium-binding proteins and unique EF-hand homologs.  4  2.  Yields and calcium release characteristics of fractionated crude SR microsomes  85  3.  Purification of u- and mCANP from rabbit skeletal muscle  105  4.  Calcium: APIII Dissociation constants.  119  5.  Calcium release from uCANP treated vesicles  172  6.  Calcium-ATPase activity of CANP treated HSR membranes.  178  7.  Effect of CANP proteolysis upon ryanodine induced calcium release.  184  8.  PEST regions in the calcium release channel sorted by score.  218  LIST OF ABBREVIATIONS  %  percent  ADP  adenosine 5'-diphosphate  AMP  adenosine 5-monophate  APIII  Antipyrylazo III  ATP  adenosine 5'-triphosphate  p  beta  Ca  2 +  calcium free ion  Ca ATPase calcium stimulated, magnesium dependent 2+  ATPase pump protein CaM  calmodulin  CANP  calcium activated neutral protease  CBP  calcium binding protein  CHAPS  3-[(3-cholamidopropyl)dimethylammonio]-lpropanesulphonate  CP  creatine phosphate  CPK  creatine phosphokinase  CPP  calcium pump protein  CRC  calcium release channel  8  delta  AA  difference absorbance  DEAE  diethylaminoethyl  DTT  dithiothreitol  EGTA  ethyleneglycol bis(b-aminoethylether) N,N,N',N'-tetraacetic acid  g  gram  xix  HEPES  4-(2-hydroxyethyl)-l-piperazineethane-sulphonic acid  HSR  heavy sarcoplasmic reticulum  IP3  myoinositol 1,4,5 triphosphate  ISR  intermediate sarcoplasmic reticulum  K'  first order dissociation constant  K"  second order dissociation constant  K  potassium free ion  +  Ka  association constant  kDa  kilodalton  Km  michaelis menten constant  L  litre  u.  micro  m  milli  M  molar  mg  milligram  Mg  2 +  magnesium free ion  min  minute  mol  mole  MOPS  morpholinopropanesulphonic acid  M  relative molecular mass  r  n Na  nano +  sodium free ion  p  pico  PAGE  polyacrylamide gel electrophoresis  Pi  inorganic phosphate  PIPES  1,4,-piperazinediethanesulphonic acid  pmol  picomole  XX  PMSF  phenylmethylsulphonyl fluoride  pS  picosiemens  s  second  S  svedberg  SR  sarcoplasmic reticulum  TCA  trichloroacetic acid  TEMED  N^N^N'-tetramethylethyldiamine  TLCK  N-a-p-tosyl-L-lysine chloromethyl ketone  Tris  tris (hy droxymethy 1) aminomethane  CO  omega  1  INTRODUCTION.  I. Role of C a l c i u m i n L i v i n g Organisms  The first discovery of the existence of calcium was made nearly two centuries ago by Humphry Davy in 1808. Early recognition of the biological importance of calcium (Ca ) is often attributed to the pioneering work of Ringer (1883) who 2+  discovered that constituents of tap water rather than distilled water during the preparation of physiological saline, activated the sustained beating of the frog heart.  Ringer later determined that the constituent was C a  2 +  and was an  absolute requirement for contraction. Since that time it has become clear that Ca  2+  is extensively distributed throughout the entire organism and performs a  wide variety of diverse functions.  These range from the regulation of cell  division and growth (including formation and maintenance of the skeletal structures) to the regulation of synaptosomal and neuromuscular transmitter release (Rubin, 1970), stimulus-secretion coupling from endocrine cells (Douglas 1968), mast cell histamine release (Foreman, 1981) and excitation contraction coupling (Heilbrunn and Wiercinski, 1947; Hill, 1949; Marsh, 1952; Ebashi and Lipmann, 1962; Winegrad, 1965; Fabiato, 1983). Vertebrate C a  2+  exists largely immobilised as hydroxyapatite in the presence  of collagen matrix (i.e. bone). In relation, a small amount of residual C a  2 +  is  distributed between extracellular and intracellular compartments. Within the extracellular compartment total C a  2+  concentration ([Ca ]) is relatively constant 2+  with estimates ranging between 2-3 mM (Fabiato 1983, Carifoli, 1987) half of which exists within an ionised or unbound form. specific distribution of C a requirement for C a very little C a  2+  2+  2+  Intracellularly, the tissue  is quite varied and is dependent upon the particular  in mediating intracellular processes. Erythrocytes contain  (20uM) (Long and Mouat, 1972). On the other hand, cardiac  2  muscle cells, which utilise C a  2+  processes, contain ImM total C a  in the regulation of contractile and oxidative 2+  accounting for total cell water (Fabiato, 1983).  Estimated intracellular free [Ca ] in resting or relaxed cells, however, is 2+  maintained close to O.luM. The remainder of C a  2+  is either a) sequestered within  intracellular compartments (e.g. endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, and cell nuclei) or b) bound to a variety of intracellular C a binding proteins.  Maintenance of 1,000-2,000 fold C a  2 +  2 +  electrochemical  gradients that necessarily results from compartmentation is achieved by a variety of energy dependent protein pumps (e.g. Ca -ATPases) and cation antiporters 2+  (e.g. Na/Ca antiporters) which operate, ideally, in dynamic equilibrium with a multitude of other intracellular and extracellular processes. Compartmentation of C a the cell.  2+  in this manner has two important consequences for  The first is that calcium effects upon a variety of molecular and  biochemical processes can be made to be selective, transient, and reversible (and therefore regulatory in nature) through the controlled release and re-uptake of Ca  2+  into and from, respectively, the cytosol. The second is that C a  2 +  effects can  be tolerated within an enviroment of phosphate driven metabolism (Weber, 1976). Intracellular energy dependent processes utilise the free energy liberation from the hydrolysis of mainly ATP. At rest, Pi concentrations are as high as 12mM in the fast twitch skeletal muscle and 5mM in cardiac cells (Ackerman et al., 1980; Meyer et al., 1982). With repetitive contractions Pi levels may be 15 fold elevated (see Meyer et al., 1984). The high solubility product of various forms of apatite would lead to cytosolic calcium phosphate precipitation and eventually cell death in the absence of precise control of intracellular C a  2+  distribution.  In view of the precarious co-existence of phosphate and calcium metabolism it is remarkable that C a  2+  is central to the regulation of a multitude of cellular  events in various cell types. Although the evolutionary choice of C a  2 +  in this  3  regard is not completely clear (see Carafoli, 1987) it's suitability is vested in the complex electronic structure and binding chemistry of C a flexible coordination number (Ca  2+  2+  (Williams, 1976). The  can bind between 6 to 8 electrons, usually  from oxygen) and the relatively large size of C a  2 +  permits tight protein binding  with variable bonding distances. This reduces the configurational requirements of protein binding sites that for binding of the smaller more regularly shaped Mg  2 +  are more specific. C a  2+  can therefore bind to a wide variety of proteins  with high affinity and specificity (in that it can exclude Mg ). 2+  Compartmentation of C a  2+  within subcellular organelles provides both a  source and sink of C a . It is the binding of cytosolic C a 2+  2 +  to proteins and the  subsequent induction of conformational change that provides the basis by which Ca  2+  modulates cellular function. Carafoli (1987) has catagorised these proteins  into two groups. In the first group, intrinsic membrane proteins (see later), initially bind C a  with various affinities and translocate it from the cytosol  2+  across compartment boundries (i.e. membranes) whereupon the protein is free to accept another C a  2+  ion. Proteins of the second group largely form the super  family of calcium-modulated proteins (Kretsinger, 1975). The calci-forms of this group (Kd(c )=10 M" ) are either active enzymes or modulate the function of 6  1  a  other enzymes.  The majority of these proteins (see Table 1) are evolutionary  homologs and can be categorised into sub-families each containing between two to eight copies of a conserved structural region known as an E-F hand (Moncrief et al., 1990). This structural motif was first deduced from x-ray diffraction studies of parvalbumin (Moews and Kretsinger, 1975). As shown in Figure 1, 29 amino acids are arranged in a helix-loop-helix configuration with formation of a thermodynamically stable pocket upon interaction of paired E-F hand units. Ca  2+  therefore functions as an intermediary signal or second messenger  mediating  the  trafficking  of  T a b l e 1. Subfamilies of C a l c i u m - b i n d i n g proteins and unique EF-hand homologs.  #Ca binding EF-hand domains z+  Calmodulin Troponin C Essential light chain of myosin Regulatory light chain of myosin Sarcoplasmic calcium-binding protein Calpain Aequorin Strongylocentrotus purpuratus ectodermal protein Calbinclin Parvalbumin a-actinin SI00 Calcineurin B (Bos) Tropinin C (Astacus) Calcium vector protein (Branchiostoma) Caltractin (Chlamydomonas) CDC31 (Saccharomyces) 10-kD protein (Tetahymena) Eight-aomain protein (Lytechinus) Cal cium-binding protein (Streptomyces)  Table adapted from Moncrief et al., 1990.  2-4 2-4 1-3 1 1-4 2-4 3 3-4 4 2 0-2 0-2 4 2 2 4 2 2 7 4  5  Fig. 1. The EF-hand or calmodulin fold. EF-hands consist of an ct-helix (symbolised by the forefinger of a right hand), a loop around the C a  2 +  ion  (represented by the clenched middle finger), and a second cc-helix (symbolised by the thumb). Amino acids 1-11 comprise the first cc-helix; 19-29 the second. The stipled ct-carbons-2, 5, 6, 9, 22, 25, 26, and 29- usually have hydrophobic side chains, (from Moncrief et al., 1990)  6  Figure 1.  7  biological information encoded in one form (e.g. receptor-ligand binding) through its conversion to another form ( e.g. enzyme activation). This form of intra- and intercellular communication is known as stimulus-response coupling or biological signal transduction in which tight control of C a  2 +  movements  coupled to other biological processes is essential for cell viability.  II. Control of Calcium Movements Regulation of the second messenger function of C a  2 +  can be described in two  ways. The first in terms of receptor mediated regulation of C a  2 +  movements.  The second in terms of specific devices controlling C a compartmentation. 2 +  1. Receptor Mediated Control Ca  2+  movements can be activated in two ways. In the first, binding of a ligand  to a receptor molecule located on the surface of the plasma membrane induces a change in membrane potential (e.g. acetylcholine binding to acetylcholone receptor) and leads to i) activation of voltage dependent C a release of C a  2+  2 +  channels or ii)  from sarcoplasmic reticulum (see later). In the second, more  universal type, ligand-receptor binding activates several intracellular regulatory cascades with production of additional second messengers (e.g. 3',5'-adenosine (guanosine) monophosphate or cAMP (cGMP), myoinositol l',4',5'-triphosphate (IP3) and diacylglycerol (DAG). The activity of a second messenger metabolic enzyme e.g. adenylate cyclase, guanylate cyclase or phosphoinositide phosphodiesterase (phospholipase C) is regulated by the ligand (agonist)-bound receptor via a family of coupling proteins, known as G-proteins, located within the membrane interior (see  8  Gilman, 1987). The 3 components (receptor, G-protein, enzyme) constitute the transduction domain (see Figure 2 ). As heterotrimers with a, p* and y subunits, G-proteins are regulated by the cyclical binding (activation) and hydrolysis (deactivation) of GTP (to GDP + Pi) upon the a subunit. G-protein activation may then lead to activation or inhibition of the metabolic enzyme. Myocardial adenylate cyclase for example is activated by stimulating G-proteins (G ) with s  production of cAMP by receptor binding of P-adrenergic agonists (epinephrine, norepinephrine, isoproterenol).  Inhibition occurs upon muscarinic receptor  binding of acetylcholine and activation of inhibitory G-proteins (Gj) (Josepheson and Speraklis, 1982). Similarly, production of IP3 and DAG form the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C involves the intermediary action of G (Litosch and Fain, 1986, Taylor and Merrit, 1986; Fain s  et al., 1988).  a. Phosphorylation Effects Production of the second messengers cAMP, cGMP, and DAG results in phosphorylation of specific cellular proteins and represents the most common consequence of second messenger effects. Of importance in the control of C a fluxes are phosphorylation of several proteins regulating C a  2 +  2 +  transport. The  myocardial SR Ca -ATPase regulatory protein (phospholamban) and the 2+  sarcolemmal Ca -ATPase are both phosphorylated and activated by cAMP2+  dependent protein kinase (Caroni and Carifoli, 1981, Neyses et al., 1985; Tada et al, 1975; James et al, 1989, Vittone et al., 1990). Similarly protein kinase C was shown to phosphorylate phospholamban (Movesian et al., 1984) and the C a 2+  ATPase from erythrocytes (Smallwood et al., 1988). dihydropyridine binding channels (165 and 185 kDa  In addition both the  subunits of skeletal and cardiac muscle, L-type C a  2 +  9  Fig. 2. Schematic representation of signal cascades. The transduction domain (membrane) consists of receptors, G-proteins, and metabolic enzymes. The translation domain consists of signal cascades and the modulation domain consists of responses to second messenger effects, (from Mooibroek and Wang, 1988)  10  Figure 2.  TIMUIATORY  INHIBITORY  SIGNAL  SIGNAL  | RECEPTOR*]  {  RECEPTOR*)  L.4  A  CYCI ASE  ATP  \  r  pip  ij  I P , > ^  CAMP Translation  REC£PTOR"7  \  'GTP  ®*i ?ENVLAT£  n  SIGNAL  PHOSPHOUPA^E  /  c v  OAG  Ca*  Domain  Protein  Protein - ( p )  Modulation Oomain  —1  Cellular Response  t  Kinase  A  Protain  C a  2  *  Binding Protein*  Protein  Protein-(?)  I Cellular Response  Cellular Response  Kinase  C  Protein  11  respectively) and the 52kDa (3-subunit have been shown to be phosphorylated by both protein-kinase C and cAMP activated protein kinase A (O'Callahan and Hosey, 1988, Rohrkasten et al 1988, O'Callahan et al., 1988). Phosphorylation v  increased the mean open time of cardiac L-type channels (Reuter et al., 1982) and therefore increased the slow inward C a  2+  current upon t-tubule membrane  depolarisation. Evidence suggests that cAMP dependent phosphorylation of cardiac and skeletal L-type channels may occur by direct activation of a closely associated G of adenylyl cyclase (Yatani et al., 1987,1988). s  b. Myoinositol Triphosphate Effects Regulation of C a  movements by IP3 occurs independently of  2+  phosphorylation and involves the direct binding of IP3 to receptors associated with C a  2+  channels that access a IP3 sensitive C a  1989). The location of this C a  2 +  2 +  pool (Berridge and Irvine,  pool is within the endoplasmic reticulum and  other related membrane structures (see later). The function of the IP3 sensitive Ca  2+  pool is unclear although identity of a GTP-activated IP3 insensitive  pool and its communication with the IP3 sensitive C a  2 +  Ca  2 +  pool in smooth muscle  cells (Ghosh et al., 1989) suggests a complex IP3 mediated control of intracellular Ca  2+  fluxes.  Such control mechanisms may mediate intracellular C a  2 +  oscillations which appear to propagate in the form of waves (Berridge and Irvine, 1989). It is further unclear to what extent these oscillations are associated with the linked oscillations of free C a  2+  and glycolytic flux observed in skeletal  muscle extracts and insulinoma cells (Corkey et al., 1988, Tornheim, 1988). Berridge and Irvine (1989) have suggested that information may be encoded in Ca  2+  waves as a frequency modulated signal.  amplitude modulatation of C a  2+  This is quite different from  signals (transients) associated with C a  2 +  release  from the sarcoplasmic reticulum membranes to initiate muscle contraction.  12  2. Compartment Mediated Control of Calcium Movements With the exception of erythrocytes, eukaryotic cells possess several C a  2+  transporting membrane systems that utilise the electrochemical energy stored within transmembrane C a activated and C a  2+  2 +  gradients to perform a variety of specific C a  2 +  regulated functions intracellularly. Principle membrane  systems include plasma membrane, mitochondria, endoplasmic reticulum and sarcoplasmic reticulum. In addition, membrane systems of the Golgi apparatus (Virk et al., 1985), lysosomes (Klempner, 1985), adrenal chromaffin cells (Burgoyne et al., 1989), and the nuclear envelope (Hashimoto et al., 1989) all demonstrate Ca -ATPase activity. Recent studies also indicate the presence of 2+  IP3 receptors upon the surface of nuclear membranes (Ross et al., 1989) suggesting the presence of concerted C a  2+  control mechanisms within this  membrane system.  a. Mitochondria The principle function of the mitochondria is the regulation of oxidative energy metabolism as first suggested by Lardy and Wellman (1952) and later confirmed by Chance and Williams (1955). observations of the C a  2+  However, earlier erroneous  uptake capacity of the mitochondria (Vasington and  Murphy, 1962) suggested that these organelles may participate as important regulators of cytosolic C a  2+  buffering. More recent X-ray microanalysis (Somlyo  and Walz, 1985) indicated that under normal conditions the intramitochondrial Ca  2+  pool would contribute little to the cytosolic C a  conditions where cytosolic C a associated with matrix C a  2+  2+  2+  pool. Under pathological  may be elevated, mitochondrial C a  2 +  uptake is  phosphate precipitation (Carafoli et al., 1965).  13  According to Carafoli (1987) such "matrix loading" may constitute a safety device for cellular C a  2 +  control.  The principle function of intramitochondrial C a  is believed to be the  2+  regulation of Ca -sensitive dehydrogenases which catalyse rate limiting 2+  production of reducing equivalents during tricarboxylic acid cycle flux (Denton and McCormack, 1985, 1989; Hansford, 1985). Transmitochondrial C a  2 +  flux  (and therefore matrix [Ca ]) is regulated by two independent pathways. C a 2+  2 +  uptake is mediated by a low affinity (Km=30uM at 3mM Mg ) transporter and 2+  is driven by an inside negative membrane potential (A*?) across the inner mitochondrial membrane established by H mediated electron transfer. C a  2 +  +  efflux coupled to cytochrome  efflux occurs via a specific N a / C a +  2 +  which mediates electroneutral exchange of two N a ions for one C a +  exchange is  driven by  an inwardly directed  Na  +  exchanger  2 +  gradient  ion. The (lOmM  extramitochondrial Na ) that is maintained by a N a / H exchanger coupled to +  +  +  the inwardly directed proton electrochemical gradient (Au. ). H+  b. Plasma Membrane The plasma membrane possess essentially 3 different types of C a devices: a Ca -ATPase; a Na+/Ca2+ exchanger; and C a 2+  2+  2 +  handling  channels.  (i). Calcium ATPase The Ca -ATPase is a high affinity low capacity C a 2+  cytosolic C a  2+  2 +  pump that extrudes  into the extracellular space. In erythrocytes and skeletal muscle  membranes Km values for C a  2+  of lu,M or less have been reported (Caroni and  Carafoli, 1981; Hidalgo et al., 1986) with 2-3 fold increased sensitivity in the presence of calmodulin. The Ca -ATPase of plasma membranes appear to be 2+  similar within a variety of different excitable and non-excitable cells including  14  erythrocytes (Penniston, 1982), squid axons (Post et al., 1969), smooth muscle (White and Blostein, 1982), t-tubules (Hidalgo et al., 1986) and cardiac and skeletal sarcolemma (Caroni and Carafoli, 1981; Mickelson et al., 1985; Michalak et al., 1984). Plasma membrane C a low cytosolic C a  2+  pumps are important in the maintenance of  2+  levels. Carafoli (1987) has suggested that this function, other  than in erythrocytes, may be supplemented by N a / C a +  owing to the low capacity C a  2+  exchange mechanisms  2 +  handling characteristics (10-30nmol.mg protein"  l.min'l). However, Hidalgo et al. (1986) argued that the relatively large surface area of t-tubule membranes implicates a major role for C a  2+  extrusion by cardiac  and skeletal plasma membrane/t-tubule C a pumps. 2+  (ii). Sodium-Calcium Exchange The exchange of cytosolic C a  2+  for extracellular N a is a carrier-mediated +  process which is especially prevalent in excitable tissues. Although N a / C a +  2 +  exchange is reversible (Reuter and Seitz, 1968) the main function of the carrier is the maintenance of low cytosolic C a  2+  levels. The electrogenic character of the  exchanger (3Na for lCa ) was demonstrated in the earlier vesicle studies of +  2+  intralumenal N a dependent C a +  2+  uptake with accumulation of the lipophilic  cation tetraphenylphosphonium in response to generation of an inside negative AY (Reeves and Sutko, 1980). The exchanger in contrast to the Ca -ATPase is a 2+  low C a  2+  affinity high capacity exchanger of C a  2 +  with reported Km values that  vary widely in vesicle (1.5 to 140uM) and intact tissue (180-350uM) studies (see Reeves, 1985).  In contrast, good agreement upon C a  2 +  transport rates (5-  30nmol.mg protein"^ .s.-i) by the exchanger (Reeves and Sutko, 1983; Philipson and  Nishimoto, 1983; Caroni and Carafoli, 1983) provided estimates of  transmembrane C a  2+  flux in intact tissue of 3-16pmol/cm /sec. 2  A low site  density was reported for the exchanger with turnover number estimated as high  15  as 1000 s" or more (Cheon and Reeves, 1988). 1  Recently, the N a / C a +  2 +  exchanger from cardiac sarcolemma was cloned with cDNA encoding a protein of 970 residues (Nicoll et al., 1990). N a / C a +  2+  was expressed when RNA,  synthesised from cDNA, was injected into Xenopus oocytes.  Structural  similarity was noted between the exchanger and the N a / K ATPase (Nicoll et +  +  al., 1990).  (iii). Calcium Channels In excitable tissues an additional pathway for C a membranes are a family of gated C a extracellular C a  2 +  2+  2 +  flux across the plasma  channels allowing the entry of  into the cytosol in response to membrane depolarisation.  These channels were originally categorised into low and high threshold C a  2 +  conductance pathways based upon voltage dependent gating properties (Llinas and Yarom, 1981). More recently, in accordance with the nomenclature adopted by Nowycky et al. (1985), C a  2+  channels in neuronal and muscle plasma  membranes are categorised as N,T,L, and P type channels (Llinas et al., 1989). In heart muscle L and T channels have been described (Bean, 1989). L-type channels can be distinguished by i) their dihydropyridine binding properties ii) large depolarisation requirement for activation iii) slow inactivation leading to long lasting C a  2 +  currents and iv) relatively large conductances (~15-25 ps with  lOOmM Ba carrier current) (Bean 1985, Mitra and Morad 1986). Converse properties characterise T-type channels which unlike L-type channels contribute little to the main C a  2 +  current (IQJ) during membrane depolarisation. The L-type  channel or dihydropyridine receptor is also preferentially localised to the Ttubules of skeletal muscle. (Hosey et al., 1989). However, less than 5% appear to function as C a  2+  channels (Schwartz et al., 1985).  16  It is now believed that skeletal muscle dihydropyridine receptors function as voltage sensors mediating responses of the SR ryanodine receptor to the t-tubule depolarisation (Leung et al 1988; Catteral et al., 1989; Tanabe et al., 1989; Adams v  et al., 1989). N-type (high threshold) C a  2+  channels are found largely in neuronal  tissue in addition to L- and T-types (Nowycky et al., 1985). A P-type C a  2 +  channel has also been described in Purkinje cells (Llinas et al., 1989) and bears some resemblance to N-type C a  2+  channels.  c. Endoplasmic Reticulum and Related Structures The endoplasmic reticulum (ER) of non-muscle cells share several of the C a  2 +  transporting structural features of sarcoplasmic reticulum. Liver and pancreas ER demonstrate Ca -ATPase activity with similar kinetic properties described 2+  for skeletal muscle SR (Bayerdorffer et al., 1984; Kraus-Freidmann al., 1985). In addition an intraluminal C a  2+  binding protein, calregulin, has been identified in  supernatants of bovine liver-homogenates  that although immunologically  unrelated to SR calsequestrin share similar C a 4 5  2 +  binding, Stains-All staining  and apparent molecular weight (63kDa) characteristics with the latter protein (Waisman et al., 1985; Khanna and Waisman, 1986; Damiani et al., 1988). A similar protein, chromogranin A, was identified in C a  2 +  releasing chromaffin  vesicles and microsomes isolated from bovine adrenal medulla (You and Albanesi, 1990). The release of C a  2+  from ER is stimulated by binding of TP to a specific 3  receptor protein. Recent cloning and expression (Furuichi et al., 1989) of IP3 receptor  (P400  protein) with a relative molecular mass of 250,000 (Ross et al.,  1989) reveals a remarkable sequence homology at the COOH region with the 565kDa ryanodine receptor in skeletal muscle Reconstruction of the  P400  (Mignery et al., 1989).  protein into vesicles demonstrated that the IP  3  17  receptor is likely the C a  2+  permeable pore mediating IP3 induced C a  2 +  release, in  vivo (Ferris et al., 1989). Recent immunological studies suggest that the IP C a  2 +  3  sensitive pool is localised to the ribosomes bearing endoplasmic reticulum the nuclear envelope and portions of the smooth endoplasmic reticulum (Ross etal., 1989). The relationship between these subcellular fractions and the smooth surfaced IP sensitive C a 3  2+  releasing "Calcisome" structures (Volpe et al., 1988) is  unclear, however.  d. Sarcoplasmic Reticulum The Sarcoplasmic Reticulum is an intracellular membrane structure in smooth, cardiac and skeletal muscle that regulates the pool of C a  2+  associated with the  transient triggering of muscle contraction. From intralumenal stores, C a transiently released and rapidly re-accumulated via a C a  2 +  2 +  is  channel and a C a 2+  ATPase, respectively. This structure is the focus of the current work and will be reviewed in more detail below.  III. Sarcoplasmic Reticulum Structure and Function  1. Historical Overview  The presence of an intracellular organelle with fine ultrastructure that closely associated with myofibrils was first described by Retzius (1890). Sarcoplasmic reticulum was originally prepared as a crude skeletal muscle particulate fraction by Kielley arid Mayerhof (1948) who demonstrated the presence of M g 2+  stimulated ATPase activity. Hill (1949) calculated that the presence of an internal Ca  2+  store was necessary to account for rapid contraction of fast-twitch skeletal  18  muscle. Later, Marsh (1951,1952) demonstrated that "relaxing factor" isolated from muscle extracts stimulated relaxation of actomyosin. Kumagai et al. (1955) demonstrated that the "relaxing factor" was particulate rather than soluble and Ebashi (1958) showed that the relaxing activity of the factor correlated with the associated ATP hydrolytic activity.  Concurrently, Hasselbach and Makinose  (1961) and Ebashi and Lipmann (1962) demonstrated that the particulate fraction was vesicular in nature and contained an ATPase that in the presence of both Mg  2+  and ATP could remove extravesicular C a . 2+  Description by electron  microscopy of an endoplasmic reticulum in muscle was initiated by Porter and Palade (1957). This was extended to a 3-dimensional reconstruction from serial sections demonstrating a close association of the reticulum with the t-tubule region of skeletal muscle (Anderson-Cedergren, 1959).  Later, Revel (1962)  identified the presence of junctional processes linking the t-tubule to the terminal cisternae of the reticulum. Autoradiographic demonstration of calcium release from the terminal cisternae and calcium sequestration by the longitudinal reticulum (Winegrad, 1965) supported an earlier speculation (Huxley and Taylor, 1958) that muscle contraction was stimulated by events initiated from the triads of t-tubule/reticulum junction. It is now clear that the sarcoplasmic reticulum is a closed intracellular membranous network in close association with the myofibrils and t-tubular membranes of muscle.  In response to t-tubule  membrane depolarisation the SR regulates muscle contraction and relaxation through the cyclical release and re-uptake, respectively, of C a the cytoplasmic space.  2. Morphology of SR  2 +  into and from  19  Sarcoplasmic Reticulum (SR) is an enclosed intracellular membrane system with two morphologically, and functionally distinct regions (Peachey and Franzini-Armstrong, 1983). The junctional region of SR (JSR) form cisternae in close proximity to the transverse tubular system (t-tubules). The free SR (FSR) forms the longitudinal and fenestrated regions of the reticulum connecting a pair of cisternae. The freeze-fractured FSR is densely populated with 85-90 A particles on the cytoplasmic fracture face.  The membrane is asymmetric with hardly  discernable indentations and few particles on the lumenal face (Deamer and Baskin, 1969; MacLennan et al., 1971; Franzini-Armstrong, 1975, 1980). The intramembraneous particles appear to represent the C a A particle density well correlated to the rate of C a  2+  2+  pump protein with 85  influx, the C a  2 +  stimulated  ATPase activity, and the relaxation rate of various muscles (Baskin, 1971; Rayns et al., 1975; Devine and Rayns, 1975; Martonosi, 1980). In fast twitch muscle 85 A particle density was earlier estimated with unidirectional shadowing of freezefractured replicas at 3,000-5,000.(im" across the FSR (Franzini-Armstrong, 1975). 2  More recently, rotary shadowing techniques estimated a 3 to 5 times higher particle density (Napolitano et al., 1983; Franzini-Armstrong and Ferguson, 1985). Slow twitch muscle particle density is approximately half that of fast twitch muscle (Jorgensen et al., 1983). The C a  2+  pump protein is transmembranous (Hidalgo and Ikemoto, 1977)  and protrudes from the cytoplasmic face as 40 A particles (Inesi and Scales, 1974; Saito et al., 1978). The discrepent density distribution of the cytoplasmic and intramembranous particles was suggested to be due to formation of clustered ATPase oligomers within the membrane (Vanderkooi et al., 1977). This notion received earlier support from observation of cross-linked oligomers (200 and 400kDa) in solubilised membranes (Le Maire et al., 1976; Chyn and Martonosi,  20  1977). However, Gingold et al. (1981) suggested that thefluorescenceenergy transfer technique of Vanderkooi et al. (1977) provided an unreliable estimate of the oligomeric state of the Ca -ATPase. In addition, Andersen et al. (1986) 2+  pointed out that oligomer formation in solubilised membranes depends upon the relative concentration of lipid, detergent and protein. Given the potential for methodological artifact, it remains uncertain to what extent intramembranous particles visualised from freeze-fracture replicas actually represent Ca -ATPase 2+  moleclues (see Heegaard et al., 1990). A pronounced morphological transition from the FSR to the JSR is observed with absence of 85 A particles and appearance of larger and less densely packed particles on the freeze-fractured cytoplasmic leaflet (Martonosi, 1968) and immunoferritin labelled membranes (Jorgensen et al., 1983). The sharp transition between the FSR and JSR was suggested to create an intralumenal free diffusional barrier to C a  2+  (see Martonosi, 1984).  The close apposition of the JSR and the t-tubules forms the triad structure. Flattened surfaces of 2 cisternal sacs of the JSR flank the junctional surface of a single t-tubule. In the myocardium and invertebrate muscle, a single element of the JSR occasionally forms a junction or diad with a t-tubule (Martonosi and Beeler, 1983). The junctional gap between the t-tubule and the junctional face membrane (JFM) of JSR is approximately 100-200 A with dense projections or "feet" connecting the cytoplasmic leaflets of the two membranous systems (Franzini-Armstrong, 1980). The feet are spaced with regular periodicity at 300 A intervals and arranged in rows of 2 or 3 on each t-tubule face (Jewett et al., 1971). The "feet" are associated with the JSR and are attached to the fragmented JSR membrane (Campbell et al., 1980). Additional junction spanning structures or "bridges" were earlier identified after tannic acid staining and were suggested to represent phospholipid (Somlyo, 1979). Later, junctional spanning "pillars"  21  were observed the density of which reportedly increased with the contractile state of the muscle (Eisenberg and Eisenberg, 1982).  Dulhunty (1989) has  suggested that "feet", "bridges", and "pillars" represent different images of the same junctional spanning structure. Recent evidence suggests that the junctional spanning material represents a complex of proteins including the dihydropydine receptor, the ryanodine receptor and, possibly, aldolase, phosphoglyceraldehyde dehydrogenase, and an, as yet, unidentified 95 kDa protein (Brandt et al., 1990; Kim et al., 1990). Additional features of the JSR/t-tubule region include (a) periodically spaced surface indentations on the cisternae close to the junctional gap (Dulhunty et al., 1983; Dulhunty and Valois, 1983) and (b) "tethers" which radially span the ttubule lumen and are aligned with "feet" and calsequestrin (see later) of a pair SR cisternae.  The function of the tethers is unclear although Dulhunty (1989)  suggested they may function as C a  2 +  buffers within the t-tubule lumen where  total [Ca ] may be 28mM. 2+  3. Protein Composition of SR  Mechanical disruption of muscle in salt or sucrose containing solutions fragments the SR membrane which forms sealed spherical vesicles or microsomes (mean diameter = 80-100nm). Meissner (1975) initially separated crude SR microsomes into populations of "light" and "heavy" SR vesicles. It is now well recognised that "light" SR (LSR) is composed largely of Ca -ATPase 2+  (over 90% of the total protein) and is referable to the longitudinal and fenestrated SR. "Heavy" SR is derived from the terminal cisternae and in addition to the Ca -ATPase (55-60% total protein) is (a) enriched in the electron dense acidic 2+  Ca  2+  binding calsequestrin and (b) contains much of the "feet" protein, in  22  particular the ryanodine receptor. The SR proteins comprise, approximately, 65% of the microsomal dry weight (Inesi, 1981) and, in addition to the above, contain several other proteins.  The best characterised of these include  sarcalumenin, a 53 kDa glycoprotein, calreticulin, and a 55kDa multifunctional thyroid hormone binding protein.  a. Calcium-ATPase Earlier studies showed that mild tryptic digest cleaves the Ca -ATPase 2+  into 55kDA and 45kDa peptides; fragments A and B, respectively (ThorleyLawson and Green, 1973; Inesi and Scales, 1974). Ca -ATPase activity was 2+  preserved and both fragments remained associated with the membrane. Incorporation of  3 2  P into fragment A localised the site of ATP hydrolysis  (Stewart et al., 1976). Extended digestion of fragment A resulted in 2 smaller subfragments; a 33kDa (Aj) fragment and a 22kDa (A ) fragment. Fragment B 2  remained intact.  The P label incorporated into the Ca -ATPase prior to 3 2  2+  extended digest was recovered from fragment A j .  Stewart et al. (1976)  sequenced fragment Aj and identified the active site as a phosphoaspartate residue.  Allen et al., (1980) deduced that the alignment of the fragments was  A - A | - B with A possessing the N-Ac-Met terminal sequence. 2  More recently,  2  Garcia de Ancos and Inesi (1988) suggested that the A j and A fragments form 2  the C a  2+  binding domain of the Ca -ATPase. Earlier, cDNA clones encoding 2+  two forms of rabbit skeletal muscle SR Ca -ATPase were obtained (MacLennan 2+  et al., 1985).  Consistent with tryptic digest studies secondary structural  predictions suggested the protein possessed 3 cytoplasmic domains. MacLennan et al. (1985) identified 10 transmembrane helices linked to the bulky cytoplasmic domains via a penta-helical stalk (see Figure 3). One domain (B) was identified as  the  nucleotide  binding  site  which  is  separate  from  the  23  Fig. 3. Structural diagram of the SR Calcium-ATPase protein (Calcium Pump).  The structural prediction of the SR Ca -ATPase is based upon hydrophobicity 2+  plots of the primary sequence.  Tj and T refer to the typtic cleavage sites 2  producing fragments A and B, and A | and A , respectively. S refers to the 2  pentahelical stalk sectors; M refers to the membrane spanning segments (taken from Brandl et al., 1986).  Figure 3.  25  phosphoaspartyl site (Aj) located upon an adjoining domain.  The third  cytoplasmic domain (A ) was suggested to be a transduction domain as cleavage 2  at the second tryptic site (located within this domain) uncoupled C a from ATPase activity which remains intact. suggested as the high affinity C a  2+  2 +  transport  The penta-helical stalk was  binding site due to enrichment of  amphipathic a helical segments with polar glutamate residues. Very little of the protein is located upon the lumenal face of the membrane. However, on the loop between the first and second transmembrane helices a group of 4 glutamic acid residues was suggested by MacLennan et al. (1985) to be the low affinity C a  2+  binding site. More recently Leberer et al. (1990) suggested that these residues may form a C a  2+  bridging structure with the acidic COOH domain of  sarcalumenin (see below).  Site directed mutagenesis of several polar  transmembranous residues revealed important contributions of 3 glutamic acids, one aspartate, one asparagine and one threonine residue in putative transmembrane segments M4, M5, M6 and M8 to the formation of high affinity Ca  2+  binding sites of the Ca -ATPase (Clarke et al., 1990a). In addition, 2+  substitutions at Thr 181, Gly 182, and Glu 183 within the (3-strand domain indicated that these residues were essential for E|P to E P conformational 2  transitions (Clarke et al., 1990b).  b. Ryanodine Receptor Cadwell and Caswell (1982) initially suggested that the high molecular weight SR proteins (300-325kDa), resolved by SDS-PAGE, possibly represented the components of the "feet" or "pillar" structures that had been identified earlier (Franzini-Armstrong, 1970; Eisenberg and Eisenberg, 1982). Seiler et al. (1984) later showed that these proteins bound calmodulin, were phosphorylated by  26  cAMP and calmodulin, dependent protein kinases and were degraded by calcium activated neutral protease (CANP). Kawamoto et al. (1986) were the first to purify the high molecular weight protein (~300kDa) from Zwittergent (3-14) detergent solubilised terminal cisternae vesicles and to recognise that protein dissolution of vesicles required moderate to high salt concentrations.  With affinity purified antibodies the  300kDa (spanning) protein was localised by immulogold staining of the tissue sections and rotary shadowed isolated protein to the junctional face membrane between the terminal cisternae and the t-tubules. This was rapidly followed by reports demonstrating that the 3-[(3-cholamidopropyl) dimethylammonio]-2hydroxy-l-propanesulphonate (CHAPS) solubilised high molecular weight protein bound ryanodine specifically in skeletal (Lai et al., 1987; Inui et al., 1987) and cardiac (Inui et al., 1987) muscle. Later, Imagawa et al.(1987) demonstrated that this protein , solubilised in digitonin, possessed single channel activity in planar bilayers consistent with it's identity with the C a Ca  2+  2+  permeable pore of the  release channel. This was later confirmed by Lai et al. (1988a,b), Hymel et  al. (1988), Smith et al. (1988), Anderson et al. (1989). Subsequent studies have shown that the functional ryanodine binding C a  2 +  channel complex is a 30S  quatrefoil structure composed of four identical 9S monomers (Lai et al., 1989). Each leaflet of the 30s particle represented a single ~450kDa protein that self associated cooperatively to form a C a  2+  conducting homotetramer.  Complimentary DNA for the rabbit form of the protein has been sequenced and expressed (Takeshima et al., 1989).  These studies deduced a protein  sequence of 5,037 amino acids and a molecular weight of 565,223. Later, Zorzato et al. (1990) obtained cDNA clones for human and rabbit forms of the ryanodine receptor in which the former encoded for a protein of fewer amino acids (5032) and lower molecular weight (563,584). Otsu et al. (1990) then obtained a cDNA  27  clone encoding the rabbit cardiac form of the release channel with slightly lower molecular weight than the skeletal form (4,969 residues; Mr=564,711). A 66% sequence homology between the skeletal and cardiac proteins was reported (Otsu et al., 1990). Major differences were noted in the presence of a highly acidic glutamate rich domain (residues 1872-1923) in the skeletal form which were not well conserved in the cardiac form. Imagawa et al. (1989) showed that the cardiac form was immunologically distinct from isoforms expressed in fasttwitch and slow-twitch muscle. In both skeletal and cardiac forms a regulatory domain was identified that contained potential binding sites for calmodulin and nucleotides (Zorzata et al., 1989; Otsu et al., 1990). These domains were found to lie within a protease-sensitive region of the receptor (Marks et al., 1990). Otsu et al. (1990) suggested that the dispersed sequence differences noted between the two proteins  reflects the fact that they represent different gene products.  Mackenzie et al. (1989) showed that the human cardiac form is derived from chromosome 1 as opposed to the skeletal form located on chromosome 19. For both the cardiac and skeletal receptor protein secondary structural predictions revealed the presence of 12 potential transmembranous segments (Zorzato et al. 1989; Otsu et al., 1990). This is different from earlier predictions (Takeshima et al., 1989) of only 4 transmembrane segments. Negatively stained images of the purified receptor revealed the presence of protein "loops" surrounding a low density region or cavity (2-4nm) associated with each leaflet of the quatrefoil (Lai et al, 1989; Anderson et al., 1989). These were identified as "peripheral vestibules" (Wagenknecht et al., 1989). The apposition of each leaflet formed a "central channel" (Wagenknecht et al., 1989) of 12-14nm in diameter (Lai et al., 1989). Wagenknecht et al (1989) report apparent "radial channels" emanating from the central cavity and connecting with the "peripheral vestibules".  These authors proposed that these structures may represent  28  pathways for C a  2+  release. However, Liu et al. (1989) argued that the cavities  within each leaflet were too large to constitute ion channels. The pathway for Ca  2+  release within the ryanodine receptor molecule remains, therefore, to be  elucidated. A [ H]ryanodine binding protein (~400kDa) was recently isolated from crude 3  rabbit brain membranes (McPherson and Campbell, 1990).  This protein  sedimented as an apparent homotetramer (30s) in sucrose gradients and was immunologically cross-reactive to monoclonal antibodies raised against the rabbit skeletal muscle (fast-twitch) isoform. The precise tissue distribution of this brain protein is uncertain. However, it raises the intriguing possiblity that a Ca  2+  control mechanism similar to that found in muscle also operates in brain.  c. Calsequestrin Calsequestrin, one of the major accessorial proteins of the HSR, is an acidic glycoprotein (Maclennan and Campbell, 1979) that binds C a  2 +  with  variable affinity that is dependent upon ionic strength (Kd=lmM at lOOmM KC1) (MacLennan and Wong, 1971). Calsequestrin is localized within the terminal cisternae upon the lumenal surface (Hidalgo and Ikemoto, 1977) and is thought to be a major intralumenal C a Ca  2+  2+  buffer with a C a  2 +  binding capacity of 40-50 mol  per mol protein (Reithmeier et al, 1987). Jorgensen and Campbell (1984)  showed that calsequestrin was also present in non-junctional regions of the SR (corbular SR) that appeared to be specialised regions of the fenestrated SR localised to the I-band of chicken ventricular muscle.  The concentration of  calsequestrin within the cisternae was estimated at lOOmg per ml internal cisternal volume (Williams and Beeler, 1986) such that at saturation calsequestrin can bind lOOmM C a  2+  (or 1000nmol.mg-l protein).  The cDNA for skeletal  muscle calsequestrin encodes a protein of 367 residues with a molecular weight  29  of 42,445 (Zarain-Herzberg et al., 1988). The complete amino acid sequence has also been obtained for the cardiac isoform with a deduced molecular weight of 45,269 and 65% sequence homology with the skeletal form (Scott et al., 1988). Apparent molecular weight estimates derived from alkaline gels may be as high as 63,000 in skeletal muscle and 55,000 in cardiac muscle. This is attributable to the highly acidic character of this protein.  Cozens and Reithmeier (1984)  reported that calsequestrin is highly asymmetric with an extended structure created by the electrostatic repulsion amongst neighbouring negative charges. In addition, calsequestrin reportedly underwent large conformational transitions from an extended structure in the absence of C a  2 +  to a compact, globular  structure in it's presence (Cozens and Reithmeier, 1984).  At physiological  concentrations of calsequestrin, however, secondary structural transitions in solution were relatively invariant with alterations in C a  2 +  and ionic environment  (Williams and Beeler, 1986). On the other hand, Williams and Beeler (1986) showed that the crystalline structure of calsequestrin was sensitively dependent upon ionic conditions. Recent evidence suggests that calsequestrin may belong to a multigene family with remarkable homology to a putative laminin-binding protein, apartactin (Yazaki et al., 1990). In this regard Fliegel et al. (1987) identified the presence of a Ser-Glu-Glu sequence which is found in sarcalumenin, 53kDa glycoprotein, and the, recently identified, 165kDa histidinerich calcium and low density lipoprotein binding protein (HCP) of sarcoplasmic reticulum (Hofmann et al., 1989) together with troponin C and calmodulin (Fliegel et al., 1990). This motif appears to be important in protein-protein interactions (Earnshaw, 1987).  d. Calreticulin  30  Earlier known as the 55kDa high affinity C a  2+  binding protein (HABP),  calreticulin was found to bind 1 mol Ca /mol protein with high affinity 2+  (Kd=3|iM) and 25 mol Ca /mol protein with low affinity (MacLennan et al., 2+  1972; Ostwald and MacLennan, 1974). Recently, calreticulin was cloned with cDNA encoding for a protein of unexpectedly lower molecular weight (Mr=46,567) than was earlier determined from both acidic and alkaline gels (Fliegel et al., 1989). The carboxyl terminus of calreticulin is highly acidic and may represent the C a  2+  binding domain (Fliegel et al., 1989). The function of  calreticulin is unknown although the KDEL (Lys-Asp-Glu-Leu) structural motif at the COOH terminal suggests that it may be associated with protein retention within the SR lumen (Fliegel et al., 1989, 1990). Calreticulin was shown to be localised to the lumen of the terminal cisternae and is structurally homologous to Calregulin, an ER associated protein (Fliegel at al., 1989).  e. 53kDa Glycoprotein  Using Laemmli (Laemmli, 1970) gels to separate and identify possible contaminants within the 55kDa band of Weber and Osborne gels (Weber and Osborn, 1969), Michalak et al. (1980) discovered a glycoprotein with an apparent molecular weight of 53,000. Unlike the glycoprotein of the Na +K -ATPase, the +  +  53 kDa protein of purified SR did not form a tight association with the C a 2+  ATPase.  The 53 kDa glycoprotein was later purified and shown to be  transmembranous owing to it's reactivity with concanavalin A in leaky vesicles and cycloheptaamylose-fluorescamine complex in sealed vesicles (Campbell and MacLennan, 1981). Recently, cDNA clones were obtained that encoded a protein of 453 residues and a molecular weight of 52,421 (Leberer et al., 1989). The protein was found not to regulate ATPase activity with little evidence of C a  2 +  binding properties. The function of this protein remains unclear although the  31  COOH terminal is identical to that of the C a  2+  binding sarcalumenin (see below).  This together with the co-localisation of the 53kDa protein with sarcalumenin led Leberer et al. (1990) to suggest that this protein may be involved with C a  2+  sequestration in non-junctional SR.  f. Sarcalumenin Sarcalumenin is a 160kDA glycoprotein that was found to be immunologically related to the 53kDa glycoprotein (Leberer et al., 1989). cDNA clones obtained by Leberer et al. (1989) encoded an unprocessed protein of 889 residues, substantially less than would account for a protein of 160kDa. Transfection of COS-1 cells with the cDNA resulted in expression of the mature 160kDa protein. The COOH terminal was found to be highly acidic and to bind 400nmol Ca /mg protein with intermediate affinity (Kd=300-600uM) depending upon 2+  ionic strength (Leberer et al., 1990).  g. 55kDa Thyroid Hormone Binding Protein The presence of 55kDa multifunctional thyroid hormone binding protein (T3BP)  in cardiac and skeletal SR membranes was indicated from earlier studies  (Hiegel et al., 1989). Anti-calreticulin antibodies were found reactive to the COOH terminus of T3BP  T3BP  and enabled isolation of full length cDNA clones for  (Fliegel et al., 1990). The COOH terminus was found to highly acidic and  contained a variant, RDEL (Arg-Asp-Glu-Leu), of the KDEL retention signal found in several heat shock and glucose regulated proteins.  4. SR Calcium Uptake The function of SR in cardiac and skeletal muscle is to provide both a source and sink for C a  2+  that, upon binding to troponin-C, stimulates  32  myofilament shortening and therefore muscle contraction. The release of C a  2 +  occurs from the terminal cisternae in response to t-tubule membrane depolarisation. Relaxation of muscle occurs via Ca -ATPase mediated C a 2+  2 +  uptake into the longitudinal or FSR.  a. Ca^ transport +  Ca  2+  transport by the Ca -ATPase occurs with the binding of both the  translocated C a  2+  2 +  ion and the MgATP to specific sites located upon the  cytoplasmic face of the enzyme (Weber et al., 1966). C a  2+  binding, which has  been studied with native and reconstituted vesicles and purified Ca -ATPase, 2+  occurs with high affinity in the absence of ATP (Meissner, 1973: Ikemoto, 1974; Chiu and Hayes, 1977). Three classes of C a  2+  binding sites were identified in the  purified enzyme (Ikemoto, 1975). At 0°C all three binding sites (ce,p\y classes) were expressed with association constants (Ka) and number of sites (n) per class as follows: a sites (Ka=3xl0 M , n=l);p sites (Ka=5xl0 M" ,n=l); y sites 6  (Ka=10^M"^, n=3).  _1  4  1  Above 22°C, however, the number of a sites increased to 2  with suppression of (3 site binding and maintenance of low affinity of y site binding. Inesi et al. (1980) showed that C a  2+  binding to the high affinity sites of  Ca -ATPase is cooperative with a derived Hill coefficient of 1.82 and apparent 2+  Ka of 2.3x1 O^M"!. C a  2 +  binding to the external high affinity sites of the C a 2+  ATPase induced a slow conformational changes in the protein (Pick and Karlish, 1980) and was considered to be one of the rate limiting steps of the catalytic cycle (Dupont, 1977; Pick and Karlish, 1982). MgATP is the true substrate for translocation of C a  2 +  (Vianna, 1975). ATP  binds to the transport enzyme with high affinity (Meissner, 1973) only as a Mg.nucleotide complex (Hasselbach et al. 1981). suggested that M g  2+  Hasselbach et al. (1981)  functions both as an ionic cofactor and a charge  33  compensator for the polyphosphate residues of the unliganded nucleotide substrate.  The resulting Mg-ATPase-enzyme complex is formed by random  binding of the cofactor and substrate to the translocator. C a  2 +  and MgATP bind  in a molar ratio of 2 as determined from Hill plot coefficients under optimal conditions (Tada et al., 1978). 8-9nmol C a  2 +  can bind per mg of Ca -ATPase 2+  with 4-4.5 nmol phosphoenzyme (EP) formed with each turnover of the pump (Inesi et al., 1982). C a  2+  and Mg-ATP binding was earlier thought to occur  randomly as implied from the apparent lack of allosteric effect of the two binding sites (Vianna, 1975). However, Chiu and Haynes (1980) and Pick and Karlish (1982) showed that C a  2 +  occupation of the high affinity sites prior to  substrate binding is crucial for formation of the influx stabilized form of the enzyme. Inesi et al. (1980), and Martonosi and Beeler (1983) suggested that the active quaternary complex may occur with successive C a  2 +  binding steps which  precede and follow Mg-ATP binding. Formation of the active quaternary complex leads to hydrolysis of ATP where y phosphate is covalently attached to Asp 351 (De Ancos and Inesi, 1988) with formation of an acid stable, alkaline labile EjP enzyme (see Figure 3). C a  2 +  translocation is coincident conversion of an ADP-sensitive E^P to an ADPinsensitive E2P (Takisawa and Makinose, 1983). The energy of this isomerisation step is transferred into osmotic work and the creation of an inward C a associated with reduction of C a al., 1981). Following C a  2+  2+  2 +  gradient  affinity (Chiu and Haynes, 1980; Hasselbach et  translocation E2P decays with liberation of Pi and  formation of E2. The cycle is then repeated with binding of C a (Kawashima et al., 1990).  b. Calcium Pump Regulation  2 +  and Mg.ATP  34  (i) . Calcium Under optimal conditions of MgATP, pH and temperature, rapid C a in SR vesicles, is stimulated at C a Ca  2 +  2+  inhibit C a  2 +  2+  influx  concentrations above 0.01 uM with maximal  influx occurring in the presence of 1-10JJM C a  Hasselbach, 1979). External C a  2 +  2 +  (Weber et al., 1963;  concentrations above 29uM were found to  accumulation (Chiu and Haynes, 1980). Although prior occupation  of the external high affinity C a  2+  binding sites stabilizes the Ca -ATPase in it's 2+  influx mode (Chiu and Haynes, 1980) rising C a  2 +  concentrations form an  inhibitory Ca-ATP complex (Kanazawa et al., 1971; Hasselbach et al., 1981). CaATP, under particular conditions may serve as a substrate for the Ca -ATPase 2+  (Wakabyashi and Shigekawa, 1984) although the partial catalytic reactions were considerably slower than with Mg-ATP as the substrate (Shigekawa et al., 1983). Elevated intralumenal C a (Yamada et al., 1972).  2+  (~20mM), inhibits C a  2+  2 +  accumulation  The inhibitory mechanism is uncertain although  stimulation of Ca -ATPase mediated C a binding of C a  2 +  2+  efflux through low affinity site  may occur at elevated intralumenal C a  2+  (Hasselbach and Waas,  1982).  (ii) . Magnesium Magnesium regulation the of Ca -ATPase is complex. The complex of 2+  Mg  2 +  and ATP is the physiological substrate for the Ca -ATPase (Weber et al. 2+  1966; Makinose and Boll, 1979) with the cation acting, possibly, as an ionic cofactor or change compensator for the nucleotide (Hasselbach et al., 1981). Mg  2 +  is thought to remain bound or in an occluded state (EDTA  inaccessible) upon initial EP formation (Hasselbach and Oetliker, 1983) and has been shown to activate the reversible transphosphorylation from ATP to the enzyme (Hasselbach et al., 1981). M g  2 +  accelerated the EjP to E P transition 2  35  (Shigekawa and Dougherty, 1978) and was essential for the phosphorylation of the  Ca -ATPase  by  2+  Pi  (Masuda  and  DeMeis,  1973)  and  enzyme  dephosphorylation as monitored through Pi-P^O-exchange (Kanazawa and Boyer, 1973). M g  may act a single site although evidence is inconclusive (see  2 +  Makinose and Boll, 1979; Yamada and Tonomura, 1972; Shigekawa et al., 1983; Takakuwa and Kanazawa, 1982) Millimolar concentrations of M g and efflux, with C a Taylor, 1976). M g  2+  2 +  2 +  (l-5mM) stimulate both C a  influx inhibition at elevated M g inhibition may due to M g  2 +  2 +  2 +  influx  (lOmM) (Froelich and  competition with C a  2 +  for  binding (Yamamoto et al., 1979; Haynes, 1983)  (iii). ATP The vesicular form of the Ca -ATPase is non-linearly dependent upon 2+  ATP for both C a  2+  transport and ATPase activity (Martonosi and Beeler, 1983).  Mg-ATP, in addition to substrate provision (Hasselbach et al., 1981), stimulates a secondary activation of the Ca -ATPase at higher substrate concentrations. Up 2+  to 100|iM Mg-ATP, hydrolysis is stimulated with first order kinetics. Above O.lmM Mg-ATP a secondary activation of ATP hydrolysis is observed (Mcintosh and Boyer, 1983) with two distinct Km values of 2-3u.M and 500 u,M (Moller et al., 1980). The biphasic activation of the Ca -ATPase indicates the presence of 2 2+  classes of Mg-ATP binding sites (Hasselbach, 1979). The high affinity site is the catalytic site whereas the low affinity site has a regulatory function (Dupont, 1977). Chiu and Haynes (1980) and Haynes (1983) proposed that these effects were mediated through a single Mg-ATP binding site of variable affinity that was dependent upon cationic concentration.  36  5. SR Calcium Release  a. Overview Muscle contraction in skeletal and cardiac muscle results from relief of troponin-I inhibition of myosin and actin interaction through C a troponin-C (Leavis and Gergely, 1984). The source of C a  2 +  2 +  binding to  is the terminal  cisternae of the sarcoplasmic reticulum. In response to a depolarising action potential propagated along the t-tubule the SR releases C a  2+  via the C a  2 +  permeable pore of the ryanodine receptor with a first order rate constant between 50 and 100 s"* observed in frog skeletal muscle (Baylor et al., 1983; Melzer et al., 1984). However, the mechanism by which the electrical signal leads to the triggering of C a  2+  release remains elusive.  b. Triggering Mechanism of SR Calcium Release. Two main hypotheses have been forwarded to account for the triggering of Ca  2+  release in both skeletal and cardiac muscle.  The first proposes that a  mechanical coupling between the dihydropyridine receptor of the t-tubules and the ryanodine receptor of the terminal cisternae mediates a non-linear charge transfer across the t-tubule/SR junction. The second proposes that C a  2 +  release  is stimulated by transient local increases in the production of second messengers in response to t-tubule depolarisation (e.g. C a  2 +  and/or IP3).  (i). Charge Movement Triggered Calcium Release The charge movement theory is based upon the earlier observation (Schneider and Chandler, 1973) of extra outward and inward currents measured after voltage pulses were applied to voltage clamped frog sartorius single muscle fibres. Three key features of the charge movement were that i) the total charge  37  movement during excitation and recovery was conserved but of opposite sign ii) the amount of charge was saturable and sigmoidally related to the voltage pulse and iii) the rate constants of the outward ("on" pulse) and inward ("off" pulse) charge transients were strongly voltage dependent (range 179-249 s"^ at 80mV). Charge movement was not, therefore, rate limiting and could account for the strong voltage dependence of SR C a  2+  release. In addition the results implicated  movement of a fixed number of charges rather than movement of intra- of extracellular ions.  Chandler et al. (1976) proposed that charge movement  occurred between membrane bound charged groups which act either as a reorientable dipole or can move in response to changes in membrane electric field. Schneider and Chandler (1973) calculated that the amount of charge movement was consistent with the density distribution (700 per u ) of the t-tubule/SR 2  spanning feet protein (Franzini-Armstrong, 1970). Later Rios and Brum (1987) implicated the involvement of the L-type C a  2 +  channel /dihyropyridine receptor in mediating charge movement through demonstration of reduced charge movement and SR C a  2 +  release in response to  nifedipine at concentrations consistent with the affinity (kd=32nm) of the receptor for dihydropyridines. Similar effects upon contractility (Eisenberg et al., 1983) and charge movement (Hui et al., 1984) inhibition were also observed with A-600, a related dihydropyridine compound (Morad et al., 1983).  Further  evidence that the skeletal muscle receptor is the voltage sensor mediating intramembrane charge movement arises from demonstration that expression of skeletal muscle cDNA in dysgenic myotubes restores normal skeletal type E-C coupling (Tanabe et al., 1988, 1990; Adams et al., 1990). The critical regions appear to reside within the C a  2+  conducting aj subunit (Pelzer et al., 1990) upon  the proposed cytoplasmic domain between repeats 2 and 3 of the protein ( Tanabe et al., 1990). Fill et al. (1988,1990) also observed non-linear charge  38  movement and capacitance properties associated with the voltage dependence of the ryanodine receptor open time in channels incorporated into Mueller-Rudin bilayers.  Futhermore, Ma et al. (1988) have demonstrated gap junction  properties associated with the voltage dependence of the receptor open state probability. Such properties would be expected of a channel sensitive to "gating" currents. These studies provide some evidence toward a molecular basis for Schneider and Chandler's (1973) original hypothesis.  (ii). Calcium-Induced Calcium-Release The phenomenon of regenerative or Ca -induced Ca -release (CICR) was 2+  2+  first demonstrated by Ford and Podolsky (1970) and Endo et al. (1970) using skinned (split) fibre preparations from frog skeletal muscle. Numerous studies have since demonstrated the presence of this phenomenon in skinned fibres and isolated vesicles from both cardiac and skeletal muscle.  Ca  2 +  release from  skeletal and cardiac SR vesicles was maximally stimulated by 5-1 OuM C a  2 +  in the  presence of 5mM adenosine 5'-(P,y-methylene) triphosphate (Meissner et al., 1986; Meissner and Henderson, 1987) with release rate constants (30-100 s"^) compatible with physiological C a  2 +  release.  In the presence of nucleotides  (AMP-PCP) and M g , channel mediated C a 2+  dependent upon trigger C a  2+  2+  efflux was cooperatively  (Meissner et al., 1986). In skeletal muscle the  apparent affinity for trigger C a  2+  et al., 1989). Amounts of C a  release rather than release rate constants were a  2+  binding is approximately 10^M"^ (see Ikemoto  graded function of intralumenal C a  2+  loads (Kim et al., 1983).  Endo (1977) has consistently argued, based upon earlier observations the CICR may not constitute a physiological mechanism of C a  2+  release, in skeletal muscle  at least, due to it's observation under apparently unphysiologically exaggerated conditions (elevated M g , high intralumenal Ca ). 2+  2+  Fibre studies have  39  indicated (see Winegrad, 1982) that despite the regenerative nature of CICR, this form of C a  2 +  release does not produce maximal contractions. Furthermore,  Stanfield and Ashcroft (1982) concluded that inward C a  2 +  currents were too slow  to account for the rate of E-C coupling. However, Stephenson (1982) proposed that earlier fibre studies erroneously assumed tight control of myofilament space Ca  2+  by bath Ca -EGTA buffers. Without adequate control of C a 2+  2 +  within the  immediate vicinity of the SR, particularly within unstirred layers, description of the physiological significance of CICR is limited. The significance of CICR in cardiac muscle is more accepted due to the apparent increased sensitivity to trigger C a  2+  in this tissue with low C a  2 +  pre-  loading of the SR (Fabiato and Fabiato, 1975). Futhermore it was shown that an increase in free M g  2 +  up to a presumed physiological level (3.16mM) actually  potentiated the amplitude of SR C a  2 +  release in skinned cardiac cells and cat  caudofemoralis muscle (Fabiato, 1981, 1982). Fabiato (1983) argued that the gating stimulus is more a function of the rate of change of the free C a than the C a  2 +  2 +  rather  concentration per se. It was suggested that failure to account for  this in previous studies with skeletal muscle fibres may explain why CICR has been poorly demonstrated in this model. Assessment of the physiological role of CICR from studies upon vesicles depends critically upon estimates of intracellular free M g . 2+  Estimates using  different metallochromic dyes varied between 200nM and 6mM free [Mg ] 2+  depending upon assignments of intracellular pH (Baylor et al., 1982). In vesicle studies (Meissner et al., 1986) M g  2 +  concentrations approximating those found  intracellularly (0.2-4mM) reduced significantly the first order rate constant (at 5(iM Ca ) for CICR although rapid releases were still observed. The conditions 2+  under which releases were observed in vitro (22 °C) may have underestimated the true release rates in vivo at 37 °C.  40  More recent studies with intact cardiac cells using fluorometric dyes Indo-1 and Fura-2 demonstrated that the voltage dependent C a  2 +  current (Ic ) fr° L" m  a  type channels is well correlated with the transient rise of myoplasmic C a  2 +  (Ca  2+  transient) released from the SR (Callewaert et al., 1988; Barcenas-Ruiz and Wier, 1987; Beucklemann and Weir, 1988; Nabauer et al., 1989). These observations clearly support a mechanism of C a  2+  induced C a  2 +  release in the heart.  However, recent studies (Cannell et al., 1987; Cohen and Lederer, 1987, 1988; Lederer et al., 1989) have demonstrated that the voltage dependent peak C a transient precedes the voltage dependent IQ indicating stimulation of C a  2 +  2 +  &  release prior to activation of trigger C a  2+  flux. These observations which support  the involvement of charge movement have been criticised (see Fabiato, 1989) on the basis of potential methodological artifacts triggering cardiac SR C a Therefore, it remains unclear whether SR C a  2 +  2 +  release.  release from either cardiac or  skeletal muscle involves a single triggering mechanism (i.e. CICR vs charge movement) or whether SR C a  2+  release is triggered by both mechanisms in a  highly complex manner. Recently, Nesterov (1989) has postulated that an initial Ca  2+  release from channels within the immediate vicinity of the t-tubule/SR  junction is dependent upon ion or charge movements may initiate a regenerative or C a  2+  induced C a  2 +  release from more laterally disposed C a  2 +  channels.  c. Modification of Calcium Release  (i). Nucleotides Rapid kinetic studies of C a  2+  release (Meissner et al., 1986; Morii et al., 1986;  Moutin and Dupont, 1988) from SR vesicles passively loaded with C a  2 +  demonstrated up to 25 fold increases in the release rate constant in the presence of adenine nucleotide (5mM AMP-PCP or ATP).  In the cardiac SR, C a  2 +  41  triggered release rates were stimulated by over 2 orders of magnitude in the presence of nucleotide (Meissner and Henderson, 1987). The amount of C a triggered release of C a  2+  2 +  was relatively unaffected by nucleotide which  stimulated release in the following order of effectiveness; AMP-PCP > cAMP > AMP > ADP > adenine > adenosine (Meissner, 1984). With depolarisation induced C a  2 +  release, nucleotides elevated amounts of release without effects  upon the first order rate constant (Kim et al., 1983; Ikemoto et al., 1984, 1985). The effects of nucleotides appear to be mediated by direct action upon the channel itself (Lui et al., 1989).  (ii) . Extravesicular Calcium Ca  2+  release induced by t-tubule depolarisation was shown not to require  extravesicular C a  2+  in both fibre studies (Endo and Nakajima, 1973; Stephenson,  1981) and vesicle studies (Ikemoto et al, 1985). However, the C a depolarisation and C a  2 +  2 +  stimulation of  induced release was shown to be similar (Ikemoto et al.,  1985) exhibiting a bell shaped dependence upon extravesicular C a  2 +  between  pCa 6 to 5 in agreement with similar reports (Moutin and Dupont, 1988; Meissner et al., 1986). These observations indicated that both forms of release were mediated by the same C a  2+  channel (see Ikemoto et al., 1989).  (iii) . Drugs In numerous studies with whole cells, peeled fibres, vesicles and release channels incorporated into bilayers, a variety of different drugs have been shown to activate SR C a  2+  release directly.  Caffeine was earlier shown to stimulate C a Ca  2+  2+  release at sub-threshold cytosolic  (Ogawa, 1970, Weber and Hertz, 1968) and to decrease the C a  for Ca -induced C a 2+  2 +  2 +  threshold  release (see Endo, 1977). In both cardiac (Meissner and  42  Henderson, 1987) and skeletal vesicles (Katz et al., 1977; Kim et al., 1983; Su and Hasselbach, 1984; Rousseau et al., 1988) caffeine stimulated C a  2 +  release with a  reported Km value of 200 uM (Kim et al., 1983). Recent evidence suggested that a 170kDa glycoprotein may be a common receptor for C a  2 +  and caffeine  mediated effects (Rubtsov and Murphy, 1988). This is at variance with a direct effect of caffeine to increase the open state probability of isolated channels incorporated into bilayers (Lui et al., 1989). Quercetin, an inhibitor of the Ca -ATPase (Shoshan et al., 1980; Shoshan and 2+  MacLennan, 1981; Gilchrist et al., 1990) has been shown to stimulate (Palade et al., 1983) SR C a  2+  release (Palade, 1987; Ikemoto et al., 1983) at low concentration  (Km ~3u.M). Doxorubicin (Adriamycin) was also shown to stimulate unidirectional C a efflux (8 fold) with inhibition of SR C a stimulation of C a  2 +  2+  2 +  uptake and greater than 2 fold  dependent ATPase activity (Zorzato et al., 1985). Part of the  cardiotoxic effects of doxorubicin (Caroni et al., 1981) may be due, in part, to its' action upon the SR C a  2+  release channel.  Another drug with toxic effects upon skeletal, cardiac and smooth muscle is the alkaloid ryanodine. Ryanodine exhibits complex inhibitory and stimulatory effects upon SR C a  2 +  release channel function (Jenden and Fairhurst, 1969). It is  now known that ryanodine binds to the release channel at both low and high affinity sites the expression of which is dependent upon assay conditions (see Lai and Meissner, 1989). Ryanodine binding affinity is elevated by micromolar C a , 2+  and millimolar concentrations of adenine nucleotides and caffeine (Lai and Meissner, 1989; Lai et al., 1989; Chu et al., 1990). Ryanodine binding was also dependent upon osmolarity with optimum binding observed in the presence of IM NaCl (or KC1) (Pessah et al., 1987; Inui et al., 1987; Michilak et al., 1988; Chu et al., 1990; Ogawa and Harafuji, 1990).  43  The correspondence between ligand effects upon ryanodine binding and the Ca  2+  releasing behaviour of the SR suggest that ryanodine binds preferentially to  the open state C a  2+  channel (Pessah et al 1986; Lai et al., 1989; Chu et al., 1990). v  Consequently, ryanodine binding to SR membranes and ryanodine effects upon Ca  2+  fluxes may reflect C a  ryanodine upon C a  2 +  2+  channel conformation(s). The inhibitory effects of  uptake in sarcotubular fragments (crude SR) were first  observed by Fairhurst and Jenden (1966) and Jenden and Fairhurst (1969). Fairhurst and Hasselbach (1970) observed that ryanodine (100|iM) preferentially increased C a  efflux from the heavy SR C a  2 +  2 +  loaded fraction with increased  Ca -ATPase activity. More recently, studies upon junctional SR vesicles and 2+  Ca  2+  channels incorporated into bilayer have demonstrated that with low  micromolar concentrations, at least, ryanodine inhibits the ATP dependent uptake of C a passive C a 4 5  (Lattanzio et al., 1987; Fleischer et al., 1985 and Inui et al., 1988)  2+  2 +  loading of vesicles (Meissner, 1986) and maintains the open state  of the high conductance C a  2+  channel (Nagasaki and Fleischer, 1988; Lai and  Meissner, 1989). These effects have been attributed to high affinity ryanodine binding resulting in maintenance of open channel states (Fleischer et al., 1985). The C a  2 +  releasing action of ryanodine was also observed by Hansford and  Lakatta (1987) in cardiac myocytes. Contrary effects of ryanodine upon SR function (i.e. inhibition of C a opening or C a  2 +  release) have also been reported.  2 +  channel  Within smooth muscle  preparations ryanodine (~10^iM) was shown to diminish depolarisation (Marban et al., 1985; Sutko and Kenyon, 1983), K and norepinephrine (Ashida et al., 1988; +  Iko et al, 1986), and caffeine and carbachol evoked contractures (lino et al., 1988; Iko et al., 1986). These observations parallel similar ryanodine effects upon tension and C a  2+  transients in cardiac and purkinje fibres (Cannell et al., 1985;  Wier et al., 1985) and skinned cardiac cells (Fabiato, 1985; Sutko et al., 1985).  44  Ryanodine also markedly stimulated C a  2 +  accumulation and inhibited C a  2 +  release in heavy SR vesicles derived from the terminal cisternae of cardiac and skeletal muscle (Jones and Besch, 1979; Jones et al., 1979; Chamberlain et al., 1983; Hasselbach and Migala, 1986; Meissner, 1986 and Lattanzio et al., 1987). Meissner (1986) suggested the contrary effects of ryanodine on SR C a  2 +  handling may reflect differential action of more than one ryanodine binding site. Moreover, the expression of these effects was dependent upon temperature, assay conditions and time of exposure to ryanodine (Meissner, 1986; Lattanzio et al., 1987). Although it is clear that C a  2+  influences the expression of the number  of ryanodine binding sites (Lai et al, 1989) it is unclear whether this reflects an effect of C a  2+  upon extraluminal or intraluminal C a sites. 2 +  (iv) . Magnesium Numerous studies have shown that at millimolar (1-lOmM free Mg ) 2+  concentrations (Meissner, 1984; Meissner et al. 1986; Meissner and Henderson 1987; and Gilchrist, et al., 1990) inhibits the release of C a skeletal SR vesicles. Similarly, M g  2+  2 +  from cardiac and  reduced the open state probabilities of  purified and vesicle associated channels incorporated into bilayers (Liu et al., 1989; Rousseau et al., 1986; and Smith et al., 1985). The mechanism by which Mg  2+  acts remains unclear (Meissner et al., 1986) although the above studies  indicate a direct effect upon the release channel.  (v) . Anaesthetics In skinned skeletal fibre studies procaine (Thorens and Endo, 1975; Ford and Podolsky, 1970) and tetracaine (Thorens and Endo, 1975) inhibited C a Ca  2+  release and caffeine induced (Feinstein, 1963) contractures.  2 +  induced Similar  inhibition of CICR was observed in isolated cardiac and skeletal muscle vesicles  45  with SKF 525-A and procaine (Chamberlain et al., 1984) and tetracaine (Ohnishi, 1979; Antoniu et al., 1985; Morii et al., 1986; Meissner and Henderson, 1987). Conversely, halothane has been shown to activate SR C a subthreshold extraluminal C a  2 +  2+  release directly at  levels required for CICR (Ohnishi, 1979; Ohnishi  et al., 1983; Kim et al, 1984, Palade, 1987) and to increase both the amount and rate constant for CICR from skeletal SR vesicles (Kim et al., 1984; Mickelson et al., 1986, 1988).  In addition, low doses of halothane (0.47-1.89mM) reversibly  stimulated SR C a  2 +  efflux directly in chemically skinned rat tuberculae (Herland  et al., 1990). The mechanism of anaesthetic mediated effects are unknown although a hydrophic interaction with some component of the release channel complex has been suggested (Chamberlain et al., 1984). The hypersensitivity of Malignant Hyperthermia (MH) patients to halothane and the decreased intraluminal C a  requirement for both halothane induced and C a  2 +  release of SR C a  2+  2 +  induced  together with the probable mutation of the M H SR C a  2 +  release channel (MacLennan et al., 1990; Knudson et al., 1990; Zorzato et al., 1990) suggests that halothane interacts with an important aspect of the triggering mechanism of the C a  2+  release channel.  (vi). Organic Polycations Numerous studies have shown that ruthenium red ([(NH^ RuORu (NH3) ORu (NH ) ] ), which was originally shown to inhibit mitochondrial C a 5  6+  4  2 +  3  uptake through C a stimulates C a  2 +  2 +  channel inhibition (Rahamimoff and Alnaes, 1973),  accumulation in SR vesicles (see Ikemoto et al., 1989). The action  of ruthenium red is via direct inhibition of C a efflux.  2+  release channel mediated C a  2 +  In a detailed study Palade (1987) demonstrated that this and other  polyamines including i) antibiotics (neomycin, gentamycin, streptomycin, clindamycin, kanamycin, tobramycin) ii) naturally occuring polyamines  46  (spermine, spermidine) and iii) basic proteins and polypeptides (polylysine, polyarginine, some histones, protamine) were effective in blocking the skeletal SR C a  2+  release channels.  Similar inhibitory effects and of neomycin were  reported in cardiac SR (Meissner and Henderson, 1987). More recently, complex effects of polylysine (Cifuentes et al., 1989) upon stimulation and inhibition of SR Ca  2+  release at low (0.3|iM) and high (30u,M) concentrations, respectively, were  observed. Cifuentes et al. (1989) demonstrated direct binding of polylysine to the release channel with variable binding to calsequestrin and/or a 100 kDa protein. Ruthenium red inhibited polylysine-induced C a  2 +  release but did not displace  the latter from the release channel. This suggests the presence of multiple classes of polyamine binding sites upon the channel.  (vii). Protons Earlier studies showed that a rapid increase in extravesicular pH initiated a rapid release of C a  2+  from skeletal (Nakamura and Schwartz 1970, 1972) and  cardiac SR (Nakamura and Schwartz, 1970). Similar results were reported with submaximally C a Naylor, 1979).  2 +  loaded (Ca -ATPase mediated) vesicles (Dunnet and  Subsequent  2+  studies indicated that an inward protein  transmembrane gradient (>0.2 pH units) rather than a finite extraluminal pH level was critical in demonstrating pH jump induction of C a  2 +  release (Shoshan  et al., 1981; MacLennan et al., 1982). Similar results have been demonstrated more recently (Meissner, 1984; Sumbilla and Inesi 1987) in both SR vesicles (Argaman and Shoshan-Barmatz 1988; Shoshan-Barmatz, 1988) and skinned fibres (Shoshan et al., 1981). The pH jump induced release of C a  2 +  was inhibited  by dicyclohexylcarbodiimide (DCCD) which evidently bound to the C a release 2 +  channel with inhibition of ryanodine binding.  47  (viii). Reactive Group Modification DCCD inhibition of pH induced C a  2 +  release was suggested to involve free  carboxyl residues of either glutamate or aspartate (Argaman and ShoshanBarmatz, 1988). These putative carboxy residues which were reactive to acetic anhydride but not the more hydrophilic carboxyl reagents N-ethyl-5phenlyisoxazolium-3-sulphonate  (WRK) and N-ethoxycarboxyl-2-efhoxy-l,2-  dihydroquinoline (EEDQ) were suggested to lie within a hydrophobic domain of the protein(s) regulating C a  2+  release. Studies from the same group (Shoshan-  Barmatz, 1986) suggested that acetic or maleic anhydride may also react with positively charged amino residues located on the SR lumen to activate C a  2 +  release. Interest has also been generated in role of sulphydryl (SH) groups in the modification of SR C a  2 +  release. Earlier studies demonstrated induction of C a  2 +  release in skeletal SR by microtubular concentrations of heavy metals (Cu , 2+  H g , Ag ) and para-chloromercuribenzenesulphonate (Abramson et al., 1982; 2+  2+  1983; Bindoli and Fleischer, 1983).  Release was mediated by SH group  modification (Salama and Abramson, 1984) and could be blocked by known inhibitiors (e.g. tetracaine, procaine and Ruthenium Red) of C a mediated C a  2 +  2 +  channel  efflux (Trimm et al., 1986).  Oxidation of SH groups to initiate C a  release was found to be reversible  2 +  upon addition of dithiothreitol in both skeletal and cardiac SR (Abramson et al., 1988; Zaida et al., 1989; Prabhu and Salama, 1990). SR vesicles fused with planner bilayers exhibited increased C a elevated choline permeability and M g  2+  al., 1988). Rapid kinetic studies of A g  2 +  channel open probabilities with  efflux in intact SR vesicles (Nagura et 2 +  induced C a  2 +  release (Moutin and  Dupont, 1988; Moutin et al., 1989) estimated release rate constants, in the absence of M g  2 +  (between 6 and 24s"^) were increased by the presence of KC1. SH  48  induced C a  2 +  release was inhibited by the prior addition of nucleotide (Zaidi et  al 1989) while release rates were increased by nucleotides subsequent to SH v  group modification (Stuart and Abramson, 1988; Zaidi et al, 1989).  These  complex effects of nucleotide are difficult to reconcile with modification of a single reactive SH site on the SR. compounds in stimulating C a compounds  2 +  The site of action of thiol containing  release is unclear.  Concentrations of  (5-50uM) most effective in stimulating C a  2 +  release  (4,4-  dithiopyridine, 2,2'-dithiopyridine) inhibited [ H]ryanodine binding to SR 3  vesicles. This is inconsistent with the view that ryanodine binding is favoured in the open state of the C a  2+  release channel (Lai et al., 1989; Chu et al., 1990).  Abramson and Salama (1989) suggested the SH effects may be mediated via a thiol-disulphide exchange trigger reaction (Figure 4). The site of thiol mediated effects may not be the ryanodine receptor, as earlier thought, but a novel 106kDa Ca  2+  channel protein distinct from the Ca -ATPase (Zaidi et al., 1990). 2+  However, the physiological triggering of C a  2+  release involving such a  mechanism remains controversial. Brunder et al. (1988) found that in split fibres, concentrations of glutathione and DTT which inhibited heavy metal induced Ca  2+  release did not inhibit C a  2+  transients induced by electrical stimulation.  (ix). Myoinositol Triphosphate The potential role of IP in mediating SR C a 3  described earlier IP mobilises C a 3  2+  2+  release is controversial. As  from several intracellular stores in a variety  of cell types. The ER and/or ER associated structures are the main store of C a . 2+  Studies on skinned fibres from cardiac (Nosek et al., 1986) and skeletal muscle (Vergara et al., 1985; Volpe et al., 1985; Walker et al., 1987; Donaldson et al., 1987) and cardiac cells (Kentish et al., 1990) demonstrated either direct IP activation of 3  Ca  2+  release  or  49  Fig. 4. Gating of the Ca^ release channel. +  A hypothetical model of Ca^  +  release channel gating involving thiol-disulphide exchange between closely apposed thiol groups, (from Abramson and Salama, 1989).  50  51  IP potentiation of CICR (Movesian et al., 1985). However, Lea et al. (1986) using 3  skinned frog muscle fibres, Movesian et al. (1985) using myocytes, Mikos and Snow (1987) using skeletal SR vesicles and Rousseau et al. (1986) using cardiac SR vesicles fused into bilayers did not observe IP induction or potentiation of 3  Ca  2+  release or channel opening.  SR containing vesicles (from frog muscle)  fused into bilayers did exhibit D? stimulation of C a  2 +  3  channel open probability  (Suarez-Ilsa et al., 1988). In this study the unexpected inhibition of channel activity by sub-micromolar ryanodine (200nm) suggests the presence of other membranes which may have been sensitive to IP3.  (x). Calmodulin Several proteins other than the C a important in the regulation of C a  2+  2+  release channel are known to be  release. Calmodulin, an ubiquitous E-F hand  containing protein has been shown to inhibit the release of SR C a  2 +  in passively  and actively C a  2+  passively C a  loaded cardiac vesicles (Meissner and Henderson, 1987). The  2 +  loaded skeletal vesicles (Plank et al., 1988; Meissner, 1986) and  effect of calmodulin appeared to be due to direct binding effects rather than due to calmodulin mediated phosphorylation of the C a  2 +  channel or associated  protein(s) (Plank et al., 1988). Calmodulin did not completely inhibit C a release (Meissner, 1986) and did not decrease C a  2 +  2 +  channel conductance (Smith  et al., 1989). Rather, calmodulin decreased single channel mean open times by accelerating the open to closed state transitions in cardiac and skeletal vesicles fused into bilayers (Smith et al., 1989). Schneider and Simon (1988) proposed a two step model for calmodulin mediated channel inactivation in which C a  2 +  bound calmodulin could directly bind to the channel at an inactivation site. However, Smith et al., (1989) and Meissner (1986) demonstrated partial antagonism of calmodulin effects by nucleotides (5mM AMP-PCP) and elevated  52  cytosolic C a  2+  (50u.M).  Smith et al. (1989) proposed that calmodulin may  function to counteract the effects of elevated cytosolic C a  2 +  upon C a  2 +  channel  opening during sustained muscle activity  (xi). Intralumenal Calcium In contrast to the well documented effects of extralumenal effectors and inhibitors of C a  channel mediated C a  2 +  2+  release from the SR, relatively little is  known of the role (if any) of intralumenal C a opening and closing.  2 +  in regulating C a  2+  release C a  channel  This has been largely a problem of access to the  intralumenal compartment. The issue of how intralumenal C a Ca  2 +  2 +  might regulate  be traced back to the contrary views of Weber et al. (1966) and  2 +  Ebashi and Endo (1968) in accounting for the distribution of intralumenal C a subsequent to C a  2+  2 +  uptake. Weber et al. (1966) suggested that intralumenal C a  was largely bound to a low affinity C a  2+  2 +  binding site. Ebashi and Endo (1968)  argued that in order to account for the rapid uptake of a large pool of cytosolic Ca  2+  during relaxation (700nmoles/ml cytosol) high affinity C a  located either in the Ca -ATPase and/or intralumenal C a 2+  2 +  2 +  binding sites  binding proteins.  The corollary to this latter view was that saturation of this site would lead to back inhibition of the ATPase (see Endo, 1977). intralumenal C a  2 +  Ca -induced C a 2+  Ca -induced C a 2+  2+  2+  This is important since  inhibition was forwarded to account for the mechanism of release. As Endo et al. (1981) noted "the ease of evoking release is dependent on the level of loading of the SR".  The phenomenon of intralumenal threshold C a  2 +  requirement for C a  2 +  triggered release has been demonstrated in a limited number of studies with SR vesicles (see Ohnishi, 1979; Nelson and Nelson, 1990). The conclusion of these studies was that loading of SR to a critical level of C a  2+  regulated the sensitivity  of the triggering mechanism to extralumenal C a . A similar phenomenon has 2+  53  been observed with forms of spontaneous C a However, in Endo's (1977) view, C a  2+  2+  release (Palade et al., 1983).  loading to a threshold level would serve to  inhibit the Ca -ATPase and prevent accumulation of the trigger C a . 2+  induced C a  2+  Ca -  2+  release would be strictly a function of extralumenal C a  2 +  2+  binding  the sensitivity of which was presumed to be unaffected by intralumenal C a loads.  This is quite different from the conclusions of later studies that the  intralumenal C a  2 +  threshold requirement for Ca -induced C a 2+  a role of intralumenal C a  2+  2 +  release reflects  in directly regulating this form of C a release. 2 +  Miyamato and Kasai (1979) demonstrated an assymetric distribution of C a binding sites in crude (unfractionated) SR vesicles. affinity (Kd=2.7xl0  -7  3.8xlO" M) C a 2  3  2 +  2+  2 +  Intralumenally, 2 high  and 4.7xlO M) and 2 low affinity (Kd=1.05xl0 _6  -3  and  binding sites were identified. One low affinity site (kd=1.05xl0~  ) was found to bind ~45 nmol Ca .mg HSR"* binding sites consistent with it's 2+  identity as calsequestrin (Cozens and Reithmeier, 1984). With a Kd of ~lmM and a physiological concentration of calsequestrin of ~2.6mM or 104mg/ml SR (Mr~40,000) at an internal volume of ~3|il per mg SR (Williams and Beeler, 1986) it can be calculated that about 90% of C a  accumulated into the terminal  2 +  cisternae (100-150nmol.mg~l SR protein) would be bound to calsequestrin. Most of the releasable C a  2+  pool would then be derived from a bound store. Williams  and Beeler (1986) showed that at physiological concentrations, calsequestrin undergoes little secondary structural changes as monitored by circular dichroism with manipulations in C a  2 +  and ionic conditions contrary previous observations  with dilute concentrations of calsequestrin (Cozens and Reithmeier, 1984). However, Ikemoto and Koshita (1988) reported that the rapid biphasic kinetics of arsenazo IH difference absorbance during C a  2+  uptake paralleled the biphasic  kinetics of intrinsic tryptophan fluorescence of calsequestrin upon C a  2 +  binding.  54  These studies together with the observation that i) removal of intralumenal calsequestrin reversibly abolished caffeine induced C a  2 +  release (Ronjat and  Ikemoto, 1989) and ii) that fluoresence of DACM, incorporated into the C a release channel, suggested an important role for intralumenal C a  2 +  2 +  in regulating  structural states of the release channel indirectly through effects upon calsequestrin. Recent studies identified a critical binding region on calsequestrin (between Lys 86 and Lys 191) that interacted with the C a  2 +  channel rich  junctional face membranes of the SR cisternae (Collins et al., 1990). Collins et al. (1990) suggested that C a  2+  binding to calsequestrin may induce a coil-helix  transition which may be directly or indirectly an important regulator of C a  2 +  channel states.  (xii). Proteases The extreme protease sensitivity of the ryanodine receptor was first recognised by Seiler et al., (1984) who showed that treatment of SR membranes with CANP led to extensive fragmentation of the C a upon the remaining SR proteins.  2+  channel with no discernable effect  Prior to identification of the C a  channel  2+  constituents Shoshan-Barmatz et al. (1987) reported that tryptic digest of SR vesicles uncoupled ATP hydrolysis from C a  2+  transport. They suggested the  presence of an additional trypsin sensitive protein other than the Ca -ATPase 2+  which regulated SR C a  2+  transport.  demonstrated small increases in SR C a  Shoshan-Barmatz and Zarka (1988) 2+  accumulation with low level trypsin  exposure; conditions which led to formation of a 135kDa digest product retention of which correlated with maintenance of [ H]ryanodine 3  binding.  Limited tryptic digest was also reported to stimulate the activation of C a by cAMP, H g , and doxorubicin from passively C a 2+  al., 1988). In ascribing a modified pathway for C a  2 +  2 +  2 +  efflux  loaded vesicles (Trimm et  release these studies could  55  not discriminate between effects of trypsin upon the Ca -ATPase and the C a 2+  2 +  release channel. Chu et al. (1988) noted little effect of extensive tryptic fragmentation of the release channel upon functional properties. Moreover, no significant loss of electron dense material from the channel rich junctional face domain of cisternal vesicles was observed. presence  Chu et al. (1988) suggested that this reflected the  of mutiple non-covalent interactions maintaining the channel  ultrastructure. Rapid kinetic studies of C a  2+  release from C a  2 +  loaded vesicles  revealed a bifunctional effect of trypsin exposure (Meissner et al., 1989). Mild tryptic digest resulted in slight increases in passive loading with little effect upon the rate of release.  Extensive tryptic digest led to loss of C a  properties of vesicles and increase in the rate of C a  2+  2 +  retaining  release. These latter effects  were paralled by loss of low affinity ryanodine binding and irreversible loss of channel activity from bilayer recordings (Meissner et al., 1989). Recently, Rardon et al. (1990) reported the effects of millimolar C a  2 +  sensitive  CANP from smooth muscle upon cardiac and skeletal muscle SR. A discrete peptide pattern was observed with production of 315kDa and 150kDa products under mild proteolytic conditions. Extensive proteolysis removed the 315kDa product leaving an apparent 150kDa limiting digest product. Increases and decreases in single site [ H]ryanodine binding were observed for the skeletal and 3  cardiac vesicles, respectively. However, for both skeletal and cardiac SR CANP treatment resulted in loss of channel inactivation in planar bilayer recordings. Gilchrist et al. (1990b) reported a similar pattern of CANP mediated proteolysis in cardiac and skeletal SR vesicles. Comparison was made, however, between the effects of skeletal muscle micromolar and millimolar C a  2 +  sensitive CANP  after which subtle differences were noted in the production of peptides by each isoform.  The effect of CANP was to elevate (15-20%) the amount of C a  2+  56  retained after passive loading, to increase the rate of slow phase ATP dependent Ca  2+  accumulation by vesicles and to in crease low and high affinity  [ H] ryanodine binding capacity. These results reflected an apparent modification 3  of the sensitivity of channel opening to either intralumenal or extralumenal C a . 2+  IV. Calcium Activated Neutral Protease.  Calcium Activated Neutral Protease (CANP) is an ubiquitous intracellular cysteine protease found within a variety of cells across a number of animal species (see Suzuki et al., 1984). The proteinase bears a remarkable homology to papain and is optimally active within a pH range of 7-8 (Murachi et al., 1979). In most tissues CANP exists in two isoforms. One isoform requires uM C a activation (uCANP) whereas mCANP is activated by mM C a  2 +  2 +  for  concentrations,  m CANP is the predominant isoform is most mammalian tissues although U.CANP is the only isoform found in erythrocytes (Wang et al., 1989). Both isoforms of CANP exist as heterodimers. The large subunit (80-82kDa) is the catalytic subunit whereas the small 29kDa subunit is believed to serve a regulatory function (Suzuki et al., 1987). The large subunit has four domains (I, II, III, IV). The catalytic domain (domain II) is the cysteine protease domain and shows a high degree of sequence homology to cathepsins B and H, Papain and Actinidin (Suzuki et al., 1987). Domain IV, at the -COOH terminal has been identified as a CaM like C a  2 +  binding domain with 4 EF-hand structures. This  region appears to define the C a isoform.  2 +  dependence of CANP activation in each  Sequence comparison between u,- and mCANP reveals increased  hydrophobicity in domain IV of the U.CANP isoform (Imajoh et al., 1988). The active sites of CANP are located at Cys-108 and His-265 and are found to be  57  conserved residues within the proteases listed above (Suzuki et al., 1984). Barret (1986), has proposed that nitrogens in the imidazole ring of His-265 initiates proton abstruction of the Cys-108 sulfer atom. The resultant thiyl radical then attacks the amide carbonyl group of the protein substrate. The function of the remaining domains remains obscure. Suzuki et al., (1987) have suggested that these domains may be important for interaction of the large subunit with the small subunit. CANP exhibits the property of autolytic activation for expression of full activity. Suzuki et al. (1987) suggested that CANP activity may be repressed by interaction of domain II with domains I and III. Autolytic activation leads to production of a partially cleaved large subunit (~78-76kDa in erythrocyte uCANP) that exhibits an increased sensitivity for C a  2 +  activation (Pontremoli  and Melloni, 1986). However, further auto-proteolysis results in inactivation of CANP. Autolysis is also associated with cleavage of the small subunit with production of a 20kDa product from the 29kDa parent peptide (Suzuki et al., 1988; DeMartino et al, 1986; Croall, 1989).  1. Regulation of CANP  a. Phospholipid Although CANP is often considered to exist largely within the cytosol (Suzuki et al., 1988) is has been proposed that autolytic activation of CANP occurs through  plasma  membrane  association  (Mellgren,  1987).  The  membrane/autolysis hypothesis was proposed through observation that both membrane associated isoforms of CANP rapidly autolyse in the presence of Ca  2 +  and that autolysis reduces the C a  2 +  dependence of CANP activation. In  particular, phosphatidyl inositol has been shown to markedly increase the C a  2 +  58  sensitivity of CANP (Cong et al., 1989). Imajoh et al. (1986) have shown that the hydrophobic glycine rich N-terminal region (domain V) of the small subunit is required for the membrane interaction and activation of the small subunit with membrane bound phosphatidyl inositol mediated the autolytic activation of CANP.  b. Calpastatin Calpastatin is an endogenous protein inhibitor of CANP that exists as two distinct types.  Liver calpastatin has a molecular weight of 107,000 while  erythrocyte calpastatin exhibits a molecular weight of 70,000 (Murachi, 1983). One liver CANP molecule., binds 4-10 molecules of CANP whereas erythrocyte CANP is associated with the binding of 3-5 CANP molecules. The mechanism of calpastatin inhibition of CANP involves a C a  2 +  dependent association of  calpastatin and CANP (Imajoh and Suzuki, 1985). Four internal repeat sequences within the primary structure of calpastatin appear to be important in the expression of inhibitory activity (Maki et al., 1987).  2. Role of CANP The intracellular role of CANP remains unclear. CANP appears to target specific substrates that are primarily located either at the plasma membrane, the cytoskeleton or within the cytosol.  Localisation of CANP within these  compartments has been deduced from several immunolocalisation studies within a variety of tissues (Murachi, 1989; Goll et al., 1989).  It has been  considered that localisation of CANP to plasma membranes may mediate proteolytic modification of membrane associated proteins in response to increases in local concentrations of C a  2 +  close to C a  2 +  channels (Belles et al.,  1988; Goll et al., 1989). These responses are numerous. Mellgren (1987) has  59  proposed that CANP effects upon cytoskeletal structures may be directed toward the restructuring of membranes that would be necessary for membrane rapair and growth. In this regard, Mellgren (1987) has suggested that CANP may be involved in the remodelling of postsynaptic membranes resulting in proliferation of dendrite spines.  It is this involvement of CANP which is thought to be  involved with long term potentiation in hippocampal neurons after repetitive stimulation. CANP, therefore, appears to play an important role im memory retention.  In many cases where the substrate is an enzyme limited CANP  proteolysis leads to activation of function, prefering to cleave between the domains rather within (Suzuki et al., 1987). In this respect, Suzuki and Ohno (1990), have suggested that CANP may act as a "biotransformer" mediating alterations in function, rather than attenuation, in response to alterations in cellular metabolic activity and, ultimately, C a evident that the C a  2 +  2 +  metabolism.  It has become  release channel of sarcoplasmic reticulum is also sensitive  to limited CANP mediated proteolysis (Seiler et al., 1984). The central role of the Ca  2 +  release channel in regulation of intracellular C a  2 +  levels in smooth and  striated muscle implicates an interesting role for CANP in the regulation of C a  2 +  metabolism in muscle.  V. Thesis Rational  Recent studies have demonstrated that regenerative HSR C a regulated through an intimate C a  2 +  2 +  release is  mediated structural and functional  association of calsequestrin with the ryanodine receptor.  Calsequestrin was  shown to be attached to the junctional face of the cisternal lumen (FranziniArmstrong et al., 1987) as filamentous strands aligned with the ryanodine  60  receptor/dihydropyridine complex  and intra t-tubular "tether" proteins  (Dulhunty, 1989). In the presence of mM C a , calsequestrin formed dense 2 +  intralumenal aggregates adjacent to the junctional face of HSR vesicles (Saito et al., 1984).  Circular dichroism studies with soluble calsequestrin also revealed  Ca  2 +  dependent formation of globular structures from extended forms of the  Ca  2 +  depleted protein (Cozens and Reithmeier, 1984). More recently, Collins et  al. (1990) identified a critical C a  2 +  channel interaction domain on calsequestrin  (between Lys 86 and Lys 191) that was suggested to undergo a coil-helix transition upon C a  2 +  binding. Functional studies revealed the relative intensity  of the fluorescent conformational probe, DACM, incorporated into the C a release channel, was well correlated with C a et al., 1989).  2 +  binding to calsequestrin (Ikemoto  Additionally, removal of intralumenal calsequestrin reversibly  abolished caffeine induced C a  2 +  release (Ronjat and Ikemoto, 1989). This was  consistent with an observed correlation between the intralumenal C a dependence of the caffeine induced C a Ca  2 +  Ca  2 +  2 +  2 +  2 +  release rate constant and the degree of  binding to calsequestrin (Ikemoto et al., 1989). These studies suggest that occupied states of calsequestrin are important in the regulation of C a  z +  release. This implicates complex formation between the release channel and calsequestrin in which functional regulation may be, conceivably, reciprocal. In many different myopathies (eg diabetes, muscular dystrophy, starvation, ischemia, chronic fatique, malignant hyperthermia) SR C a  2 +  release is often  impaired. The impairment is generally associated with increased C a leakiness which may contribute to elevated cytosolic C a . 2 +  2 +  channel  With malignant  hyperthermia the impaired SR function is due to elevated sensitivity to C a 2 +  induced and caffeine-induced C a  2 +  release due to molecular differences in C a  2 +  channel structure arising from genetic mutation (O'Brien, 1990). In other cases defective C a  2 +  release arises secondarily to primary defects of specific origin. In  61  addition, different myopathies are commonly characterised by disrupted energy metabolism, K loss, thiol group oxidation, intracellular proton accumulation, +  vacuolation of triadic and mitochondrial membranes and disruption of myofibril ultrastructural organisation. Since the defect is often preserved through vesicle purification, release modification must arise from some structural or conformational alteration in the isolated membranes. It is not clear to what extent a common basis for the specific secondary defect exists among the different myopathies. However, a common consequence of intracellular C a  2 +  overload that may result in modification of SR C a  release is  2 +  the activation of Calpains (CANP). Although the specific mechanism for CANP activation is uncertain, it is clear that elevated cytosolic C a  2 +  alone leads to  intracellular proteolysis characteristic of the action of CANP. In vitro, CANP activation has been shown to result in limited proteolysis of the C a  2 +  channel.  The functional consequences of CANP action appear to be modified channel gating as determined from bilayer recordings (Rardon et al., 1990). How CANP structural modification of the channel affects the C a  2 +  handling characteristics of  the SR is unknown, however. It would be interesting for example to determine whether  Ca  2 +  channel  structural alterations  results  in  alteration in  channel/calsequestrin interaction and, possibly, intralumenal C a Could the action of CANP upon the C a SR C a  2 +  2 +  2 +  handling.  channel account for the alteration of  handling observed in vivo? There is currently no direct evidence to  support or refute this notion. In the case of SR membranes, intralumenal C a shown to be associated with impaired C a  2 +  2 +  accumulation has been  release (Gonzales-Serratos et al.,  1978) after prolonged contraction. However, it is not clear whether (a) release inhibition and cisternal C a  2 +  accumulation results from channel impairment or  (b) impaired release results from intralumenal C a  2 +  accumulation. Preliminary  62  studies (Gilchrist et al., 1990) indicated that extralumenal ligand modification of Ca  2 +  release modifies the intralumenal C a  dependence of C a  2 +  Elevated M g , in addition to reducing the extralumenal C a 2 +  increased the intralumenal C a  2 +  threshold for C a  2 +  release.  2 +  sensivity of CICR,  release (Gilchrist et al.,  2 +  1990). These observations suggested that effector modification of C a states may modify the functional coupling to calsequestrin.  2 +  channel  This concept has  received little attention but may be important in understanding the regulation of SR C a  2 +  release.  In this regard, the plant alkaloid ryanodine might be a  potentially valuable probe of the relationship between C a intralumenal C a  2 +  2 +  channel states and  binding. Ryanodine binds specifically to the C a  2 +  and mediates complex activatory and inhibitory effects upon HSR C a  channel  2 +  release  (Meissner, 1986b; Lattanzio et al., 1987) that are dependent upon the existing state of the channel. Demonstration that dual effects of ryanodine depend upon the state of intralumenal C a  2 +  loading would provide evidence that transitions  between functional states of the channel and calsequestrin are reciprocally interdependent. If these events are linked, could structural modification of the channel by CANP alter the intralumenal C a  2 +  depedence of C a  manner that may account for the altered C a  2 +  2 +  release in a  handling and  Ca  2 +  compartmentation associated with myopathy?  V I . Objectives of this study.  It is hypothesised that intralumenal C a  2 +  within the terminal cisternaeregion  of the SR is important in the regulation of the C a  2 +  release channel. It is further  hypothesised that structural and pharmacological perturbation of the release channel will alter the intralumenal C a following objectives were established.  2 +  dependence of SR C a  2 +  release. The  63  1) To purify skeletal and cardiac SR membranes referable to the terminal cisternae. 2) To develop a C a  2 +  transport assay method that will probe intralumenal C a  2 +  compartment. 3) To purify p.- and mCANP from skeletal muscle. 4) To investigate the role of intralumenal C a  2 +  upon C a  2 +  induced C a  5) To investigate the effect of ryanodine upon the intralumenal C a of C a  2 +  induced C a  2 +  2 +  2 +  release.  dependence  release.  6) To characterise the structural and functional effects of CANP upon HSR C a release.  2 +  64  MATERIALS AND METHODS I. Materials The chemicals and/or proteins were purchased from the following sources. 1. Sigma Chemical Co. A23187 2-mercaptoethanol ATP (Na and TRIS salt) +  Antipyrylazo HI Aprotinin Bovine serum albumin Bromophenol blue Calmodulin-agarose Casein CHAPS Citric acid Copper sulphate CPK CP EDTA EGTA Folin reagent Formamide Hepes Imidazole Isopropanol Leupeptin Magnesium chloride  Quercetin PIPES PMSF Sodium tartrate Stains-All TEMED TLCK TPCK Trichloroacetic acid Tris base Tris-HCl Trypsin inhibitor (soybean, type IV) Omega-aminohexyl-agarose 2. BDH Chemicals Ammonium sulphate Calcium chloride Dimethyl dichlorosilane Ethanol Glacial acetic acid Glycerol Glycine Hydrochloric acid Magnesium chloride Methanol Potassium chloride Potassium hydrogen phosphate  66  Potassium hydroxide Sodium carbonate Sodium chloride Sodium hydroxide SDS Sucrose 3. Bio-Rad Laboratories Acrylamide Ammonium persulfate Coomassie brilliant blue R-250 N,N'-methlene-bisacrylamide SDS-PAGE high molecular weight standards SDS-PAGE low molecular weight standards 4. Pharmacia DEAE-sepharose CL-4B Gel filtration molecular weight standards 5. LKB Ultrogel AcA34 6. New England Nuclear Aquasol (liquid scintillation fluid) [ H]Ryanodine (specific activity=60Ci.mmor^) 3  [ ^Ca]Calcium chloride (specific activity=125Ci.mmol"^) 4  7. Serdary Research Products Phosphatidylcholine (Pig liver) 8. Progressive Agri Systems Inc.  67  Ryanodine  68  II. Methods  1. Isolation of HSR membranes. HSR membranes were prepared from fast twitch rabbit skeletal muscle using the buffer systems described by Chu et al. (1986) with several modifications to their protocol.  Back and hindlimb muscle was rapidly excised, trimmed of  excess fat and connective tissue and placed in 0.9% saline on ice within 10 minutes.  Muscle was cut and weighed into 20 gram portions and was  immediately freeze-clamped in liquid N between aluminium tongs into 20 gram 2  discs, wrapped in aluminium foil and stored at -70°C for up to 6 months. Frozen muscle was ground to a fine powder under liquid nitrogen using a porcelain pestle and mortar packed in ice.  The powdered material was added to 5  volumes (w/v) of homogenisation buffer containing 300mM sucrose, 20mM imidazole, 0.5mM EGTA, ImM DTT, 2mM PMSF, lOuM leupeptin (pH 7.4) and was thawed (0-5°C) with constant stirring. The protein slurry was homogenised in a 125ml Waring blendor bottle at slow speed (15,000 rpm) for two 40 second bursts with a 40 second interval. The homogenates were combined and were centrifuged for 10 minutes in a Beckman JA-14 rotor (Beckman Instruments, Palo Alto, CA) at 11,500 rpm. The supernatant was decanted and filtered through 4 ply cheese-cloth and centrifuged at 45,000 rpm in a Beckman 50.2 rotor for 90 minutes. Pelleted material was suspended in homogenisation buffer to 40mg.ml" 1 and centrifuged at 27,000 rpm (Beckman SW 28 rotor) for 16 hrs through a linear 25-45% (w/w) sucrose gradient containing 5mM imidazole, ImM DTT, 2mM PMSF, lOuM leupeptin (pH 7.4). HSR membranes enriched in ryanodine receptor protein were collected from the 38-40% region, slowly diluted (1:4) to 10% sucrose with buffer containing 150mM KC1, and 20mM imidazole, pelleted as above, and resuspended in homogenisation buffer containing O.OluM  69  leupeptin and ImM DTT to a final concentration of 40-50mg.mr.  All  procedures were conducted in the cold room and, typically, 200 grams (10 discs) of muscle were used per isolation. Cardiac SR was prepared from frozen dog hearts using the same procedure with modifications. In addition to the protease inhibitors leupeptin and PMSF, all buffers contained Sug-ml" aprotinin, lOOug.ml" TLCK, lOOug.ml" TPCK, 1  1  and 50|ig.ml"l soyabean trypsin inhibitor.  1  After sucrose density gradient  fractionation of SR membranes, protein was diluted to 10% sucrose (as above) with the inclusion of 400mM KC1 to remove accessorial protein. Membranes were incubated with constant stirring for 2 hrs at 4°C and were pelleted and resuspended in homogenisation to ~30mg.ml~l.  2. Ryanodine Receptor Purification. Ten ml of skeletal muscle junctional SR (~25mg.ml) was thawed and diluted to a final concentration of lOmg.ml"^ in buffer containing IM NaCl, 20mM tris-OH (pH 7.4), 2mM DTT, and lOug.ml" leupeptin (at 22°C). After 20 mins, protein 1  was solubilised with addition of an equal volume of buffer containing 2% CHAPS, 1% asolectin, 20mM tris-OH (pH 7.4), and 2mM DTT. C a from stock solution to a final concentration of 200u.M.  2 +  was added  After 30 mins  solubilisation at room temperature, CHAPS-insoluble material was sedimented at 45,000 rpm in a Beckman 50.2 rotor for 30 mins. The soluble supernatant was decanted and diluted ten fold in buffer containing 50mM KC1, 20mM tris-OH (pH 7.4), 2mM DTT, 200uM C a , 5 ug.ml" leupeptin (4°C). The insoluble pellet 2+  1  was resuspended in muscle homogenisation buffer to an approximate concentration of lmg.ml"^. Solubilised membranes were applied to a calmodulin (CaM)-agarose affinity column (10 cm x 1.6 cm), pre-equilibrated at cold cabinet temperature (4°C) with 0.1% CHAPS, 0.05% asolectin, 20 mM Tris-OH (pH 7.4),  70  200uM C a , SLtg.ml" , 2mM DTT (column buffer), at a flow rate of 0.5 ml.min" . 2+  1  1  The column was washed exhaustively at 0.5 ml.min"! jth column buffer and w  protein was eluted across a 150 ml linear EGTA (lOmM) gradient from 200uM Ca  2+  at a flow rate of 0.5ml.min"^ with 4ml fractions collected. For SDS-PAGE  analysis, aliquots (1ml) were added to 0.5ml of ice-cold TCA (10%) in an eppendorf microcentrifuge tube to preciptate protein. Protein was sedimented at 14,000 rpm in a Beckman microcentrifuge and pellets were resuspended directly in SDS-PAGE sample buffer.  3. Purification of CANP. The purification of both micromolar and millimolar C a  2 +  sensitive CANP  (uCANP and mCANP, respectively) from frozen rabbit skeletal muscle was performed as described (Tan et al., 1988) with several modifications. Frozen muscle was ground as described for the preparation of HSR membranes and homogenised in low salt buffer (LSB) containing 2mM EDTA, 2mM EGTA, 50 mM HEPES, ImM DTT (pH 7.4). Muscle homogenate was centrifuge at 11,500 rpm in a JA-14 rotor for 10 minutes. The supernatant was decanted, filtered across 4 ply cheese-cloth and made to 25% saturated ammonium sulphate by the addition of solid salt. The solution was constantly stirred in the cold room for 30 mins and precipitated material was sedimented in at 13,500 rpm for 30 mins in a Beckman JA-14 rotor. The supernatant was decanted and the solution made to 65% ammonium sulphate with a supplemental addition of salt. Stirring and sedimentation was repeated as above. After each addition of salt the pH was adjusted to 7.4 with the addition of tris base.  Precipitated material was  resuspended in 0.5 volumes (of starting muscle weight) of LSB and the suspended protein was dialysed four times at 2 hour intervals (4 °C) across 34mm diameter (3,500 mol. wt. cutoff) Spectraphor membrane tubing (Spectrum  71  Medical Industries, Los Angeles, CA) against 40 volumes of LSB followed by final overnight dialysis. The ammonium sulphate free protein suspension was diluted with an equal volume of LSB and applied to a 200ml DEAE-Sepharose CL-4B anion exchange column (2.6cm x 35cm), pre-equilibrated with LSB, at 75ml.min" . 1  After  extensive washing (10 bed volumes) with LSB, protein was eluted (75ml.min ) _1  with a 400ml salt gradient from zero to 400mM KC1 in LSB (High salt buffer or HSB).  Fractions were  assayed  for CANP  activity  (see  later) and,  characteristically, uCANP eluted between 120 and 180mM KC1 while mCANP eluted later as a distinct peak between 250 and 330mM KC1 (see results). The ionic strength of each fraction (10ml) was measured with a hand-held ion conductivity meter. The |i- and mCANP containing fractions were each pooled and separately applied at 75ml.hr" to a Phenyl-Sepharose CL-4B column (1.6cm 1  x 25cm) pre-equilibrated with 200mM KC1 in LSB (LSB-200). In the case of U.CANP, the ionic strength was raised to an equivalent 250mM KC1 with addition of HSB. The column was washed (5 bed vols) with LSB-200 and protein was eluted in 4ml fractions with LSB at 75ml.hr" . CANP fractions were pooled and 1  loaded onto a co-Hexylamine-agarose bi-functional affinity column (1.6cm x 12 cm) at 75ml.hr" . Protein was eluted with a 100ml linear gradient of LSB and 1  HSB. Active CANP fractions were pooled and concentrated to 3ml in an Amicon N pressure cell across YM-10 membranes (at 4°C). Each CANP isoform was 2  loaded onto an Ultrogel AcA 34 gel filtration column (1.6cm x 80cm), preequilibrated with LSB, at 6ml.hr" . The protein was eluted with LSB and 1  collected in 3ml fractions. Purified CANP was concentrated in LSB to 100 u.g.ml" 1  in an Amincon N2 pressure cell across YM-10 membranes (0-4 °C). When  required, CANP was further concentrated to lmg.ml" with Centricon 1  72  microconcentrators (PM-10 membranes) in 50mM HEPES, 0.25mM EGTA and ImM DTT (pH 7.4,0-4 °C).  4. Protein Determination.  Protein was assayed using a modification of the Lowry method (Lowry et al., 1951). 400 ul of protein solution (20-lOOug.ml in H20) was added to 3ml of -1  solution containing 1.785% (w/v) Na C03, 0.893M NaOH, 0.893% SDS, 0.0089% 2  CuS0 , 0.0178% N a C H 0 and was incubated for 10 mins. 300 ul of 0.5N Folin 4  4  4  6  reagent was added to each tube to initiate colour development. After 45 mins, a blue colour was visualised and absorbance was determined spectroscopically at 750nM against a blank solution prepared identically to the above except for the addition of distilled-deionised water instead of protein.  Relative protein  concentration was determined from the aborbances of the standard curve using BSA (Fraction V) as the protein standard.  5. Assay of C A N P Proteolytic Activity.  CANP activity was standardised to the proteolysis of casein substrate and was assayed at 37°C in the presence of 2mg.rn.rl casein, 5mM DTT, 2.5mM C a (unless otherwise stated) and 50mM HEPES (pH 7.4).  2 +  After 30 minutes,  caseinolysis was quenched upon addition of an equal volume of ice cold 5% TCA. A unit of CANP activity is defined as the amount of TCA soluble product resulting in an absorbance increase of 1 unit at 280nm after CANP digest of casein substrate.  Estimates of CANP activity were performed with triplicate  determinations upon 3 serial dilutions of each isoform.  Due to potentially  significant non-linearities of this assay, resulting from substrate limitations, quantitative comparison of u- and mCANP functional effects required restriction of difference absorbance measurements at 280nm (after background subtraction)  73  to less than 0.3 absorbance units. The C a  2 +  dependence of CANP activity was  determined as above except with the addition of lOOpM EGTA (added in conjunction with CANP) and various concentrations of C a  2 +  required to obtain  desired free C a . 2+  6. A n t i p y r y l a z o III Purification.  Antipyrylazo III (AP III) was purchased from Sigma (Sigma Chemical Co., St. Louis, MO) and purified by double re-crystallisation as described (Scarpa, 1978). In a typical preparation, 500mg AP III was added to 12.5ml 40% ethanol (4% dye solution w/v) in a borosilicate test tube and heated to 60 °C in a water bath for lhr to solubilise the dye. The tube was removed from the water bath and the interior bottom of the tube was scratched with a glass pipette to initiate crystallisation. The solution was cooled and allowed to stand overnight in the dark at room temperature. Crystals of AP III were collected by filtration across Whatman (#3) filter paper and were dried in a warming oven (60 °C) for 3hrs. Dried  crystals  were  harvested  and  weighed  and  the  solubilisation/crystallisation/filtration procedure then repeated at the same ratio „ of dye and ethanol.  .7. Double-Beam Spectroscopy.  Double beam difference spectra (between 640nm and 790nm) were obtained using a SLM Aminco DW2C spectrophotometer on-line with a SLM Aminco MID AN II kinetic processor/controller. The optical chopper speed was 270 Hz with a monochromator bandpass set to 3nm and a light path of 1cm (3ml quartz cuvettes). The buffer for spectral records contained 300mM sucrose, 150mM KC1, 20mM PIPES (pH 7.0), 50uM AP III (Buffer A) and was titrated with concentrated stocks of Mg.ATP, M g  2+  and, C a  2+  (prepared in Buffer A) as described in the  74  figure legends. All cuvette solutions were thermostatically controlled (27°C) and continually mixed with a magnetic flea stirring accessory.  Baseline spectral  records were obtained with a full chart scale difference absorbance (AA) of 0.1 absorbance units at the slowest scanning rate (0.5nm per second). Baselines were collected and stored on-line by the processor and were automatically subtracted from subsequent dyedigand difference spectral records.  8. Calculation of CalciumrDye (CaD ) Dissociation Constants. 2  The molar extinction coefficients (e) of the C a  free AP IB (eD) and the  2 +  Ca :AP III complex (eCaD2) were obtained using procedures described for 2+  Arsenazo III (Kendrick et al., 1977).  All spectroscopic procedures were  performed using a Shimadzu UV-160 spectrophotometer and 1ml quartz cuvettes. eD was found from the absorbance of the Ca -free dye (Buffer A plus 2+  200uM EGTA) read against a blank cuvette containing Buffer A prepared without addition of dye. For calculation of eCaD , the dye concentration in 2  Buffer A was increased to 2mM AP III. Upon titration with fiM C a all of the C a  2+  2 +  (10-50uM)  would be bound to AP III and eCaD could be found from the 2  slope of AA vs [Ca ] and was determined in the presence and absence of ImM 2+  Mg . 2+  The value for [CaD ] was obtained from AA=(eCaD -2eD).[CaD ] as 2  2  decribed (Rios and Schneider, 1981) and the free C a  2 +  in the cuvette could then  be obtained from Caf=Ca -[CaD ] where Caf=free C a t  2  2+  2  and Ca =total C a . 2+  t  Apparent first order dissociation constants (K'^ ) for CaD in the absence and aD  presence of ImM M g  2 +  were obtained by C a  2+  2  titration of Buffer A. The value  was calculated from the negative reciprocal abscissa intercept obtained from double reciprocal plots of AA vs Caf. Each plot was a linear least-squares fit to the data.  Second order dissociation constants (K"c ) were obtained, as aD  75  described (Abramcheck and Best, 1989), from the relation K"c =Caf.(D aD  t  2CaD ) /CaD where D is the total. [AP III] (50uM). 2  2  2  t  9. Spectroscopic Determination of HSR Calcium Transport Spectroscopic determinations of HSR C a  2+  uptake and C a  2 +  release were  monitored in the dual-wavelength mode of the SLM Aminco DW2C spectrophotometer. The recording wavelength pairs were 675-790nm and 720790nm as indicated in the figure legends. HSR vesicles from freshly thawed stock (40 to 50 mg.ml" ) were pre-incubated to final concentrations of 0.5 to 1  lmg.ml for 3 minutes in 2.75ml transport media containing 300mM sucrose, -1  lOOmM KC1, 20mM PIPES (pH 7.0), 50uM AP III (25°C) and, various concentrations of C a  2+  and M g , as indicated in the figure legends. C a 2+  2 +  uptake  was initiated by addition of stock 50mM Mg.ATP to a final concentration of ImM. Mg.ATP and other reagents were introduced to the sample compartment with a Unimetrics microlitre syringe through a customised polystyrene diaphragm that completely excluded the entry of external light upon initiation of and during C a  2+  transport. When required, ATP regeneration was supported by  the inclusion of 5mM creatine phosphate (PC) and 12.5 units.ml" creatine 1  phosphokinase (CPK: EC 2.7.3.2; from rabbit skeletal muscle) during preincubation.  10. Determination of Calcium Stimulated ATPase Activity. HSR vesicles were pre-incubated to a final concentration of 10u.g.ml in 380|il _1  buffer containing 300mM sucrose, lOOmM KC1, 20mM PIPES (pH 7.0, at 23°C), 100|iM EGTA, and various concentrations of C a  2+  and M g  2 +  to obtain the  desired free concentrations of divalent cation taking into account the presence of ATP (as indicated in the figure legends). ATPase activity was initiated upon  76  addition of 20ul Mg.ATP from 130mM stock. Assays were run in the presence and absence of lOul A23187 and 50uM Ruthenium Red. Pilot studies indicated that linear reactions could be obtained for up to 10 mins without back inhibition of ATPase activity due to accumulation of ADP. ATPase activity was quenched upon addition of 200ul of 10% SDS (w/v).  11. Determination of Inorganic Phosphate. Liberation of inorganic phosphate (Pi) resulting from Ca -ATPase-mediated 2+  ATP turnover was determined spectrophotometrically by the method of Raess and Vincenzi (1980). After reactions were quenched with 200ul of 10% SDS, 200ul of 9% (w/v) ascorbic acid (in H 0) and 200ul of 1.25 (w/v) ammonium 2  molybdate in 6.5% H S 0 2  4  (v/v) were added to initiate formatiom of a  phosphomolybdate complex. Absorbance at 660nm was recorded after 30 mins of colour development. Absorbance blanks were prepared by addition of SDS to reaction tubes prior to addition of Mg.ATP. Absorbance values for the blanks were subtracted from all sample absorbances and the amount of Pi liberated was determined using K H P 0 2  4  as a standard.  absorbance at around 600nm.  Ruthenium red exhibited peak  Therefore, separate ruthenium red containing  blanks and standard curves were also prepared.  12. Calcium Release from Passsively Loaded Vesicles. HSR vesicles from frozen stock were freshly thawed and incubated at a concentration of 10mg.rn.rl for 24hrs at 4°C in the presence of 300mM sucrose, lOOmM KC1, 50mM HEPES, lOmM  4 5  Ca  2 +  (specific activity=l0,000-15,000  dpm.nmol" ), 5mM DTT (pH 7.4). Membranes were incubated for a further 2hrs 1  at 23°C and C a  2 +  loading and C a  2 +  release were assayed as follows:  Five  microlitres of incubated membranes were rapidly diluted 250 fold into 300mM  77  sucrose, lOOmM KC1 and 50mM HEPES containing either lOmM M g , 50uM 2+  ruthenium red and lOOuM EGTA to inhibit C a 4.9mM C a  2+  2 +  release or 5mM EGTA and  (free Ca =10uM) to stimulate the release of intravesicular C a . 2+  2 +  100(0.1 aliquots of diluted membrane suspension were vacuum filtered across 13mm diameter 0.45u. nitrocellulose niters (HAWP type) pre-soaked in release inhibiting buffer and were washed with 5 volumes of the same cold buffer. A customised filtration manifold consisted of base units from 13mm diameter polypropylene Swinnex filter holders with each luer slip outlet inserted into the top luer lock inlet of stainless-steel Becton-Dickinson 3-way stop-cocks. Four to six such units were attached end to end to provide individual and consistent liquid flow control for each unit at each time point. Wetted filters, when placed on the polypropylene support screen, self-sealed with the applied vacuum and could thus be removed immediately after washing to minimise non-specific 4 5  Ca  2 +  leaks. Filters were air-dried and  4 5  Ca  2 +  retained upon the filters was  determined by liquid scintillation in a Packard Tricarb 4530 liquid scintillation counter.  13. Assay of Calcium transport in CANP Treated HSR. The effect of CANP upon HSR C a  2+  transport was evaluated by spectroscopic  and radiometric methods.  a. Spectroscopic analysis: Various amounts (20-30ui) of concentrated CANP (100-250U.mrl) suspended in 300mM sucrose, 250|iM EGTA, 50mM HEPES (pH 7.4), and ImM DTT were added directly to 2.75mg HSR membranes (50-60ul) in fixed CANP/HSR ratios (0.5-2.5U/mg HSR protein). Immediately after CANP addition, various volumes of C a  2+  (1.5-3u.l) were added from stock to stimulate proteolysis (25°C).  78  Proteolysis was quenched after various times upon addition of concentrated leupeptin (lOmg.ml" ) to a final concentration of 250uM. The HSR/CANP/Ca 1  mixture was then added directly to 2.75ml of AP III C a cuvette and C a  2 +  uptake and C a  2+  2 +  2+  transport buffer in the  release were assayed as described earlier.  Controls were run with leupeptin addition made prior to CANP addition.  b. Radiometric analysis: HSR membranes (5mg.ml~l) were proteolysed for 20 mins (23°C) at a CANP/HSR ratio of 5U/mg in the presence of 300mM sucrose, lOOmM KC1, 50mM PIPES (pH 7.4), 5mM C a 4 5  2 +  (specific activity=15,000 dpm.nmol" ). 1  Proteolysis was quenched with an equal volume buffer containing 300mM sucrose, lOOmM KC1, 50mM PIPES (pH 7.4), and 5mM EGTA. Membranes were diluted 50 fold into C a  transport buffer containing 300mM sucrose, lOOmM  2+  KC1, 50mM PIPES, 5.5mM M g , 187.68uM 2+  4 5  Ca  (specific activity=15,000  2 +  dpm.nmol"l) and 50uM EGTA (pH 7.4 at 23°C) in the presence or absence of 50uM ruthenium red. The supplemental C a  2 +  and EGTA added with the protein  resulted in final total concentrations of 237.68(iM and lOOuM, respectively. ATP was added to a final concentration of 6.107mM to initiate C a Final free concentrations of C a , Mg.ATP and M g 2+  2 +  2 +  accumulation.  were 25uM, 5mM and  0.5mM, respectively. Aliquots (150ml) of uptake media were vacuum filtered as above at various time points and filters were washed with 5 volumes of cold Ca  2+  ruthenium red/Mg containing release inhibiting buffer with radioactivity 2+  determined as above (see earlier section).  14. [ H]Ryanodine Purification. 3  Commercially available ryanodine was purchased from Agri-Systems International as a tan coloured powder (lot # 8E09). This batch which was  79  poorly soluble above 5mM, in 10% methanol, is known to contain non-UV absorbing impurities (Humerickhouse et al., 1989) and was further purified by flash chromatography (Still et al., 1978) as described (Ruest et al., 1985). Ryanodine (50mg) was dissolved in 3ml chloroform and was applied to a glass column (12mm x 150mm) packed with Silica Gel 60 (230-400 mesh). Ryanodine was eluted with 40ml CHCl /MeOH/40% aqueous C H N H 3  3  2  (90:10:1.5) and  identified with formation of a brown chromophore on heated H SC>4 sprayed 2  TLC plates (Whatman 250uM layer #4420 222).  Fractions were pooled and  vacuum dried with formation of a colourless translucent ryanodine residue which was suspended in distilled-deionised  to a final concentration of  20mM. This procedure resulted in 50% recovery of the starting material weight. [ H]ryanodine (60 Ci/mmol) was purchased from DuPont and used without 3  further purification.  15. [ H]Ryanodine Binding. 3  HSR vesicles (400u,g.ml~l final concentration) were incubated at 22°C for 24 hours in media containing 300mM sucrose, 150mM KC1, 5mM AMP, 100u,M leupeptin, 8nM-lmM [ H]ryanodine and 50mM HEPES (pH 7.4). 3  Stock  [ H] ryanodine was added to a final concentration of 8nM with increasing 3  concentrations of total ryanodine made from the addition of unlabelled ryanodine. 100u.l aliquots were vacuum filtered across 0.45|i (13mm diameter) nitrocellulose filters which were rinsed with 10 volumes of ice cold buffer containing 300mM sucrose, 150mM KC1 and 50mM HEPES (pH 7.4). Specific binding was obtained by assuming non-specific [ H]ryanodine binding to be a 3  constant proportion of total binding relative to [ H]ryanodine binding observed 3  at ImM ryanodine. Radioactivity was assayed by liquid scintillation methods as described above.  80  16. SDS-Polyacrylamide Gel Electrophoresis. HSR proteins were electrophoretically resolved across 10% uniform and 5-15% or mostly 3-13% linear polyacrylamide gradient resolving slab gels [37.5:1 acrylamide/N,N'-methylenebis-(acrylamide)] essentially as described (Laemmli, 1971). The resolving gel contained 375mM tris-OH (pH 8.8), 0.1% (w/v) SDS, and a 0-25% (v/v) glycerol gradient. The stacking gel (2.5% acrylamide (w/v)) contained 125mM tris-OH (pH 6.8), and 0.1% (w/v) SDS. Gels were polymerised with 5ul TEMED per 15ml gel solution and 150ul, 45ul, and 9ul additions of 10% (w/v) ammonium persulphate to the stacking, low acrylamide, and high acrylamide gel solutions, respectively. Protein samples were digested for 10 mins at room temperature to a final concentration of lmg.ml" in 187.5mM tris-OH, 1  2% SDS, 10% glycerol, 5% B-mercaptoethanol and 0.2% bromophenol blue. The tank buffer contained 250mM tris-OH (pH 8.3), 192mM glycine, and 0.1% SDS. Electrophoresis was performed using the Bio-Rad Protean II or the Hoeffer Mighty Small slab gel apparatus.  Proteins were resolved overnight at  12.5mA/slab for large gels or 20mA/slab for mini-gels.  17. Staining of Proteins Resolved by SDS-PAGE.  a. Coomassie Blue. Gels were placed directly into solvent containing 0.275% Coomassie Brilliant Blue  R-250  (Bio-Rad Laboratories, Richmond, CA) in  MeOH/glacial  C H C O O H / H 0 (5:1:5) and protein was stained for lhr. Gels were destained 3  2  briefly in MeOH/glacial C H C O O H / H 0 (5:1:5) and then extensively with 3  2  several changes of the above solvent (4:1:15).  81  b. Stains-All. Ca  2 +  binding proteins in HSR fractions resolved by SDS-PAGE were identified  by their staining properties with the carbocyanine dye, Stains-All (3,3'-Diethyl-9Methyl-4,5,4',5'-Dibenzothia carbocyanine), using the modifications (Campbell et al., 1983) of the original procedure by King and Morrison (1976).  After  electrophoresis the gel was dialysed in 25% 2-propanol with several changes of solvent during a 24 hr period to remove SDS which precipitates the stain. 5ml of 0.1% (w/v) Stains-All formamide was added to 195ml of solution containing 15ml formamide, 50ml 2-propanol, 130ml 46mM tris-base (pH 8.8). The pH of the final stain solution was adjusted to 8.8. The gel was placed in the stain for at least 24 hours after which highly acidic proteins and glycoproteins were visualised as blue or purple staining proteins. Proteolipids and lipid stained yellow while the majority of remaining protein stained pink.  18.CANP Proteolysis of HSR Resolved by SDS-PAGE. HSR membranes (4mg.rn.rl) were exposed to CANP at various CANP/HSR ratios (0.1-10units/mg) in buffer containing 300mM sucrose, 50mM HEPES (pH 7.4) and various free [Ca ] (5uM-20mM) for 20 minutes at 23°C. Proteolysis 2+  was quenched upon addition of an equal volume of identical buffer containing 50mM EGTA and lOOuM leupeptin. Samples were diluted with an equal volume of 2 times concentrated SDS-PAGE sample buffer (see earlier) and incubated for 10 mins.  82  19. Detergent Solubilisation of HSR Membranes for Immunolocalisation of Membrane Associated CANP. Freshly thawed HSR membranes were incubated for 10 mins on ice at a concentration of Smg.ml" in 2.5 ml of 2M NaCl, 50mM HEPES, 500uM C a 1  2 +  (pH  7.4) in the presence or absence of 200uM leupeptin. Membranes were solubilised by addition of an equal volume of buffer containing 3% CHAPS, 1% pig brain phosphatidylcholine (Serdary Research Laboratory), 50mM HEPES and lOmM DTT (pH 7.4). Solubilsed membranes were further incubated at 23°C for 2.5 hrs and  insoluble  material  was  sedimented  in  5ml  Beckman G-max  minicentrifugation tubes at 45,000 rpm for 35 mins in a Ti 50 rotor. Pelleted material was suspended in 50mM HEPES (pH 7.4) in the presence or absence of 50uM leupeptin. Soluble and insoluble material was assayed for protein (see earlier) and samples were prepared for SDS-PAGE  20. Immunostaining of HSR Membranes Western transfer of proteins resolved by SDS-PAGE was performed using the buffer system of Towbin et al. (1979). Proteins were electroblotted onto 0.45u nitrocellulose membranes at 250mA/gel at 100V for 2hrs. Membranes were then incubated for 2hrs in blocking buffer containing 5% fat-free skim milk in trisbuffered saline or TBS (0.5M NaCl, 20mM tris-OH pH 7.4 at 23°C). Monospecific polyclonal Calpain antibodies were a generous gift from Dr. T.S. Kuo (Dept. Pathology, Wayne State Univ., Detroit, MI). Polyclonal antisera which was crossreactive to both (i and mCANP was raised in rabbits against purified mCANP isolated from rat hearts.  Membranes were incubated overnight in antisera  containing blocking buffer and washed once in TBS for 10 mins and then twice for 10 mins in TBS containing 0.2% Tween 20 followed by one 10 min wash in TBS. Membranes were incubated with the secondary goat anti-rabbit antibody  83  conjugated with horseradish peroxidase for 2hrs in blocking buffer. Washing of membranes was then repeated as above and horseradish peroxidase was detected with 4-chloro-l-naphthol and hydrogen peroxide using the Bio-Rad Immuno-Blot Assay  System  (cat# 170-6534) in  accordance  with  the  manufacturer's instructions.  21. Calculation of free i o n concentrations.  Free C a , M g , ATP, and Mg.ATP concentrations were calculated using a 2 +  2 +  Fortran IV based hand-held calculator (Texas Instrument TI-59 programmable calculator) program as described (Fabiato and Fabiato, 1979). Absolute stability constants used, were those tabulated in the original article by Fabiato and Fabiato (1979).  22. Data Analysis. Where appropriate, all data points from a single experiment represent means of triplicate observations. Data is expressed as means +/- standard error the mean (SEM). Unless otherwise indicated the data presented was derived from one entire experiment  and was  representative  of highly reproducable  observations from atleast three separate experiments. For relative quantitation of proteins, gels were scanned at 633nm with a LKB 2202 Ultroscan Neon-Helium Laser Densitometer (LKB Pharmacia, Uppsala, Sweden) with protein peak integration determined using a LKB 2221 Integrator/Recorder. Extent of CANP proteolysis of the high molecular weight calcium release channel was calculated from the absorbance ratio (R633) at 633nm of the cleaved protein (A ) versus 633  control protein (A' ) (ie. R633=A6 /A' ) where 1-R6 633  fractional loss of the channel.  33  633  33  represents the  84  RESULTS  I. Protein purification  1. Purification of HSR membranes. Figure 5 (lanes C and D) shows that membranes prepared by freeze/grinding of rabbit skeletal muscle results in membrane preparation enriched in 550kDa and 57kDa proteins.  These proteins, the ryanodine receptor/Ca release 2+  channel and calsequestrin, respectively, are localised to the junctional terminal cisternae of the SR as dissused in the introduction. The yields of the membranes harvested from skeletal muscle are shown in Table 2.  An additional major  protein was found at ~105kDa. This protein was present in all of the membranes obtained from the sucrose gradients and is likely the Ca -ATPase (Ca pump). 2+  2+  Not well resolved in this figure (see figures 29 and 30) is the presence of an additional protein banding at ~95kDa which was more evident in proteins resolved in lane A. Proteins in lane A contained minor amounts of 550 and 57kDa protein and are possibly a mixture of lighter density membranes derived from t-tubules, sarcolemma, and longitudinal SR. Enzyme marker studies were not performed upon this fraction. Table 2 shows that protein in lane B was the major membrane fraction in the sucrose gradient and contained small amounts of 550 and 57kDa protein. These membranes were shown to release lower proportions of intralumenal C a  2 +  contents after C a  2 +  induced C a  2 +  release than  the fractions harvested from the 35-38% sucrose region of the gradient. The fractions from the 29-33% sucrose region are possibly a mixture of membranes derived  from  the  longitudinal  and  cisternal  SR  and  Table 2: Y i e l d s and calcium release characteristics of fractionated crude S R microsomes.  Sucrose Gradient Region (%)  Yield (mg.g-1)  25-27 29-33 35-38 39-41  0.1 (5) 1.37+/-0.2 (29) 0.52+/-0.07 (29) 0.05+/-0.001 (4)  a  Ca  2 +  release  b  (%)  0 10 35-40 30-35  a. Values are averages (+/-S.D.) for membrane mg protein per gram muscle. Bracketted value = number of preparations.  b. C a  2 +  release was assessed spectrophotometrically from the  amount of C a in Figure 21.  2 +  release in actively loaded vesicles as described  86  Fig. 5. Purification of HSR membranes from rabbit skeletal muscle. Membranes sedimenting in the 25-27% (lane a), 29-33% (lane b), 35-38% (lane c) and 39-41% (lane d) regions of the 25-45% linear sucrose gradient were separated and resolved by SDS-PAGE on a 3-13% linear acrylamide gradient. 25|il of membranes (2mg.ml ) were diluted by an equal volume of 2X sample buffer -1  (see methods) and layered on top of a 2.5% acrylamide stacking gel. A constant current of 12mA was applied across the gel and the running time was 12-15 hrs. Proteins were stained in 0.275% (w/v) Coomassie Brilliant Blue R-250 and gels were destained in MeOH/glacial acetic a c i d / ^ O (5:1:5). The scale for protein molecular weight is on the left of the figure as determined by migration of the following protein standards: O/? macroglobulin (dimer; 358kDa); myosin heavy chain (205kDa); (3-galactosidase (116kDa); phosphorylase b (97kDa); bovine serum albumin (66kDa); ovalbumin (43kDa); carbonic anhydrase (31kDa). CRC, CPP, and CBP denote the position of the Calcium Release Channel (ryanodine receptor), the Calcium Pump Protein (Ca -ATPase), and the Calcium Binding 2+  Protein (calsequestrin), respectively.  88  will be referred to as intermediate SR or ISR. Figure 6 shows that calsequestrin stains an intense blue colour with the carbocyanine dye, Stains-All, and provides a further indication that the protein fractions in lane C of Figure 5 are highly enriched in membranes derived from the terminal cisternae. The calsequestrin content of ISR was much reduced (lane B) and was present in minor amounts in the light membrane fractions. Also evident in the original coloured gels but poorly visualised in non-colour photographs was a lighter blue staining 165/175kDa doublet. This doublet was more evident in lanes C and D than lane B and was absent in lane A. The 105kDa Ca -ATPase and the 550kDa ryanodine receptor stained pink in Stains2+  All stained gels and are seen in the black and white prints as lighter intensity bands. Densitometric scanning of several Coomassie Blue stained gels revealed a HSR 550/105/57kDa ratio of 1:5:2. These membranes are hereafter referred to as heavy SR or HSR and were used in all further structure/function studies. Membranes in lane D were also referable to HSR. In view of the low yield (see Table 2) and presence of contaminating myosin (205kDa) these membranes were not used in the study. Figure 7 shows that HSR can be similarly purified from frozen rat (lane B) and dog (lane C) hearts by identical procedures used for rabbit skeletal HSR (lane A). The advantage with this method over conventional mincing of tissue is that isolated organs need not be used immediately but can be freeze clamped in liquid N and stored at below -70°C for at least six months. Inclusion of a 2  cocktail of protease inhibitors was found to be essential to the maintenance of protein structural integrity, particularly in the case of the extremely protease sensitive ryanodine receptor (~500kDa protein in lanes B and C). Figure 6 also shows  the  presence  of  a  55kDa  calequetrin  isoform  89  Fig. 6. Stairts-All staining of skeletal and cardiac HSR proteins resolved b y SDS-PAGE. 50u.g of skeletal microsomal protein harvested from 25-27% (lane A), 29-33% (lane B) and 35-38% (lane C) sucrose gradients were resolved on 313% linear gradient SDS-PAGE gels (see methods). Lane D contains 50|ig of cardiac SR protein. The open arrow indicates the banding position (~57kDa) of skeletal calsequestrin (dark blue staining). The solid arrow indicates the banding position of cardiac calsequestrin (~53kDa). The single arrowhead denotes the position of a light blue staining 165/175kDa glycoprotein doublet in lanes B and C. The double arrowhead indicates the position of a 145/155kDa light blue staining glycoprotein doublet. Molecular weight markers are as indicated in Figure 5.  90  Figure 6  91  Fig. 7. S D S - P A G E comparison of skeletal and cardiac HSR. Comparison of rabbit skeletal HSR proteins (lane A) with rat (lane B) and canine (lane C) cardiac HSR proteins resolved on 3-13% linear gradient polyacrylamide gels (50ug per well). Molecular weight markers (kDa) are as indicated on the left of the figure. RyR = ryanodine receptor; CPP = C a Ca  2 +  2 +  binding protein (calsequestrin).  pump protein (Ca -ATPase); CBP = 2+  The arrows indicate the different  molecular weights of RyR and CBP in skeletal (upper) and cardiac (lower) fractions.  92  Figure 7  A B C . »— tm RyR  93  in cardiac HSR, albeit to a lesser degree of enrichment than in skeletal HSR (compare lanes C an D).  2. Ryanodine receptor purification  The ryanodine receptor was purified to examine the effect of CANP upon the solubilised form of this intrinsic membrane protein. The isolation protocol was based upon the premise that the ryanodine receptor is a calmodulin (CaM) binding protein (Seiler et al., 1984). Figure 8 shows the elution profile of CHAPS solubilised proteins bound to the CaM-agarose affinity column. Protein eluted with an initial sharp peak corresponding to the protein in lanes 11-14 of Figure 9. This elution was followed by a broad shoulder elution of purified ryanodine receptor protein (lanes 15-26). These latter fractions were pooled and used for further analysis. It is noteworthy that although the unbound or flow through fraction in lane D of figure 9 shows that most of the ryanodine receptor associated with the column, the yield of purified protein was very low for a single step procedure (-1%). This was observed in over 15 preparations. Most of the protein appeared to "bleed" off the column during the wash stage. Therefore, although specific C a  2 +  dependent binding to CaM was observed, this binding  appeared to be weak under the conditions employed.  3. Purification of C A N P  During preliminary studies of CANP effects upon HSR protein structure and function, uCANP (a generous gift from Dr. K. Wang) purified from the cytosolic fraction of human erythrocytes, was used. In addition, commercially available mCANP was also used.  The questionable purity and cost of the latter  preparation  the  and  undetermined  species  and  94  Fig. 8. Calmodulin (CaM)-agarose affinity chromatography of CHAPS solubilised HSR protein. 500ml of solubilised HSR membranes (~250|ig) in lOOmM KC1, 20mM tris-OH (pH 7.4), 200uM C a , Sug.mr leupeptin, 0.1% 2 +  1  CHAPS, 0.05% asolectin and 2mM DTT were loaded onto a CaM-agarose column (10cm x 1.6cm) at 30ml.hr" . The column was pre-equilibrated in the above 1  buffer and protein was eluted after extensive washing with a linear (0-5mM) EGTA gradient (dotted line) in the same buffer. 4ml fractions were collected and the absorbance (A280) traces (solid line) were obtained from a flow through UVmonitor. Protein in 1ml fractions was precipitated with addition or 0.5ml icecold 10% (w/v) TCA. Sedimented protein was suspended in SDS-PAGE sample buffer and resolved across 5-15% acrylamide gradient gels.  96  Fig. 9. S D S - P A G E resolution of CaM-agarose affinity p u r i f i e d H S R proteins.  lml of protein collected in each fraction tube after C a / E G T A gradient elution 2+  (as described in Figure 8) was denatured in 3.3% (V/V) TCA on ice. Sedimented protein was resuspended in 50ul SDS-PAGE sample buffer (see methods) and resolved on 5-15% linear gradient SDS-PAGE gels. Protein was stained with Coomassie-brilliant blue as described in the methods.  Each lane (1-27)  corresponds to each fraction obtained in Figure 8. Lanes A to D correspond to untreated HSR, CHAPS soluble fraction, CHAPS insoluble fraction and flow through (unbound to CaM column) fractions respectively. The positions of the Ca  2 +  channel, Ca -ATPase and calsequestrin are indicated to the right of the 2+  Figure at 550,105 and 55kDa, respectively.  97  Figure 9  1  2  3  4  5  6  7  8  9  10 11 12 13 14 15 16 550  105  55  17 18  19 20 21 22 23 24 25 26 27 28 A  B  C  D  550  98  tissue specific effects of CANP emphasized the need to purify endogenous CANP in the present study. Both U.CANP and mCANP was purified from the cytosolic fraction of rabbit skeletal muscle homogenates using standard chromatographic procedures (Figures 10-13). Both isoforms were conveniently well separated after the first ion-exchange column step (Figure 10) and could be further purified individually. The co-elution of calpastatin, the endogenous Calcium Activated Protease Inhibitor (CDPI), resulted in a masking of U.CANP activity. As shown in Table 3, CDPI was separated from uCANP during phenylsepharose chromatography with a 1.5 fold elevation of total uCANP activity. It is of interest to note that the decreased retention of U.CANP vs mCANP upon hydrophilic ion-exchange columns is opposite to their respective interactions with the hydrophobic phenyl-sepharose column. This was consistently observed and may reflect the decreased overall hydrophobicity of mCANP vs u,CANP (Imajoh et al., 1988) particularly within domain IV, the C a  2 +  binding region.  Figure 14 shows the protein profile after each purification step. Significant purification was acheived after the phenyl-sepharose and gel-permeation steps. Lanes D and D' show the u.- and mCANP fractions used in this study with the respective 82 and 80kDa large subunits prepared intact and un-autolysed. Copurifying with each CANP fraction, in addition to 30kDa small subunits, were single proteins of different molecular weights.  Whether these proteins  represented contaminants of specific CANP binding proteins was unclear and was not investigated further. However, each was present in stoichiometric amounts in every CANP fraction obtained after gel-filtration.  II. Functional Characterisation of HSR membranes  99  Fig.  10. DEAE-sepharose CL-4B  anion exchange chromatography of  ammonium sulphate precipitated u- and mCANP from rabbit skeletal muscle homogenates.  Protein from 25-65% ammonium sulphate was loaded onto the  column after extensive dialysis. After column washing the protein was eluted with a linear salt gradient (0-400mM KC1) in LSB (see methods). The separate elution profiles of u- and mCANP are as indicated by the horizontal bars. Coelution of calpastatin (CDPI) with uCANP masked the activity of CANP and created the apparent double elution peak in this region. Protein absorbance at 280nm (  ) and CANP activity (  ) was measured for each tube. The elution  profile was obtained after isolation of CANP from 300g skeletal muscle and is representative of 4 preparations.  (A  A)Absorbance (280nm)  01 wnSij  101  Fig. 11. Phenyl-sepharose CL-4B chromatography of DEAE separated u_- and mCANP. In (A) ionic strength of the pooled DEAE-sepharose fraction |iCANP was elevated to 250mM KC1 with HSB (see methods) and loaded onto a phenylsepharose column pre-equilibrated with LSB-200. The column was washed with LSB-200 and CANP was eluted with LSB. CANP activity ( 280nm (  ) and absorbance at  ) were determined as described in the methods. In (B) ionic strength  of the pooled DEAE-sepharose mCANP fraction was not adjusted and protein was loaded directly onto the column. All other procedures were as described for uCANP.  102 Figure 11  A  Fraction Number  103  Fig. 12. Omega-hexylamine-agarose chromatography of phenyl-sepharose C L 4B isolated J I - and mCANP. phenyl-sepharose  In (A) the pooled uCANP fractions from the  column were loaded onto  an co-hexylamine column  bifunctional column (1.6cm x 12cm) pre-incubated with LSB as described in the methods. After extensive washing with LSB the protein was eluted across a 0400mM KC1 (180ml) linear salt gradient in LSB. Absorbance at 280nm ( uCANP activity ( methods.  ) and  ) was monitored for each fraction (4ml) as described in the  Each tube was measured for ion conductivity to identify the KC1  gradient as shown.  In (B) the corresponding elution for mCANP is shown.  Identical procedures to that described for uCANP were adopted.  F i g u r e 12  B  Fraction Number  105  Fig. 13. Gel permeation (Ultrogel AcA 34) chromatography of co-hexylamineagarose isolated CANP. In (A) protein eluting from the co-hexylamine column were pooled and concentrated to 3ml in an Amicon N2 pressure cell across YM10 membranes on ice. For each column run 1.5ml of U.CANP was loaded onto the column (1.6cm x 80cm) at 6ml.hr" . The column was pre-equilibrated with LSB 1  which was also used as the elutant. Protein was collected in 3ml fractions each of which was monitored for absorbance at 280nm (  ) and activity (  ). In (B)  identical procedures to that in (A) were employed for mCANP gel filtration. The void volume as determined from prior Blue Dextran-200 chromatography was 60ml.  106 Figure 13  A  B  107  Fig. 14. Purification scheme of CANP purification followed by SDS-PAGE. CANP containing fractions were pooled after DEAE-sepharose CL-4B (lanes A, A'), phenyl-sepharose (lanes B, B'), co-hexylamine (lanes C, C), and AcA 34 Ultragel gel permeation (lanes D, D') column chromatography. 25ul of each pool was dissolved in 25ul of 2x sample buffer (see methods) and applied directly to the sample wells of Hoeffer Mighty Small electrophoresis gels. The resolving gel was 10% acrylamide and the stacking gel was 2.5% acrylamide. Proteins were stained with Coomassie-R250. Molecular weight marker proteins (MfXlO^) on the left of the panel are as indicated in the legend to Figure 5. 82, 80, and 30kDa proteins refer to large subunits of uCANP and mCANP and the small subunit, respectively.  108  Figure 14  Table 3: Purification of u- and m C A N P from rabbit skeletal muscle  Fraction  Total Protein (mg)  Dialysate DEAE-sepharose(m) DEAE-sepharose(u) Phenyl-sepharose(m) Phenyl-sepharose(u) co-hexylamine(m) u>hexylamine(u) Ultragel-AcA34(m) Ulrragel-AcA34(Lt)  a  15,000 240 210 40 36 18 15 4 3  Specific Activity (Units.mg- ) 1  0.0209 1.84 0.8 5.5 6.56 11.36 10.9 32.5 35.0  a. Bracketted symbols (u, m) refer to u C A N P and m C A N P , respectively.  Purification (fold) 1.0 8.8 3.8 263.0 313.0 544.0 522.0 1555.0 1674.0  110  1. C a  2 +  dependent activation of membrane b o u n d C a - A T P a s e activity. 2+  The procedures used in this study for isolating HSR membranes were novel in that frozen rather than fresh muscle was used as a source of tissue. It was important, therefore, to determine whether HSR membrane integrity and the functional properties of the C a the presence various free C a  2+  2+  pump were preserved. Figure 15 shows that in concentrations, peak C a  2 +  stimulated ATPase  activity (650nmol Pi.mg HSR'l.min *) was observed at 40-50uM free C a -  native membranes. A classic bell shaped C a  2+  2 +  in  activation profile was observed  with inhibition of ATPase activity between 50 and lOOuM C a . In the presence 2+  of lOuM ruthenium red, a blocker of the C a  channel, a 2.8 fold suppression of  2 +  ATPase activity was observed with a dramatic reduction of the C a profile of ATPase activity.  2 +  activation  This result indicates that expression of ATPase  activity is in part, determined by a C a preparations is likely the C a  2+  2+  leak pathway which in these  release channel. On the other hand, a 2-fold  stimulation of peak ATPase activity (1375nmol Pi.mg HSR"l.min"l) was observed at ~5uM free C a A23187.  2+  upon addition of 10uM of the C a  2+  ionophore  The ionophore uncouples ATPase activity from intralumenal C a  regulation and expresses solely the extralumenal C a  2+  2 +  regulation of ATPase  activity. In this regard it is interesting that in the presence of ionophore the pCa is reduced (0.6uM) whereas in its absence the observed pCasg was much 50  higher (4uM). These differential effects of the C a  2+  channel blocker and the C a  2 +  ionophore indicated that the HSR Ca -ATPase was under tight control of both 2+  extralumenal C a  and intralumenal C a  2+  permeability to C a  2+  2+  and that non-specific membrane  in these membranes was very small.  Ill  Fig. 15. Calcium dependent stimulation of HSR calcium stimulated ATPase activity.  HSR vesicles (10u.g.ml~l) were assayed for ATPase activity in the  presence of 300mM sucrose, lOOmM KC1, 20mM PIPES (pH 7.0), 1.25mM f r e e Mg  2 +  and 0.1 to 100u,M free C a  2 +  (22°C). ATPase turnover was stimulated by  addition of 5mM free Mg-ATP and reactions were quenched by addition of SDS (see methods).  Reactions were run in the presence and absence of lOuM  Ruthenium Red and 10uM A23187. Stability constants used for calculation of total M g , C a 2 +  2 +  and ATP required for desired free concentrations were  obtained from a calculator program described by Fabiato and Fabiato (1979). Ca  2 +  stimulated ATPase activity was obtained by subtraction of ATPase activity  observed in the presence of lOOuM EGTA. This was less than 10% of the total activity at pCa 4.3.  Data points are means of 4 observations from a single  experiment (sem less than +/-5%). The experiment was repeated on 3 separate occassions with identical results.  Figure 1 5  113  2. Passive C a  2 +  loading and C a  In Figure 16 the C a  2 +  2+  release.  release characteristics of HSR vesicles was examined as a  function of intralumenal C a concentrations of C a  2 +  load. HSR vesicles were incubated with various  2+  and aliquots of the loaded vesicles were rapidly diluted  into iso-osmotic media containing either (a) C a red (50uM) and elevated M g lOuM free C a  2+  channel blockers, ruthenium  (lOmM), designed to inhibit C a  (5mM EGTA, 4.9mM Ca ) to stimulate C a 2+  shows that intralumenal C a Ca  2+  2+  2+  2+  2 +  release or (b)  release. Figure 16A  loading was steeply dependent upon extralumenal the increase in the C a  2 +  retained was much more gradual and non-saturating. The total amount of C a  2 +  2+  up to ~2mM C a . Between 2mM and lOmM C a 2+  2+  released within 15 seconds increased with elevated intralumenal C a However, as shown in Figure 16B, the fractional C a intralumenal C a  2 +  2 +  2 +  load.  release was greatest at low  (86% at 500uM extralumenal Ca ) and progressively  decreased with increased intralumenal C a  2+  2 +  (60% at lOmM extralumenal Ca ). 2+  It is worth noting at this point that the design of the filtration device allowed rapid removal of niters from support grids during time dependent filtration assays.  Preliminary investigations showed that when filters remained on  support grids, as in the case when a 12 port Millipore filtration drum was used, the C a  2+  contents of the vesicles would leak out. Because of this a large constant  error was found between the first filter (lowest counts) and the twelfth filter (highest counts). In most cases with the customised filtration device, the within sample standard error of counting at each point was less than 2.5%. This apparatus was, therefore, used in all radiometric determinations of ligand binding and C a  2+  flux kinetics performed in this study.  114  Fig. 16. Effects of varying extralumenal calcium upon HSR calcium loading and calcium-induced calcium release. loaded in various amounts of ^ C a 4  2 +  lOmg.ml" vesicles were passively 1  as indicated. After maximum loading,  vesicles were rapidly diluted (250 fold) into iso-osmotic release quench buffer (filled squares) of release stimulating buffer (open squares).  Aliquots were  rapidly removed at 15, 30, 60, 120 second intervals and filters were rinsed with quench buffer (see methods). The data represent C a  2 +  retained by the vesicles  15 seconds after dilution. The inset compares the C a  2 +  release profile after  loading in 0.5mM (A) and lOmM (B) C a . The ordinate represents % maximum 2 +  Ca  2 +  loaded and the abscissa represents time (seconds). Each data point is a  mean of 3 observations from a single experiment (sem less than +/-5%). The entire experiment was repeated twice with similar results.  115  Figure 16  116  3. Spectroscopy of H S R C a  2 +  transport.  a. C a : A P III difference spectral characteristics. 2 +  Antipyrylazo III (AP III) is a C a  2+  sensitive metallochromic dye (1:2 Ca :AP 2+  III binding stoichiometry) that is linearly responsive to C a concentration range appropriate for the study of HSR C a al., 1979).  2 +  over a C a  2 +  transport (Scarpa et  2 +  Ca :dye (CaD ) difference absorbance (AA) is very sensitive, 2+  2  however, to optical interferences from M g  2 +  and ATP. The use of AP IH in dual  wavelength studies is a potentially powerful means of investigating HSR C a  2 +  transport. It was important, therefore, to investigate the limitations and spectral characteristics of this dye and to establish conditions for the accurate quantitation of HSR C a  (i). M g  2 +  2 +  fluxes.  effects u p o n C a D : A P III difference absorbance. 2  The CaD difference spectrum between 640nm and 790nm is shown in Figure 2  17A (trace a). Absorbance maxima (A. ) were observed at 652nm and 716nm max  with an isosbestic point at the 790nm reference wavelength. Table 4 also shows that with the purified dye there was good agreement between apparent first order (K' =257u.M) and second order (K" =24789uM ) dissociation 2  CaD  CaD  constants, fixed by the relationship K' D=K"caD/2E) where D is the total dye Ca  concentration (Rios and Schneider, 1981). Addition of M g solutions containing 50uM C a  2 +  decreased dyedigand AA particularly at  wavelengths above 700nm with formation of a single A , 17A).  Part of the M g  2 +  to sample cuvette  2 +  at 685nm (Figure  m a x  effect is via a reduction of the CaD stability constant 2  with formation of a 1:1 complex (MgD) with the pure dye (Baylor et al., 1982). The presence of M g K'CaD  a n  d K"  C a D  .  2 +  resulted in 1.24 and 1.26 fold increases, respectively, in  Isosbestic points for the MgD spectra were recorded  117  Fig. 17. Double beam spectroscopy of AP IHrdivalent cation AA spectra. (A). Effect of Mg " " upon difference spectra in the presence of C a . Trace a is the 2  1  2 +  Ca :AP III difference spectrum obtained with 50uM C a 2+  Mg  2 +  2 +  and 50uM AP III.  was added in 500uM increments (traces b to g) to a final concentration of  3mM. (B). Mg :AP III difference spectra. M g 2+  2 +  was added in ImM increments  (traces a to e) to 5mM (trace e). (C) Effect of Tris-ATP (traces b to e) upon difference spectra in the presence of 5mM M g  2 +  (trace a). Nucleotide was added  in 250uM increments to a final concentration of ImM. Both reference and sample cuvettes contained basic solution (see Methods). C a , M g 2 +  2 +  and nucleotide  were added to the sample cuvette from stock (2.5mM, lOOmM and 50mM, respectively) prepared in basic solution. Spectra (between 640 and 790 nm) were obtained after baseline subtraction. Scales for AA are as indicated with positive and  negative absorbance positioned above and below, respectively, the  horizontal baseline in each figure.  Figure 17  Wavelength Inml  Table 4. CalciumrAPIII Dissociation constants.  eCaD  First Order Dissociation  a 2  Second Order Dissociation  (mM^.cm" )  (uM)  (uM )  8.33 7.14  257 318  24789 31234  1  2  a. eD, the molar extinction coefficient for the C a , 2 +  Mg  2 +  free dyes was calculated at 0.7 (mM"^.cm^).  120  at 670nm and 705nm with an observed Xmax with A ^ a x recorded at 3mM M g  2 +  a t  685nm (Figure 17B) that coincides  in the presence of C a  2 +  (Figure 17A). The  MgD difference spectrum was itself modified by the addition of ATP (tris salt) with elevated absorbance below 720nm and shift of  to 675nm (Figure 17C).  (ii). Mg.ATP effects upon divalent cation:AP III difference absorbance. In the presence of both C a  2+  and M g , nucleotide addition altered the spectral 2+  waveform in a manner that was dependent upon the relative proportion of divalent cation.  At high C a  2+  (50u.M) and ImM M g , Mg.ATP addition 2+  decreased A A above 700nm with small effects at 670nm (Figure 18A). At low Ca  2+  (20uM) and ImM M g  2 +  the addition of Mg.ATP decreased and increased  AA at 700nm and 685nm respectively (Figure 18B). Nucleotide addition with both elevated M g  2 +  and C a  2+  modified the AA spectra (Figure 18C) in a manner  similar to that observed in the absence of added C a  2 +  (Figure 17C). Within the  range of divalent cations defined by Figure 18 the addition of up to ImM Mg.ATP led to the appearance of cross-over points (open arrows) which intersected the spectra obtained prior to the addition of nucleotide. The crossover point is not a true isosbestic point since the position was dependent upon the relative concentrations of divalent cation. Spectral modification was similar with tris-ATP addition although definition of the cross-over point was enhanced by Mg.ATP. Formation of cross-over points was also observed in the presence of creatine phosphate at slightly different wavelengths.  b. Spectroscopic resolution of C a  2+  uptake and release.  121  Fig. 18.  Effect of Mg.ATP upon AP IHidivalent cation difference spectra.  Nucleotide was added in 250uM increments (traces b-c) to ImM final concentration (trace e) in the presence of (A). 50uM C a , ImM M g 2 +  C a , ImM M g 2 +  2 +  2 +  (B). 20uM  (C). 50uM C a , 3mM M g . All other conditions and figure 2 +  2 +  descriptions were as described in the legend to Figure 17.  122  Wavelength [ n m j  123  (i) . Wavelength pair selection. The appropriateness of the divalent cation and nucleotide concentrations defined by Figure 18 for the study of HSR C a  2+  transport, by dual-wavelength  spectroscopy, was convenient as sample wavelengths could be set to cross-over points with elimination of ATP induced artifacts upon initiation of C a  2 +  uptake  with greater than 500uM nucleotide. In Figure 19A the decrease in A A due to ATP was estimated by pre-incubation of HSR vesicles (lmg.ml"l) with the C a  2 +  ionophore A23187. Addition of ImM Mg.ATP (solid arrows), reduced AA by 0.008 to 0.009 absorbance units, consistent with the decrease observed in the absence of protein (see Figure 18A). After subtraction of the AA artifact a substantial residual component of the rapid decrease in AA was observed in the absence of ionophore. In Figure 19B, Mg.ATP addition to HSR, pre-incubated with A23187, was without effect upon A A at 675nm in accord with Figure 18 A. At this sample wavelength C a  uptake was clearly resolved into 3 kinetically  2+  distinguishable phases: a fast initial phase (a, >650nmol.mg"l.min"l); a slow secondary phase (b, 40nmol.mg"l.min"l) and; an intermediate tertiary phase (c, 85nmol.mg~l.min~- ). After accumulation of essentially all extralumenal C a 1  2 +  (including ~10uM contaminating Ca ) a steady state was reached. Repetitive 2+  addition of 3uM free C a of intralumenal C a  2+  (open arrows) to the medium triggered a rapid release  (35nmol.mg~l).  The secondary and tertiary phases of  uptake have been attributed to C a  release channel opening and closing,  2 +  2+  respectively, and are modified by initial extravesicular C a  2 +  load (Morii et al.,  1985).  (ii) . M g  2 +  effects upon HSR C a  2+  uptake and release.  A prediction of Figure 18C is that elevated M g ATP  absorbance  artifact  2 +  at  (>3mM) would eliminate the the  customary  124  Fig. 19. Effect of wavelength pair upon spectroscopic resolution of initial H S R calcium uptake. Wavelength pairs were (A). 720-790nm and (B). 675-790nm. Ca  2 +  uptake was initiated by the rapid addition of ImM Mg.ATP (solid arrows)  in the presence of ImM M g , 12.5 units.ml" CPK and, 5mM CP under 2 +  1  conditions otherwise described in Methods. In both A and B vesicles were preincubated for 1 minute in the presence (+) or absence (-) of the C a  2 +  ionophore,  A23187 (lOuM), prior to the addition of Mg.ATP. At the indicated times (open arrows) C a  2 +  release was induced by the direct addition of 3uM C a  2 +  to the  cuvette. Downward excursions of the traces (absorbance decrease) denote C a uptake and upward excursions (absorbance increase) denote C a scales for time (abscissa) and A C a  2+  phases of uptake and release of C a  2 +  2 +  release. The  (ordinate) are as indicated. The component 2 +  are indicated on the traces (see text for  explanation). Inset: Shows the resolution of initial C a  2 +  uptake at 682.5-790nm wavelength  pair compared to Ca -ATPase inhibition by Quercetin. Vesicles were pre2+  incubated in the presence (+) and absence (-) of 100|iM Quercetin under conditions described above except for the absence of ATP regeneration. ImM Mg.ATP addition (solid arrows) initiated C a horizontal bars denote scales for A C a for A and B.  2+  2 +  uptake.  The vertical and  and time with the same dimensions as  125  Figure 19  126  720-790nm dual-wavelength pair.  As shown in Figure 20A, the absorbance  artifact was significantly reduced when vesicles were pre-incubated in the presence of 3mM M g  2 +  prior to addition of nucleotide and is completely  eliminated at lOmM total M g  (Figure 20B).  2 +  However, loss of both the  secondary and tertiary phases of uptake was also observed with elevated M g . 2+  Additionally, Ca -induced C a 2+  (Figure 20A). C a Ca  2+  2 +  release was less sensitive to trigger C a  2 +  release was not triggered by 5uM free C a  was required to minimally stimulate C a  2+  2+  2 +  and 10uM free  release. With 20uM free trigger  C a , 25-30nmol Ca .mg was released. M g , therefore, appeared under these 2+  2+  _1  2+  assay conditions to decrease the C a  2+  sensitivity of Ca -induced C a 2+  agreement with rapid kinetic studies of C a 4 5  2 +  2 +  release in  release from passivley loaded  HSR vesicles (Meissner et al., 1986). A 60% reduction in the rate of C a uptake, after 20uM C a lower M g  2+  2+  condition in Figure 19 where the sum of added and released C a 2+  2+  re-  addition, was also observed when compared to the  was comparable (~40-50nmol Ca .mg ). At lOmM M g rate of C a  2 +  _1  2+  2 +  (Figure 20B) the initial  uptake was much reduced and Ca -induced C a 2+  2 +  release was  completely abolished with a greatly extended re-uptake of added C a . These 2+  data indicate that M g  2+  may also inhibit C a  2+  channel closing subsequent to  release, although discrimination between this possibility and Ca -ATPase 2+  inhibition was not pursued. It was also evident that M g  2+  elevation decreased  the sensitivity of measurement. Under the conditions defined by Figure 19, at both wavelength pairs, AlOuM C a At lOmM total M g , A50uM C a 2+  units.  2+  2+  produced a AA of 0.0025 absorbance units. resulted in a AA of only 0.0024 absorbance  127  Fig. 20. Effect of elevated magnesium upon HSR calcium uptake.  Assay  conditions were as described in Fig. 3 except vesicles were pre-incubated in (A). 3mM M g Mg  2 +  2 +  in the presence (+) and absence (-) of 10uM A23187 and, (B). 9mM  without A23187 treatment. C a  2 +  uptake was initiated by ImM Mg.ATP  addition (solid arrows) in the presence of ATP regeneration (see Methods). C a release was induced by addition of 5, 10 and 20uM C a  2 +  as indicated (open  arrows). The horizontal and vertical bars denote scales for time and A C a indicated. The direction of C a legend to Fig. 19.  2 +  2 +  2+  as  uptake and release are as described in the  Figure 20  00 5 10  0  20  129  c. Intralumenal C a  dependence of HSR C a  2 +  (i). Ca -induced C a 2+  2 +  2 +  release.  release.  The foregoing spectroscopic studies demonstrated that the initial phase of HSR Ca  2+  uptake could be well resolved in the presence of ImM M g  2 +  by selection of  a 675-790nm dual-wavelength pair. Subsequent spectroscopic studies of HSR Ca  2+  transport were, therefore, performed in transport media containing 300mM  sucrose, lOOmM KC1, 20mM PIPES (pH 7.0), ImM M g , 50uM AP HI, 12.5 2+  units.ml"! CPK, 5-20mM CP, and various concentrations of C a . In Figure 21 2+  HSR vesicles (lmg.ml"l) were incubated for 1 minute in transport buffer containing 5mM CP. In traces A to G initial extravesicular C a by lOuM C a  2+  from 40uM C a  2+  (trace A) to lOOuM C a  2+  2+  was incremented  (trace G). Trace A  shows that lOuM pulses of Ca2+ (~5uM free) added subsequent to the initial uptake were rapidly accumulated (820 nmol.mg HSR"l.min"l) up to an intraluminal C a  2 +  load of 80 nmoles.rn.g~l. Further addition of C a  stimulation of C a  release (Ca -induced C a  2+  2+  increased to maximal C a  2+  2 +  2 +  led to partial  release) that progressively  release observed after the accumulation of  100nm.oles.mg~l C a . Subsequent traces (B to D) show that as initial C a 2+  incremented by lOuM the amount of pulse C a Ca  2+  2+  2+  was  required to elicit Ca -induced 2+  release was reduced by a corresponding amount with maintenance of the  initial fast phase of C a  2+  accumulation.  After 90 and 100nm.oles.mg~l accumulated C a , the release of C a 2+  followed by a biphasic reaccumulation of C a  2 +  2 +  was  that was resolved into an initial  slow phase (5-10nmol.mg~l.min~l) and a secondary intermediate phase (3040nmol.mg~l.min~l). In traces E to G, increasing initial C a slow phase of C a uptake.  2 +  2+  appearance of a  accumulation followed by a faster intermediate phase of  These data indicate that threshold filling of an intralumenal C a  2 +  130  Fig. 21. Intralumenal calcium requiiement for calcium-induced calcium release.  Ca  2 +  uptake was monitored by dual-wavelength spectroscopy of  Ca :APIII difference absorbance at a 675nm sample wavelength and a 790nm 2+  reference wavelength.  HSR membranes (lmg.ml" ) were pre-incubated 1  (Iminute) at 25°C in transport buffer containing 300mM sucrose, lOOmM KC1, 20mM PIPES (pH 7.0), ImM M g , 5mM CP, 12.5 units.mr CPK and 40(A), 2 +  50(B), 60(C), 70(D), 80(E), 90(F), and lOOuM C a  1  2 +  (G). Uptake was initiated by  the addition of ImM Mg-ATP as indicated (arrows) and lOuM C a  2 +  pulses were  made to the sample cuvette (arrowheads). The scales for time (minutes) and Ca  2 +  are as indicated with a downward deflection of the traces representing  uptake.  132  pool is stimulates C a release and (b) C a  2+  2+  channel opening during (a) C a  uptake when the extralumenal C a  triggering of C a  2 +  2 +  2 +  load just matches or  exceeds a specific intralumenal C a sink. 2+  (ii). Repetitive triggering of Ca -induced C a 2+  2+  release.  It was of interest to examine the effect of increasing intralumenal C a Ca -induced C a 2+  upon  release. Figure 22 (trace A) shows that repetitive addition of  2+  Ca  2+  to trigger C a  Ca  2+  and an increase in the rate of slow phase C a  2+  release led to a gradual decline in the amount of released 2 +  reuptake. HSR vesicles  eventually failed to reaccumulate all extralumenal C a application of C a  2 +  2+  pulses.  with continued  2 +  This was followed by further spontaneous C a  2 +  release and appeared to be due to depletion of ATP. With supplementation of phosphocreatine up to 20mM the extent of submaximal and maximal C a  2+  release triggered by lOuM total C a  2 +  was  unaffected and demonstrated a similar dependence upon intralumenal C a load.  However, the rate of slow phase C a  elevated with shortening of C a  2+  2+  2 +  reaccumulation was markedly  channel open time. Although released C a  2 +  could be completely reaccumulated upon induction of release, the amount of released C a  2+  declined progressively with successive C a  reasoned that this may be due to either intralumenal C a  2 +  2 +  pulses.  It was  overload or some  other time dependent phenomenon. Placement of a 5 minute interval after the initial maximal C a  2+  release (trace E, open arrow) showed that the next C a  2+  triggered release was much reduced (asterisk) and equivalent to the magnitude of release that would have been observed had C a  2 +  been added during that time  (compare with trace D). With a further 5 minute interval C a  2 +  release was  effectively abolished. However, placement of the interval prior to submaximal and  maximal  release  (trace  F)  had  little  effect  upon  these and  133  Fig. 22. Effects of creatine phosphate concentration upon HSR calcium uptake and release. C a  2 +  uptake was monitored by dual-wavelength spectroscopy of  Ca :APIII difference absorbance at a wavelength pair of 675-790nm. In (A) to 2+  (F) C a  2 +  uptake by HSR vesicles (lmg.ml"l) was initiated upon addition of ImM  MgATP (solid arrows) to the sample cuvette containing 60uM C a Ca  2 +  transport buffer (see Figure 21). C a  2 +  2 +  in APIII  transport was supported by an ATP  regenerating system consisting of 12.5 units.ml"l CPK and 5mM (A), lOmM (B), 15mM (C) and 20mM (D,E,F) CP. After accumulation of all extravesicular C a in the initial C a  2 +  load, lOuM total C a  2 +  2 +  aliquots were added as indicated  (arrowheads). In (E) and (F) the open arrows denote a 5 minute interval during which no C a  2 +  was added. Each condition (A to F) was repeated at least 3 times.  134  135  subsequent releases. This suggested that events assodated with C a  2 +  channel  opening resulted either in (a) a time dependent inactivation of Ca -induced 2+  Ca  2+  release or (b) that the compartment of intralumenal threshold C a  2 +  was  depleted.  4. Filtration studies of C a  release from actively loaded vesicles.  2 +  a. Pi accumulation during active C a  2 +  transport  The first hypothesis from the preceding was considered unlikely since preliminary studies showed that, at 2-4 fold lower protein concentrations, repetitive Ca -induced C a 2+  2+  release could be sustained without loss of C a  2 +  release. The second hypothesis was tested with the view that excess Pi generated from CP hydrolysis may accumulate intralumenally and establish a competitive Ca  2+  binding compartment.  Under identical conditions to that described in  Figure 22, vesicles were filtered at intervals corresponding to the addition and accumulation of known amounts of C a . In one set of experiments with C a 2+  2 +  (Figure 23A), filters were assayed for Pi while in a parallel set of experiments (Figure 23B) filters were assayed for C a . Accumulated C a 4 5  2 +  4 5  2 +  correlated as  expected, with that added. Accumulation of Pi, however, occurred in a biphasic manner with a steeprise of Pi accumulation observed after the accumulation of 150nmol Ca .mg HSR'l. 2+  Upon accumulation of 500nmoles Ca .mg"l 2+  intravesicular Pi contents were identical suggesting a 1:1 binding stoichiometry of C a  2+  and phosphate.  phosphate free C a Ca  2+  2+  Back extrapolation of the second Pi curve reveals a  site of 125nmol.mg"-*. This value is close to the intralumenal  threshold for Ca -induced C a release. 2+  2+  136  Fig. 23. Calcium and inorganic phosphate (Pi) retention by HSR vesicles undergoing ATP dependent calcium accumulation.  In (A) HSR vesicles  (lmg.ml" ) vesicles were pre-incubated with 60uM total ^ C a 1  4  2 +  and C a  2 +  uptake was initiated upon addition of ImM Mg-ATP in the presence of 300mM sucrose, lOOmM KC1, 20mM PIPES (pH 7.0), ImM M g , 12.5 units.mr CPK, 2 +  1  20mM CP (2ml volume). After 3 minutes 3xl00ul aliquots were withdrawn and immediately filtered and rinsed. The procedure was repeated after addition of 120nmol ^ C a 4  2 +  to the remaining vesicles.  The C a  2 +  addition/filtration  procedure was continued up to a total of six successive 120nmol  4  ^Ca  2 +  additions. In (B) the experimental protocol was replicated except for the addition of  4 0  Ca . 2 +  After rinsing, filters (13mM HAWP type) were then placed into  600uL of 6.7% (w/v) SDS and incubated overnight. Determination of Pi was performed as described in the methods.  The data represent means of 4  observations from 2 sets of experiments (sem less than +/-10%).  A  B  138  b. C a release. 2+  The possibilty that alterations in the extralumenal compartment were responsible for the inhibition of C a  2+  release was then tested through dilution of  actively C a  2+  loaded vesicles into media that either inhibited or stimulated C a  release.  Figure 24 shows that at low (60uM) C a  extralumenal C a  2+  2 +  loaded states, 10um  2+  triggered a large release of intralumenal C a  2 +  comparable to  that observed with passivley loaded vesicles (see Figure 16). With elevated ATP dependent C a inhibited.  2 +  loading in the presence of 20mM CP, C a  release was markedly  2+  It is noteworthy that direct measurements of pH during C a  transport showed that the inhibition of C a proton accumulation due to Pi release.  2+  2 +  release was not associated with  This may reflect the fact that CP  hydrolysis is a proton consuming reaction and would be expected to counter proton release during ATP hydrolysis. The data from Figures 22 to 24 indicate that intralumenal Pi accumulation may, under the conditions employed in this study, directly or indirectly inhibit C a  2+  release. During the filtration studies  above, it was observed that the region of the filter occupied by the membranes was particularly white indicating formation of Ca :Pi preciptation. 2+  suggests that Pi may compete with calsequestrin for C a  2 +  This  binding and would  implicate an important role for the Ca :calsequestrin compartment in mediating 2+  Ca  2+  channel opening.  5. Ryanodine effects upon C a  a. C a  2+  2+  uptake and C a release, 2+  uptake.  In Figure 25A the effect of vesicle pre-incubation with ryanodine prior to addition of Mg.ATP was examined. In the presence of luM-lOuM ryanodine rapid  Ca  2 +  uptake  was  139  Fig. 24. Effect of elevated (20mM) creatine phosphate (CP) upon calciuminduced calcium release from actively loaded vesicles. HSR (lmg.ml" ) were 1  initially loaded with 60u.M total 45 2 Ca  +  (SA=50000dpm.nmol ) in the presence _1  /  of 300mM sucrose, lOOmM KC1,20mM PIPES (pH 7.0), 20mM CP, 12.5 units.mr CPK, ImM M g  2 +  (2ml volume). C a  2 +  1  uptake was initiated by addition of ImM  Mg-ATP. After 3 minutes 2x5fil aliquots were withdrawn and rapidly diluted (250 fold) into either iso-osmotic C a  2 +  release buffer containing 4.9mM C a  2 +  and  5mM EGTA or quench buffer contianing lOmM M g , 50uM ruthenium red and 2 +  IOOJIM  EGTA (therefore 20ul total). 200uL aliquots from either quench (open  symbols) or release (filled symbols) buffers were rapidly filtered across 0.45|i nitrocellulose filters at the indicated times. The filters were then washed with 5 volumes of quench buffer. An amount of C a  2 +  was then added to the actively  accumulating vesicles which raised the intralumenal C a  2 +  by an additional  60nmol.mg~l. The loss of protein due to aliquoting and the dilution of remaining protein were both accounted for.  After a further 3 minutes 2x5ul aliquots  (total=4x5|il) were withdrawn and the dilution and filtration procedures described above to either induce or inhibit release were repeated. Vesicles were therefore loaded to 60 (  ), 120 (  ), 180 (  ), 240 (  ) and 300 (  nmol.mg" . The data are means of 4 observations from 2 sets of experiments. 1  )  140  141  Fig. 25. Ryanodine effects upon HSR calcium uptake. Ca  z+  transport was  assayed as described (see Figure 21). lmg.ml"l vesicles were pre-incubated for 5 minutes in the presence of 60uM C a as indicated. C a  2 +  2 +  and various concentrations of ryanodine  uptake was initiated by addition of ImM Mg-ATP to the  cuvette and unless otherwise indicated 5uM C a added to the cuvette during the steady state.  2 +  pulses (arrowheads) were  Figure 25  55 10  143  observed with slight decreases in the intralumenal C a induced C a trigger C a  2 +  requirement for C a  2 +  release. Vesicles released 30nmoles Ca .mg-l in response to 5uM 2+  (~3uM free) although C a  2 +  2 +  2+  release frequently followed a delay  (asterisk) subsequent to addition of 5uM trigger C a . The total amount of C a 2+  2 +  released at low uM ryanodine was unaltered. Pre-incubation of vesicles with elevated ryanodine (lOOuM-lmM) resulted in 65% loss of C a ability. The accumulated C a  accumulating  2 +  could then be released upon application of C a  2+  2 +  ionophore. At luM ryanodine, vesicles could accumulate the released C a sensitive to subsequent C a  2+  triggering of C a  concentrations greater than luM released C a  release.  2+  and remained  2 +  At ryanodine  was not accumulated in the  2 +  presence of 5mM initial CP. Further addition of 5mM CP stimulated a slow C a reaccumulation. However, vesicles were insensitive to repeated C a of C a  2 +  2 +  2 +  triggering  release.  b. Intralumenal C a  2+  threshold for Ca -induced C a release. 2+  2+  Figure 26A shows 'the effects of ryanodine addition after complete uptake of medium C a  2 +  upon Ca -induced C a 2+  2 +  release. As in Figure 21, C a  were added until release was observed. decrease in the intralumenal C a  2 +  2 +  threshold of Ca -induced C a 2+  triggering. The rates of initial C a  2 +  pulses  Increasing ryanodine resulted in a  2mM ryanodine, a delayed onset spontaneous C a Ca  2 +  2 +  release. At  release resulted without  2 +  release in the presence of ryanodine  (953nmol Ca .mg HSR'^) were not measurably different from controls (971nmol 2+  Ca .mg HSR-1). However, the magnitude of C a 2+  2 +  triggered release increased  with elevated ryanodine despite decreased vesicle C a low ryanodine resulted in an initial fast C a release  phase.  At  high  2 +  2 +  loading. As in figure 25  release followed by a slower ryanodine  concentrations  144  Fig. 26. Effect of ryanodine upon calcium stimulation of calcium release. In (A) Ca  2 +  uptake in HSR vesicles (lmg.ml ) was monitored as described in Fig 21. -1  Vesicles were pre-incubated in the presence of 60uM C a  2 +  for 1 minute and  uptake was initiated by the addition of ImM Mg-ATP to the cuvette. After accumulation of 60nmol Ca .mg HSR" various concentrations of ryanodine 2+  1  were added (open arrow) prior to addition of 5u.M C a (B) C a  2 +  2 +  pulses (arrowheads). In  uptake was as in (A) except for the presence of 20mM CP. Ryanodine (5  uM) was added either prior to addition of lOuM C a or prior to partial stimulation of C a  2 +  2 +  pulses (arrowheads) in (a)  release in (b). Each trace was repeated  twice with identical consequences for repetitive triggering of C a  2 +  release.  146  (100uJvl-2mM) the initial fast phase was immediately followed by a transient reaccumulation, the rate of which increased with elevated ryanodine. A further 10-15nmol Ca .mg HSR 2+  -1  of C a  2 +  re-uptake was followed in several  experiments during the subsequent 45 minutes beyond that shown. Figure 26B shows that with CP supplementation prior to initiation of C a  2 +  uptake the  released C a , after addition of ryanodine, was reaccumulated with a much 2+  slower time course than in the absence of ryanodine (compare with trace D, Figure 22). This is consistent with u,M ryanodine effects upon maintenance of the open channel state (Meissner, 1986). released C a , subsequent C a 2+  of added C a  2+  2+  However, despite re-accumulation of  releases were markedly inhibited and the uptake  also markedly slower. This suggests that the releasable pool of  intralumenal C a  was also modified either directly or indirectly by ryanodine.  2+  However, these effects of ryanodine appear to attend formation of the open channel state since ryanodine addition prior to the maximal C a modify the rate or the extent of maximal C a addition of ryanodine at more C a  2+  2+  2 +  release did not  release. On the other hand,  loaded states increased submaximal C a  2 +  releases.  c. Intralumenal C a  2+  threshold for ryanodine-induced C a release.  The intralumenal C a  2+  2 +  sensititity of ryanodine induced C a  examined in figure 27. At defined C a  2 +  2 +  release was then  loads, ryanodine was added in 5u,L  aliquots at 30 second intervals until gradual upward excursions of the absorbance traces were observed during the lag period. The predictability of Ca -induced C a 2+  2 +  release under these assay conditions permitted C a  of vesicles with pulse C a  2 +  intralumenal  loading  up to one 5u,M addition less than that required to  induce release. The amount of ryanodine induced C a the  2 +  Ca  2 +  load.  2 +  release increased with This  was  147  Fig. 27. Effect of intralumenal calcium load upon ryanodine induced calcium release. HSR vesicles (lmg.ml"l) were allowed to accumulate 60nmol Ca .mg 2+  protein"^ upon addition of ImM Mg-ATP (arrow) as in Fig. 21. As indicated at the bottom of the trace, fixed amounts of C a Ca  2 +  2 +  was added as a series of 5uM  pulses to achieve a desired level of filling. This was followed by addition  of ryanodine in 5ul aliquots (from 20mM stock at 30 second intervals). Ryanodine addition was terminated upon observation of a definite progressive rise in the absorbance trace during the lag period. In the centre of the trace are shown the amounts of ryanodine added to induce release at the corresponding intralumenal C a  2 +  load.  The C a  2 +  release traces to the right are labelled  according to the loading scheme shown. The release traces from conditions B to D are superimposed upon the entire trace obtained under loading condition A.  148  Figure 27  Added Ca  24  (nmol.mg  1.  A. 3 0 1901 B. 2 0 180)  C  DL  Added Ryanodine  luM! 145 436  1 0 [70}  873  0(60)  1454  +  CN  <3 5  Ca2+  Addition  o Ryanodine Addition  Lag Period  CM  1 min  149  different from ryanodine augmentation of C a  z +  induced C a  z +  release (see Figure  26A) which may reflect mixed effects of ryanodine induced release and ryanodine stimulation of C a Ca  2 +  2 +  induced release. The ryanodine triggering of  release at 25°C required a minimum 125uM ryanodine and was  immediately followed by partial re-uptake of released C a . At 30°C, a two fold 2 +  decrease in the amount of ryanodine was required to induce C a  2 +  release (not  shown).  III. CANP effects upon HSR structure and function.  1. CANP proteolysis of HSR proteins. In order to study the effects of CANP upon HSR C a Ca  2+  2+  transport, in particular  release, it was important to characterise the effects of CANP upon HSR  protein structure.  a. Endogenous CANP effects. Preparation of membranes without leupeptin addition to isolation buffers resulted in the appearance of a second high molecular weight peptide (Figure 28A, lane B). This common observation has been attributed to the action of CANP (Seiler et al., 1984). The inclusion of 2.5mM C a  2 +  in sucrose density  gradients during purification of the post 45,000 rpm crude microsomal pellet led to further fragmentation of the C a  2 +  release channel in HSR fractions (Figure  28A, lane C). This intially suggested that CANP co-purifies with the HSR and/or the crude microsomal fraction. With 3-13% gels the size of the C a  2 +  release channel estimated from linear log/log plots of size (m.w.) versus %T (Figure 28B) was 550 kDa. This is very close to the actual size (565 kDa) of the Ca  2 +  release  channel  determined  from  150  Fig. 28. Endogenous CANP effects upon HSR protein structure. In (A) HSR membranes were isolated (see methods) in the presence (lane a) and absence of 5uM leupeptin (lanes b and c) and resolved by sodium dodecyl sulphatepolyacrylamide gel electrophoresis on linear gradient (3-13%) gels. In lane c ImM C a  2 +  was included in the 25-45% sucrose density gradient buffers for the  16 hr sedimentation to equilibrium centrifugation step. On the left of the panel are positions of molecular weight marker proteins (o^-macroglobulin (dimer), 358 kDa; myosin heavy chain, 205 kDa; j3-galactosidase, 116 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 31 kDa). To the right of the panel are the estimated molecular weights (kDa) of the intact calcium release channel (550) and proteolytic fragments (410,150). These estimates were interpolated from the least squares fit in (B) of log molecular weight (Mol. Wt.) versus log acrylamide concentration (%T) as described ().  The filled squares mark the positions of the above  standards with the inclusion of (a) the o^-macroglobulin tetramer (725 kDa) observed under non-reducing conditions and (b) soyabean trypsin inhibitor (21 kDa).  Figure 28  0  o  1 I  O  I I  II I II 1 H  I  11  m  152  the cDNA primary sequence (Takeshima et al., 1989). From this plot the size of the high molecular weight proteolytic fragment was found to be 410 kDa. Coincident with appearance of the 410 kDa peptide was production of a 150 kDa peptide (Figure 28, lanes B and C).  b. Exogenous CANP effects. Observation of endogenous CANP mediated proteolysis of the C a channel prompted examination of the C a  2 +  2 +  release  dependence of proteolysis of HSR  proteins by two forms of skeletal muscle CANP differentially sensitive to micromolar and millimolar concentrations of C a  2 +  (uCANP and mCANP,  respectively). Figures 29A and 30A shows that a major HSR substrate for both forms of CANP was the 550 kDa ryanodine receptor/Ca release channel. As 2+  with endogenous CANP (Figure 28A) limited proteolysis of the 550 kDa peptide by both uCANP and mCANP resulted in the appearance of 410 and 150 kDa digest products (Figures 29A and 30A, respectively).  Production of these  peptides appeared to represent an initial proteolytic stage. Distinct differences in the C a  2 +  sensitivity of [i- and mCANP mediated 550 kDa fragmentation were  readily apparent. Maximal C a  2 +  at 500 jiM and 2.5 mM free C a shows that the C a  2 +  activation of 550 kDa proteolysis was observed 2 +  for u.- and mCANP respectively. Figure 31  dependence of |i- and mCANP proteolysis of the 550 kDa  peptide was similar to the corresponding profile observed for caseinolysis. Half maximal proteolysis of casein and the 550 kDa peptide was observed at 50 and 75 u,M C a , respectively, for U.CANP, whereas with mCANP the corresponding 2 +  half-maximal values were 750 and 760 |iM C a , respectively. Additionally, 2 +  U.CANP mediated 550 kDa fragmentation was inhibited at elevated (>lmM) Ca  2 +  (Figure 29A lanes I to K) as observed with caseinolysis (Figure  153  Fig. 29. Calcium and protease dependence of uXZANP mediated proteolysis of HSR proteins.  In (A) 4mg.ml~ HSR membranes were exposed to 1 unit 1  CANP/mg HSR" for 20 minutes at 23°C. 1  The following free  Ca  2 +  concentrations corresponding to lanes B to K, respectively, were employed to stimulate CANP activity: 5uM (B), lOuM (C), 50uM (D), lOOuM (E), 250uM (F), 500uM (G), ImM (H), 2.5mM (I), 5mM (J), and lOmM (K). Lane A was a control (zero added U.CANP) and each lane contained 60uL protein. In (B) 4mg.ml~  1  membranes were exposed to 0.1, 0.5, 1.0, 2.5, and 5 Units uCANP/mg HSR (Lanes A to E, respectively) for 20 minutes in the presence of ImM free C a . In 2 +  (A) the amount of total C a  2 +  added to obtain desired free C a  2 +  concentrations  was adjusted to account for the amount of EGTA containing uCANP with previously determined activity. In (B) various admixtures of EGTA were made with various amounts of uCANP to maintain a constant EGTA concentration. To the left of Figure 29A is the molecular weight scale as described in the legend to Figure 5. To the right of (A) and (B) are the substrates (550 and 88kDa) and digest products (410, 338, 286, 175, 160, 150, 137 kDa) of CANP mediated proteolysis. The open arrow to the right of Figure 29B indicates the position of uCANP.  Figure 29  £8£  CM  LU  °  )  o  )  il I  •I ) <) ) o o K co  S  5  8  as  II II  X  II  o Ll_ LU Q O  III III  II  IIIII  J  J < )  III  III  III  II  CO  r  CO LO CO  IO  o CN  ~r  CO CO  I:  "f  l T—  CO  155  Fig. 30. Calcium and protease dependence of mCANP mediated proteolysis of HSR proteins. For both (A) and (B) identical experimental conditions to that employed for uCANP mediated proteolysis of HSR proteins. For description of this figure see legend to Figure 29.  157  Fig. 31. Comparison of the calcium dependence of CANP mediated proteolysis of (A) casein and (B) the skeletal muscle ryanodine receptor. In (A) both u,- and mCANP were adjusted to 6 units.ml" in ImM EGTA, 50mM HEPES (pH7.4) 1  from a previous determination of activity (see methods).  Caseinolysis was  assayed in the presence of 2mg.ml casein, 5mM DTT, 50mM HEPES (pH 7.4) at _1  30°C, and various total concentrations of C a Ca  2 +  concentrations in the presence of  apparent C a  2 +  2 +  IOOJIM  required to obtain desired free  EGTA (added with CANP). The  /EGTA stability constant was 1.552 x 10 M" . Caseinolysis was 7  1  initiated upon addition of CANP as a 50u.L aliqout to a final 500u,L reaction volume. CANP activity was determined as described in the methods. In (B) CANP proteolysis of the 550kDa channel and electrophoretic resolution of proteins was identical to that described in Figure (x) upon a separate HSR preparation with free C a  2 +  varied between 5u.M and lOmM C a . Coomassie 2 +  blue stained 550kDa protein was densitometrically scanned and proteolysis calculated (see methods) from the fractional loss of 550 KDa protein at ImM and 5mM C a Ca  2 +  2 +  for [i- and mCANP, respectively. The insets in (A) and (B) show the  dependence of CANP mediate caseinolysis and 550kDa proteolysis,  respectively, over a narrower range of C a . Data points are means of 3 2 +  observations from a single experiment (sem less than +/-5%). representative of 3 separate experiments in (A) and five gels in (B).  Data are  Caeslnolysls (% max)  8SI  >  159  31 A). On the other hand, mCANP proteolysis of both casein and the 550 kDa substrates was relatively unaffected at high C a With elevated C a  2 +  2 +  (Figure 31B).  activation or increased CANP/HSR ratios (Figures 29B  and 30B) a secondary stage fragmentation of the release channel was evident. For both CANP isoforms, formation of major 330, 175 and 165kDa peptides appeared to coincide with loss of the 410 kDa product. Isoform specific digest products for uCANP (286kDa) and mCANP (360, 225kDa) were also observed. Secondary stage fragmentation by both CANP isoforms was also associated with further fragmentation of the 150 kDa peptide.  Again, however, subtle  differences were observed between the pattern of |i- and mCANP mediated fragmentation of this peptide.  Extensive mCANP digestion resulted in  sequential production of 145,137, and 130 kDa peptides. The latter two peptides were not further degraded at higher CANP/HSR ratios (at 10 and 20 units mg ; -1  data not shown) and appeared to represent limiting digest products. With U.CANP, however, production of the 145 kDa peptide was never observed with direct fragmentation of the 150 kDa parent peptide to a limiting 137 kDa product. In addition to the 550kDa protein, proteolysis of a major 88kDa protein was also observed.  The 88 kDa protein was cleaved in a similar C a  2 +  and protease  concentration dependent manner as seen with the 550 kDa peptide (Figures 29 and 30). The identity of this HSR associating protein and it's contribution to the regulation of HSR C a  2+  transport is unknown.  Figure 32 shows that the proteolytic pattern of the solubilised ryanodine receptor is identical to that observed with the native vesicle associated form of the protein.  With U.CANP from human erythrocytes the time dependent  production of the 410 and 150kDa major peptides within the two proteins was indistinquishable. This indicates that the effects of CANP upon C a structure  are  2 +  channel highly  160  Fig. 32. Comparison of the effects of U.CANP upon the CHAPS solubilised and vesicular 550kDa protein. In (A) 20ml (~80|ig protein) of the pooled purified Ca  2 +  channel fractions obtained from CaM-agarose affinity chromatography  were proteolysed at 25°C for various times by direct addition of 5mM C a (estimated 2.5mM free C a erythrocyte uCANP.  2 +  2 +  with ~2.5mM EGTA) and 25u. Units human  2ml aliquots were denatured in 3.3% (V/V) TCA on ice  after 0.5 (lane b), 1 (lane c), 2 (lane D), 5 (lane E), 7 (lane F), 10 (lane G), 20 (lane H), 30 (lane I), 45 (lane J), and 60 minutes (lane K). Lane A represents the control (without U.CANP addition).  Sedimented material was resuspended in 50ul  sample buffer and layered onto 5-15% gradient SDS-PAGE gels as in Figure 9. In (B) 500|ig HSR protein was exposed to CANP under identical conditions of CANP and time of proteolysis as in (A). Each lane in (B) corresponds to the same time of proteolysis as in (A). Proteolysis was stimulated by addition of 2.5mM Ca  2 +  and aliquots containing 50(ig protein were added directly to 2xSDS-PAGE  sample buffer to quench proteolysis.  100u.l samples containing 50ug protein  were resolved by SDS-PAGE as above. Proteins were stained with CoomassieR250 (see methods). The open arrows in (A) and (B) indicated the positions of CANP (~82kDa). To the right of (A) and (B) are molecular weights (M^IO ) of 3  substrates (550,88) and proteolytic products (450,410,330,150).  Figure 32  *  11  - 11  I  ^  1 n  | II  i in i »r i rn o 1 11 1 1 i 1 i n «- n i i M l i MI * in i I 1 III D  11  |  AB C  -llll x in i  1  III  1  II pin It i wia in i« (ill H pill  | ! t taim  II MM • i if 30 -00  o  0  r \ i  u-  l I I !  UJ  , |  a  I o I CO I Q  i  1  J  <1 i -  CO  -rCO  -r  3  CO  162  selective and restricted to the cytosolic domain of the protein. It is of interest to note that a transition peptide (~450kDa) was often observed in 5-15% gels for both u,- and mCANP. With 3-13% gels, this peptide was only faintly resolved. Figure 33 shows that the ryanodine receptor of canine cardiac HSR membranes was also specificly fragmented by u- and mCANP with production of 360 and 155kDa limited digestion products at 360 and 155kDa. Each peptide was further fragmented to 300kDa and 150kDa products, respectively. Eventual loss of high molecular weight protein was observed after extensive proteolysis with accumulation of minor products at ~175kDa. The 150kDa peptide was not further degraded, however, and appeared to be a limiting peptide. This pattern of fragmentation was similar to the skeletal HSR although no subtle differences between u,- and mCANP effects were observed.  2.Immunolocalisation of CANP to HSR membranes. Observation of endogenous CANP mediated proteolysis of the 550kDa peptide during purification of HSR membranes (Figure 28, lanes B and C) prompted investigation of the possible association of this protease with HSR. In the right panel of Figure 34, the 4 membrane fractions harvested after 25-45% sucrose density gradient centrifugation were immunoblotted with a polyclonal anticalpain antibody, cross-reactive to u.- and mCANP.  For reference, the  complementary Coomassie-Blue stained fractions shown in Figure 5 are reproduced here (Figure 34A). These experiments revealed the presence of a 90 kDa protein reactive to the antibody. This protein was present in all the fractions although more abundant in the lighter density membrane fractions. In preliminary studies of HSR solubilisation with CHAPS it was observed that the 550 kDa protein, present in the CHAPS insoluble fraction, was selectively fragmented when resuspended after sedimentation in buffer devoid of  163  Fig, 33. CANP proteolysis of cardiac HSR proteins. Under identical conditions to that defined for skeletal HSR proteolysis in Figures 29B and 30B, cardiac HSR membranes were treated with uCANP (A) and mCANP (B). In (A) and (B) lane A corresponds to control, untreated HSR. Lanes B to E in (A) correspond to treatment with 0.1, 0.5, 1.0 and 2.5 units CANP.mg HSR" , respectively. In (B) 1  lanes B to D correspond to treatment with 0.5, 1.0 and 2.5 units.mg" , 1  respectively.  CRC, CPP AND CBP have the same meanings as in Figure 5.  Positions of peptides (MyXlO ) are indicated to the right of each panel. 3  164  Figure 33  §88  CO  II liI I I II ' 1 II  1  <) 7%  COCO  < J  r  m o  CM  IT  L Q  o  T- r-  1.1  (  II 1 11 Si|i II 1 1 1i i ' I i Hill 1 1 1 ON i I I I it ul T P  i  I  CD  05  CO  3  165  Fig. 34. Immunostaining of SR microsomal protein reactive to CANP polyclonal antisera. Microsomes obtained from the 45,000 rpm centrifugation step were separated by density gradient centrifugation (see methods) into 4 discrete bands at 25-27%, 29-33%, 35-38% and 39-41% sucrose. In (A) 50 ug protein of each fraction (lanes a to d, respectively) was resolved by SDS-PAGE (3-13% gradient gels) and stained with Coomassie brilliant blue R-250. Identity of the calcium release channel /ryanodine receptor (CRC), the calcium pump protein/Ca  2+  ATPase (CPP) and the calcium binding protein/calsequestrin  (CBP) is as indicated on the right of the panel. Western blots of protein (50 ug per lane) from complementary gels were transfered onto 0.45u nitrocellulose membranes and were incubated with CANP polyclonal antisera for 24 hrs (see methods).  After washing, blots were incubated for a further 2 hrs with a  secondary goat anti-rabbit antibody conjugated with horseradish peroxidase. Staining of horseradish peroxidase after reaction with 4-chloro-l-naphthol and hydrogen peroxide permited identification of a 90 kDa SR/t-tubule associated protein (open arrow) reactive to CANP polyclonal antisera. Fractionation of proteins in (A) was representative of the results of over 30 separate membrane isolations.  Immunoblots in (B) were reproduced with 4 separate membrane  preparations with identical results.  166  Figure 34  B a b e d Mol. wtlkDaj  - m  a b e d -%CRC  358-i  205-1 11697664331-  CPP CBP  167  Fig. 35. Immunostaining of CHAPS solubilised protein reactive to CANP polyclonal antisera. HSR membranes were solubilised in 1.5% CHAPS: 0.5% phosphatidylcholine as described in the methods.  In (A) membranes were  solubilised in the presence (lanes A and C) and absence (lanes B and D) of lOO^iM leupeptin. 50ug of CHAPS insoluble (lanes A and B) and CHAPS soluble (lanes C and D) were resolved across 3-13% SDS-PAGE gradient gels and stained with Coomassie-R250 (see methods). In (B) complementary gels were run as in (A) and proteins were immunoblotted with CANP polyclonal antisera as in Figure 35. The solid arrow indicates the position of the 410kDa proteolytic cleavage product. The open arrow indicates the position (90kDa) of the immunoreactive protein in lane A.  Figure 35  169  leupeptin (Figure 35A, lane B). The soluble fraction was apparently resistant to endogenous proteolysis regardless of the presence or absence of leupeptin (compare lanes C and D). It was then investigated whether a specific localisation of  CANP  to HSR membranes might account ' for these observations.  Immunoblots of membranes resolved by SDS-PAGE revealed localisation of immunoreactivity toward the anti-calpain antibody solely in the pelleted fraction prepared in the presence of leupeptin (Figure 35B). Loss of immunostaining and 550 kDa proteolysis of the pelleted material in non-leupeptin containing buffers is consistent with autolytic fragmentation of CANP.  3. CANP effects upon HSR function.  a. Passive C a  2+  loading and Ca -induced C a release. 2+  2+  The functional effects of both u- and mCANP digest of HSR proteins were examined through studies of Ca -induced C a 2+  loaded with C a  2 +  2 +  release from vesicles passively  (Figure 36 and Table 5). For both isoforms, C a  2 +  loading was  elevated by 15% with mild CANP treatment as determined after rapid dilution of Ca  2 +  loaded vesicles into C a  2 +  release inhibiting media containing the C a  channel blockers ruthenium red and M g . 2 +  2 +  Extensive CANP treatment with  eventual production of limiting digest products did not markedly stimulate further increases in HSR C a  2 +  loading. Much of the modified C a  2 +  handling  appeared to be associated with initial stage production of the 410 and 150 kDa peptides.  The elevation of HSR C a  2 +  loading after CANP digest was  accompanied by 30-35% increases in fractional C a amounts of C a Ca  2+  2+  2 +  release with identical  remaining after release. A similar elevation of C a  2 +  loading and  release was also observed with CANP treatment of cardiac HSR  170  Fig. 36. Calcium loading and calcium induced calcium release and mCANP treated HSR membranes. HSR vesicles (lOmg.ml" ) were incubated at 22°C in 1  the presence of 300mM sucrose, 150mM KC1, 5mM DTT, lOmM C a , 50mM 4 5  2 +  HEPES (pH 7.4) and either 0.25, 1, or 5 units.mg" HSR for 20 minutes. 1  Proteolysis was quenched upon addition of lOOuM leupeptin.  Control  experiments were performed in the presence of 5 units.mg" CANP with prior 1  addition of lOOuM leupeptin. Membranes were subsequently incubated at 4°C for 24 hrs followed by a further 2 hr incubation at 22°C. Aliquots (5ul) were rapidly diluted (250 fold) and mixed into iso-osmotic media (300mM sucrose, 150mM KC1, 50mM HEPES, pH 7.4) containing, in addition, either 5mM EGTA and 4.9mM C a  2 +  (10uM free C a ) or lOmM M g , 50uM ruthenium red and 2+  2 +  lOOuM EGTA (Quench buffer). At the indicated time intervals 200ul aliquots were withdrawn and vacuum filtered across 13mm diameter 0.45u nitrocellulose filters which were rapidly washed with 5 volumes of Quench buffer. Filters were immediately removed from their support grids, air dried and assayed for radioactivity by liquid scintillation counting.  The data represent means  (standard error less than +/-5%) of triplicate determinations from a single experiment representative of 4 independent experiments conducted on separate HSR preparations using 3 separate CANP preparations.  171  Figure 36  Table 5. C a l c i u m release from u C A N P treated vesicles.  CANP/HSR RATIO  Ca  (Units.mg HSR )  (nmol Ca .mg HSR' )  (% max.)  Control 0.25 1.0 5.0  103+/-1.7 115.5+/-2.7 117.9+/-2.2 119.3+/-1.9  77.7 80.1 80.6 80.9  -1  a  2 +  Retained  Ca  b  2+  1  2 +  Released  a. Control vesicles were treated with leupeptin prior to addition of 5 Units CANP.mg HSR  -1  b. Determinations represents averages (+/-sem) from triplicate observations in a single experiment.  173  (Figure 37).  b. ATP dependent calcium accumulation. Consistent with the above effects was the observation of similar increases in Ca -ATPase mediated C a 2+  2 +  accumulation under conditions of C a  2 +  (25uM  free) and pH (7.4) designed to promote the open state of the channel (Figure 38). These absolute increases, which were not due to modified Ca -ATPase activity 2+  determined in the presence of A23187 (Table 6), were preserved in the presence of ruthenium red.  c. Spectroscopic studies of CANP effects upon HSR C a transport 2+  The above findings of a possible alteration of an intralumenal C a  2 +  compartment (Figures 37 and 38) were investigated further with dualwavelength spectroscopy of HSR C a  (i) . C a  2+  2 +  uptake and release.  uptake.  With limited exposure of HSR membranes to mCANP, the rates of both the secondary slow phase and tertiary fast phase of C a  2 +  accumulation were slightly  elevated (Figure 39B). With prolonged CANP treatment, the rate of the slow phase accumulation was doubled with a 50% shortening of the time required to accumulate all extravesicular C a . 2+  Similar effects were observed with  endogenous CANP (Figure 39A).  (ii) . C a release. 2+  Figure 40 shows that with extensive u- and mCANP treatment the amount of intralumenal C a  2+  slightly by 15%.  required to trigger the release of C a  2+  uptake was decreased  Similarly, the amount of ryanodine required to induce  174  Fig. 37. Effect of  JICANP  and mCANP upon passive calcium loading and  release by cardiac HSR vesicles. Vesicles (lOmg.ml" ) were exposed to 5mM 1  4  ^Ca  2 +  and 0.5 U.mg" CANP for 30 mins. 1  Proteolysis was quenched by  addition of leupeptin to a final concentration of lOOuM. Control ( were treated similarly to uCANP (  ) and mCANP (  ) vesicles  ) except that leupeptin  was added prior to CANP. Vesicles were further incubated for 20hrs at 4°C. Vesicles were diluted 250 fold into either 50uM ruthenium red containing media to obtain the zero time ^ C a 4  2 +  loading by back extrapolation, or 4.9mM  C a / 5 m M EGTA buffer (lOuM free C a ) to induce 2+  2+  45 2+ Ca  release.  175  Figure 37  15n  0  30  60  90  Time (sees)  120  150  176  Fig. 38. Effect of C A N P upon ATP dependent calcium uptake by H S R vesicles. 5mg.ml HSR was exposed to both u- (  ) and mCANP (  -1  methods.  ) as described in  Vesicles were diluted into iso-osmotic transport buffer and C a  2 +  uptake was initiated upon addition of Mg.ATP (5mM free in the presence of 25uM free  4 5  Ca  2 +  and 500uM free M g .  uptake were compared to controls (  2 +  The effects of C A N P upon C a  2 +  ) in the presence (filled symbols) and  absence (open symbols) of 50uM ruthenium red. Aliquots (150ul) were filtered at the indicated times and radioactivity determined by liquid scintillation methods.  -O  -RR »RR  O • Control • • mCANP A A //CANP 60  120  Time (seconds)  180  240  Table 6. Ca -stimulated ATPase activity of CANP treated HSR membranes. 2+  Ca -ATPase (nmol Pi.mg HSR" ^.min." )* 1  2+  uCANP mCANP  -A23187  -A23187  635+M3 641+/-50  1391+/-109 1384+/-81  a. Ca -ATPase activity was determined at 25oM free C a 2+  2 +  in the presence  of 500uM free M g . Activity observed in the presence of 500uM EGTA was 2 +  subtracted from the total activity to yield C a  2 +  stimulated activity.  179  Fig. 39. Effect of CANP mediated proteolysis of HSR upon calcium uptake. In (A) 0.5mg.ml HSR vesicles isolated in the absence (trace a) or presence (trace b) -1  of leupeptin (Leu) were incubated in the presence of transport buffer containing 60uM C a  2 +  and 5mM CP as described in Figure x. C a  2 +  uptake was initiated by  addition of ImM Mg-ATP. In (B) 1.375mg HSR (40mg.mr ) were pre-treated at 1  25°C with 1.375 units mCANP in the presence of 300mM sucrose, lOOmM KC1, 50mM HEPES (pH 7.4), 5mM DTT, 2.5mM C a  2 +  for 2 minutes (trace b) and 30  minutes (trace c). Proteolysis was terminated upon addition of leupeptin to a final concentration of lOOuM. In trace a (control) leupeptin was added prior to the addition of mCANP. Vesicles were added to the cuvette and pre-incubated for 20 minutes to allow efflux of passively loaded C a . 2 +  Ca  2 +  uptake was  initiated by ImM Mg-ATP addition and monitored by dual-wavelength spectroscopy as in (A) above.  180  181  Fig. 40. Effects of CANP upon intralumenal calcium dependence of calcium induced calcium release.  2.75mg HSR protein were exposed to 2.75 units  uCANP (A) and mCANP (B) in the presence of 2.5mM C a  2 +  for 20 minutes at  25°C. The composition of the proteolysis buffer was otherwise, identical to that described in Figure 39B. C a with addition of 5uM C a  2 +  2 +  uptake was assayed as described in Figure 21  pulses (arrowheads) to stimulate C a  2 +  release. The  final EGTA concentration in the cuvette was ~3-5uM. In (A) and (B) controls (trace b) were treated identically to CANP proteolysed HSR (trace a) except for addition of lOOuM leupeptin prior to addition of CANP. Solid arrows indicate addition of ImM Mg-ATP.  182  Figure 40  A.  AA AAA AAA A A A  183  Ca  2+  release was also decreased (Table 7). The amount of C a  assay system was unaltered although the rate of C a  2+  2 +  released in this  re-accumulation was  decreased. These effects were only observed after extensive CANP mediated proteolysis of the channel. Although these were small changes in HSR function the titration of intralumenal C a  2+  sites in this manner was highly predictable.  4. CANP effects upon [ H]ryanodine binding. 3  In view of the specific CANP effects upon C a  2+  channel structure it was  appropriate to examine these effects upon ryanodine binding. CANP mediated fragmentation of the release channel upon [ H]ryanodine binding are shown in 3  Figure 41. Incubation of membranes in the presence of 5mM AMP, 50uM free Ca  2 +  and 8nM-2.5uM [ H]ryanodine revealed the presence of both low and high 3  affinity ryanodine binding sites. Under conditions which promoted complete fragmentation of the intact 550kDa peptide by both CANP isoforms, total [ H]ryanodine binding was elevated by 25%. Scatchad analysis revealed that 3  both low and high affinity B  m a x  values were elevated after both u and mCANP  treatment (Figure 42). These data show that [ H]ryanodine binding affinity was 3  unaffected at either site by CANP treatment of HSR membranes.  T a b l e 7 . Effect of C A N P proteolysis u p o n ryanodine induced calcium release.  Added C a  A d d e d Ryanodine ( u M )  2 + b  (nmol.mg* )  Control  uCANP  60 80 100  144 396 1440  106 288 1260  1  c  b  mCANP  a  108 324 1188  a. Proteolysis was conducted under identical conditions to that described i n Figure 40. b. The experimental protocol for C a as described in Figure 27.  2 +  and ryanodine addition was performed  c. Observations are averages of two determinations at each C a  2 +  loading.  a  185  Fig. 41. Effect of uCANP and mCANP upon ryanodine binding to HSR membranes. HSR vesicles (20 mg.ml ) were exposed to CANP (1 U.mg" HSR) -1  1  for 20 minutes at room temperature (23 °C) in the presence of 300mM sucrose, 150mM KC1, 5mM C a , 5mM DTT, 50mM HEPES (pH 7.4). Proteolysis was 2 +  quenched upon addition of leupeptin to a final concentration of lOOuM. Control experiments were performed with the addition of leupeptin prior to the addition of CANP. Aliquots (lOul) of the above mixture were diluted fifty fold into media containing 300mM sucrose, 150mM KC1, 5mM AMP, 8nM-lmM [ H]ryanodine 3  and 50mM HEPES (pH 7.4) and membranes were incubated at 23 °C for 24 hours.  Final protein and C a  2 +  concentrations were 400ug.ml" and lOOuM  (50uM free) respectively. Effects of uCANP (  1  ) and mCANP (  specific [ H]ryanodine binding are compared to controls ( 3  ) HSR upon ).  At ImM  [ H]ryanodine total binding for control, uCANP and mCANP were 23.1, 23.5 3  and 23.8 nmol.mg" HSR, respectively (p>0.05). Specific binding was obtained 1  by assuming non-specific [ H]ryanodine binding to be a constant proportion of 3  total binding. The above data were means of triplicate observations in a single experiment and are representative of three independent experiments conducted on different HSR preparations (less than 5% standard error).  186 Figure 41  35-1  Ryanodine (nM)  187  Fig. 42. Scatchard analysis of ryanodine binding to HSR membranes. Data from Fig. 41 was replotted as a scatchard diagram. The table in the figure summarises the maximum [ H]ryanodine binding at each low and high affinity 3  site.  1.5 i  BINDING  CONT //CANP mCANP  Hlflh Affinity  0  5  10  15  20  25  i  Control  •  //CANP  •  mCANP  30  Bound Ryanodine (pmol/mg protein)  35  40  189  DISCUSSION  The major hypothesis within this study was that intralumenal C a  2 +  within the  terminal cisternae of sarcoplasmic reticulum is an important regulator of the Ca  2 +  release channel.  It was envisioned that the binding of C a  calsequestrin might facilitate the timing of C a order to coordinate the movements of C a  2 +  2 +  2 +  to  channel opening and closing in into and out of the SR during  isotonic contraction of muscle. Considering that the release channel and the C a -ATPase are both regulated by the same cytosolic effectors within a similar 2 +  concentration range (e.g. u.M C a , mM Mg-ATP, mM M g ) , such a signalling 2 +  2+  mechanism might be necessary. Within this scheme, the conformational changes that would be associated with C a  2 +  release channel opening and closing would  be coordinated with structural changes in calsequestrin associated with the binding and dissociation of C a .  It was then hypothesised that structural  2 +  modification of the C a  2 +  release channel by C a  during episodes of cytosolic C a association of C a compartment.  2 +  2 +  2 +  activated neutral proteases  overload would lead to alteration in the  with calsequestrin or some other intralumenal C a  2 +  binding  In exploring these hypotheses the following objectives were  established, (a) to obtain a highly purified membrane preparation, referable to the terminal cisternae of the SR, (b) to develop a C a allowed investigation of the intralumenal C a  2 +  2 +  transport assay that  compartment, (c) to obtain highly  purified preparations of CANP, (d) to assess the effect of intralumenal C a upon C a  2 +  2 +  release channel function and (e) to assess the effect of  pharmacological and structural manipulation of the C a intralumenal C a  2 +  dependence of C a  I. H S R structural characterisation  2 +  release.  2 +  release channel upon  190  In pursuit of the first objective Figures 5 and 6 show that with the novel freezing/grinding method of tissue disruption, highly purified HSR membrane preparations could be obtained. The isolated HSR membranes were enriched in 550kDa and 57kDa proteins identified, respectively, as the C a  2 +  release  channel/ryanodine receptor (Lai et al., 1987; Inui et al., 1987; Imagawa et al., 1987) and calsequestrin (Maclennan and Campbell, 1979).  The amount of  calsequestrin within these preparations was approximately 20%, as deduced by densitometric scanning of Coomassie blue stained gels. This value is, likely, an underestimate, as calsequestrin binds this dye relatively poorly in view of its high acidity (Reithmeier et al., 1987). In isolated SR, Williams and Beeler (1986) estimated that calsequestrin constituted 30-33% of the total protein within the terminal cisternae.  With Stains-All staining, the amount of evident 57kDa  protein was much increased in relation to the amount of 105kDa protein (Ca 2+  ATPase). Calsequestrin therefore appeared to represent a major protein within these preparations. The amount of 550kDa protein was estimated at 7-10% of the total SR protein from densitometry. These membranes were shown to bind a total of 30pmol ryanodine.mg HSR" (Figure 42) with identification of low and high affinity 1  ryanodine binding sites. These results are consistent with similar values (3233pmol.mg ) reported by Lai et al. (1989) who also observed dual site binding of _1  ryanodine. It is difficult, however, to make strict comparisons of data obtained from reported ryanodine binding studies since a survey of the literature reveals a diversity of findings. inhibition of C a  2 +  Ryanodine, reportedly, induces both activation and  channel function (see Meissner, 1986; Lattanzio et al., 1987).  Observation of these contrary effects appears to depend upon assay conditions. In many cases, studies report the existence of a single high affinity ryanodine binding site (e.g. Imagawa et al., 1987; Mickelson et al., 1990; Pessah et al., 1987;  191  Chu et al., 1990). It has been shown that assays conducted in the presence of high salt (IM KC1 or NaCl) result in expression of a low affinity ryanodine binding site (McGrew et al., 1989; Lai et al., 1989; Meissner et al., 1989). In the present study [ H] ryanodine binding studies were conducted in the presence of 3  150mM KC1. This is a substantially lower and more physiological ionic strength and represents conditions under which most other laboratories report single high affinity [ H] ryanodine. The basis for these differences is unclear. A curious 3  finding was that expression of low affinity [ H] ryanodine receptor sites was 3  related to the stage of development and ryanodine receptor content in dystrophic and normal chicken pectoralis muscle in the presence of physiological salt (Pessah and Scheidt, 1990). Whether this can be taken to indicate that expression of ryanodine binding site affinity is also a function of receptor density and architecture within the membrane is unclear.  However, observation of high  capacity ryanodine binding at both low and high ryanodine binding sites in the present study was taken as indicative of the enrichment of this microsomal preparation in terminal cisternae membranes.  II. HSR functional characterisation The above observations upon the structural properties of the isolated HSR membranes were consistent with the functional properties expected of terminal cisternae derived fractions.  As shown in Figure 15, C a  2 +  -stimulated ATPase  activity at p H 7.0 was markedly suppressed (2.8 fold) by the presence of ruthenium red (lOuM), an organic poly cation blocker of the C a  2 +  release channel  in HSR membranes (Palade, 1987a). This is reflected in the 1.8-2 fold elevation in 4  ^Ca  2 +  accumulation at p H 7.4 (Figure 38).  stimulated the Ca -ATPase activity. 2+  apparent C a  2 +  Conversely, the C a  2 +  ionophore  However, A23187 also increased the  sensitivity of Ca -stimulated ATPase in these membranes. The 2+  192  basis for this was not experimentally pursued. This effect may reflect uncoupling of indirect C a  2 +  channel regulation of ATPase activity. As indicated earlier, the  Ca -ATPase and the C a 2+  2 +  concentration range of C a  2 +  release channel are both activated within a similar (see Gould et al., 1987). In the absence of channel  modification, ATPase activity would be activated by both (a) a C a leak pathway (open channel) and (b) a C a of A23187, C a  2 +  2 +  2 +  activated  activated ATPase. In the presence  activation of the major leak pathway would be diminished with  expression of, solely, the C a  2 +  activated ATPase. The absolute values reported  here for ATPase activity, in the presence and absence of ruthenium red and A23187, are markedly (1.5-3 fold) lower than that reported in a comprehensive study of HSR function (Chu et al., 1986). In addition, Chu et al. (1986) observed only 1.1 to 1.45 fold A23187 stimulation of ATPase activity between 5 and O.lmM M g , for junctional terminal cisternae membranes. Conversely, ruthenium red 2 +  inhibited ATPase activity by only 16-24%. It is appropriate, at this point, to comment upon the procedures for preparation of the membranes used in this study.  Conventional protocols  commonly employ mincing of fresh muscle in preparation for homogenisation. In this study muscle was ground to a fine, electrostatically charged powder under liquid nitrogen.  Conventional homogenisation techniques were then  employed utilising buffer systems and centrifugation protocols described by Chu et al. (1986). It is possible that membranes, in this case, were freeze fractured, rather than sheared. This appears to result in retention of much of the junctional protein in these membranes, which were obtained with high yield (see Table 2). In addition to the 550kDa and 57kDa proteins, these membranes also demonstrated the marked presence a ~95kDa protein (see Figures 5, 6, 29, 30), which may be referable to the junctional protein identified by Brandt et al. (1990).  These membranes, therefore, appeared  to be highly enriched in  193  membrane derived from the terminal cisternae and were used throughout the remainder of this study.  III. CANP characterisation As discussed earlier, in order to investigate CANP effects upon HSR structure and function it was essential to obtain a purified endogenous source of this protease. Isolation of purified m- and mCANP from rabbit skeletal muscle was achieved by standard chromatographic techniques (Figures 10 to 13).  The  presence of an additional protein associated with each isoform (Figure 14, lanes D and D') was curious. For each isoform, the additional protein was present in stoichiometric  amounts  with the large subunit during gel permeation  chromatography. Subsequent repeated phenyl-sepharose chromatography did not remove this protein. Both fractions were differentially sensitive to C a , as 2 +  expected, with observed pCa5Q values of 50mM and 750mM C a  2 +  with casein  substrate for u- and mCANP respectively, (Figure 31 A). These are consistent with similar values reported by Cong et al. (1989) for bovine skeletal muscle. However, the reported C a  2 +  activation characteristics of both u- and mCANP are  varied depending upon the source of tissue and the species. Chicken gizzard mCANP was half maximally activated by 150uM C a  2 +  (Hathaway et al., 1982),  whereas Suzuki et al. (1981) reported a pCa5Q of 400uM for chicken skeletal muscle mCANP.  This variation was instrumental in identifying the need to  purify endogenous CANP. Significant variation in reported C a  2 +  activation characteristics  CANP  purified by different laboratories using the same tissue is also evident. With bovine cardiac mCANP, Clark et al. (1986) reported half maximal activation by 300uM C a , whereas Tan et al. (1988) reported a value of ImM. These 2 +  differences may be due to varying degrees of autolytic activation of C a  2 +  194  sensitivity. This may depend upon the method of isolation. It is also evident that the method of assaying CANP significantly affects the estimated activity. With casein substrate, it was observed (Gilchrist and Belcastro, 1990; unpublished observations) that the estimate of CANP activity depended nonlinearly upon CANP concentration. limitations, since  This effect was not due to substrate  increasing concentrations  of casein did not increase  caseinolysis. This peculiar behaviour of CANP is under current investigation (Gilchrist, Machan and Belcastro). The significance of this, to the present study, was that comparison of u- and mCANP effects required several repeated determinations of caseinolysis. Concentrations of CANP were adjusted from a previous determination in order to obtain identical absorbance values (A280) at each of 4 serial dilutions. CANP activity was then estimated from the average determination. This effect was earlier reported (Tan et al., 1988 cf Table 2), but the significance of this was ignored. The difficulty in obtaining quantitative estimates of CANP for comparative purposes was further exacerbated by the peculiar loss of CANP activity upon protease concentration, both in N2 pressure cells and centrifugation filtration (Gilchrist and Belcastro, 1990; unpublished observations). This phenomenon was also reported by Kenessey et al. (1989) with brain mCANP. This does not appear to be due to autolytic inactivation of CANP, since the large subunits of purified u- and mCANP fractions (see Figure 14) remained intact after concentration for gel permeation chromatography. This concentration dependent behaviour of CANP may involve hydrophobic site mediated aggregation of CANP.  The distinct elution peak after phenyl-  sepharose chromatography (Figure 11) was only observed with concentrated CANP preparations. With dilute CANP suspensions, CANP eluted much later and with a broad elution profile.  195  IV. CANP effects upon HSR structure In preliminary studies, it was found that the effectiveness of u- and mCANP in fragmenting HSR protein was particularly variable. Recognition of the above problem allowed a clear comparison of u- and mCANP effects. It was shown that both isoforms of CANP fragmented the channel in an identical protease concentration dependent manner (Figures 29A and 30A). As reported earlier (Seiler et al., 1984), both the intact 550kDa protein and the 410kDa digestion product were good CANP substrates. Subtle differences were evident, however, in the fragmentation pattern. In particular 360 and 225kDa mCANP specific peptides were observed whereas mCANP digestion resulted in production of a faint 286kDa peptide. Both proteases resulted in production of high molecular weight limiting peptides at 175, 165, and 137kDa. In this regard, a two stage fragmentation pattern was evident with both proteases. The primary stage could be identified with production of 410 and 150kDa peptides from the 550kDa parent protein. The sum molecular weight of these peptides (560kDa) suggests that initial cleavage of the ryanodine receptor by CANP was initiated at a single specific site. This was particularly evident in Figure 30A (lanes H to K) where production of the 150kDa peptide coincided with appearance of the 410kDa peptide.  This was also seen in Figure 28A, after endogenous proteolysis of  skeletal HSR and exogenous CANP treated cardiac HSR (Figure 33). Secondary stage fragmentation was coincident upon complete fragmentation of the intact 550kDa peptide. In the case of mCANP this stage was marked by the appearance of (a) a 225kDa fragment from presumably, the 410kDa fragment and (b) a 137kDa limiting peptide that resulted from progressive fragmentation of the 150kDa peptide through a 145kDa product.  With uCANP the  fragmentation of the 150kDa protein resulted in abrupt production of the 137kDa peptide. This was always observed with endogenous uCANP. With exogenous  196  human erythrocyte uCANP, fragmentation of the 150kDa fragment resembled the gradual pattern of endogenous mCANP proteolysis (Figure 32).  This  suggests that CANP species differences may determine substrate selectivity to a small degree. However, Figure 32 shows that the major cleavage products (410, 330,150kDa) are identical between CANP species and isoforms (compare Figures 29, 30, 32).  Furthermore, an identical rate and pattern of fragmentation was  obtained with the CHAPS (0.1%) solubilised and vesicular ryanodine receptor. The conditions under which this was observed appear to be dependent upon the concentration of CHAPS detergent. At higher CHAPS concentrations (1-1.5%), normally employed to solubilise the channel (see Lai et al., 1989), CANP was inactivated (D. Croall; personal communication). After the completion of the experimental work, Rardon et al. (1990) published similar observations of chicken gizzard mCANP fragmentation of the ryanodine receptor. Production of 350 and 315kDa peptides was observed after limited proteolysis with a limiting 150kDa peptide after extensive proteolysis. The high molecular weight fragments, resolved on their 5% acrylamide gels are likely equivalent to the 410 and 330kDa peptides observed in the present study using 313% gradient gels. Within these gels, linear log/log plots of %T (% acrylamide) versus M revealed that good estimates of the size of high molecular weight r  components could be obtained (Figure 29B). Several differences in the results of the 2 studies were, however, evident. Rardon et al. (1990) could not attribute initial limited proteolysis to a single site cleavage and did not observe further fragmentation of the 150kDa peptide. It is unclear whether these differences can be attributed to the different sources of CANP used in each study.  It is  interesting that the chicken gizzard mCANP employed by Rardon et al. (1990) was, earlier, reported to be half maximally activated by 150uM C a  2 +  (Hathaway  197  et al., 1982). In the present study, rabbit skeletal muscle mCANP was half maximally activated by 750uM C a  2 +  (Figure 31).  A novel contribution of this study the was demonstration of striking differences  in C a  2 +  sensitivity  fragmentation (Figures 20,30).  of u- and mCANP mediated 550kDa  The C a  2 +  sensitive fragmentation for each  isoform, obtained from densitometry of Coomassie-Blue stained gels, was identical to the observed with C a  2 +  importance of this is that the C a  stimulation of proteolysis was due to C a  2 +  activated caseinolysis (Figure 31). The  activation of CANP, rather than C a  2 +  2 +  induced conformational changes in  550kDa substrate sensitivity to proteolysis.  Belcastro, Machan and Gilchrist  (1990; submitted) have shown that CANP substrate proteolytic sensitivity in myofibrils was increased by C a  2 +  induced protein conformational changes. In  addition, the present study identified a second 88kDa substrate that was cleaved in a similar C a  2 +  and CANP concentration dependent manner to the 550kDa  protein (Figures 29,30). The identity of this HSR associating protein is unknown. However, a recent study showed a similar peptide to be phosphorylated in a Ca  2 +  /calmodulin-dependent manner (Chu et al., 1990). It is possible that this  protein may be associated with the feet structures spanning the junctional gap between the junctional face membranes of the cisternae and the t-tubules. The content of this protein was quite variable between membrane preparations and could, in some preparations, be resolved as a doublet (see Figures 5,28). In a recent study, Kim et al. (1990) showed that the connections between the ttubules and the terminal cisternae in "weak triads" could be broken by mCANP with an increase in the buoyant density of the HSR membranes in sucrose density gradients. The same group (Brandt et al., 1990) reported production of 270,  250,  220  and  190kDa mCANP  digest products  observed  after  immunoblotting of terminal cisternae enriched membranes with a polyclonal  198  antibody raised against the ryanodine receptor. The molecular weight estimates for their fragments are different from that reported here and direct comparison with the results of their study to the present is difficult. Moreover, Brandt et al. (1990) did not observe fragmentation of a ~80-90kDa protein although this may have been precluded by the resolution and reprographic quality of their gels. The following observations led to the proposal that CANP may directly associate with HSR membranes: (a) the ryanodine receptor was a good substrate for CANP (Figures 29, 30); (b) the ryanodine receptor was partially degraded when HSR membranes were isolated in the absence of leupeptin (Seiler et al., 1984; Figure 28A; lane B); (c) CANP binds hydrophobically to plasma membranes (Murachi, 1989; Mellgren et al., 1987; Suzuki et al., 1987) and subcellular organelles (Gopalakrishna and Barsky, 1986); (d) calpastatin, an endogenous inhibitor protein of CANP (Mellgren, 1988), is localised to the SR (Mellgren, 1987);  (e) prolonged incubation of HSR/t-tubule membranes in  sucrose density gradients lacking leupeptin resulted in further fragmentation of the ryanodine receptor (Figure 29A, lane C). In order to test this hypothesis, the 4 major membrane fragments obtained from sucrose density gradients (Figure 34A) were immunoblotted (Figure 34B) with an antiCANP polyclonal antibody, a generous gift from Dr. T. Kuo. Immunoreactivity toward a ~90kDa protein was observed to be strongest in the light membrane fraction which was likely derived from the t-tubule/triad region. Curiously, ISR fractions (lane C) were more immunoreactive than HSR. This might indicate the presence of undissociated ttubule/HSR membranes within this fraction. A preliminary observation, during earlier isolations of the ryanodine receptor, was that the CHAPS soluble fraction was apparently resistant to endogenous proteolysis in the absence of protease inhibitors. Conversely, the ryanodine receptor in the CHAPS insoluble pellet was highly susceptible to endogenous proteolysis.  This suggested a specific  199  compartmentation of CANP within HSR membranes. repeated and the hypothesis confirmed.  The experiment was  Figure 35B (lane A) shows  immunoreactivity toward a protein present in the leupeptin containing CHAPS insoluble pellet (open arrow). In the absence of protease inhibitor (lane B) the ryanodine receptor was cleaved (solid arrow) and immunoreactivity was absent. The latter was presumably due to CANP autolysis. Of interest is the enrichment of the CHAPS insoluble pellet in 165 and 88kDa protein relative to the CHAPS soluble membranes. These proteins were relatively more abundant in the light density membranes (Figure 5, lane A) which were of presumed t-tubule origin. The recent observation that CANP dissociates the "weak" triads (Kim et al., 1990) and the evident association of CANP with junctional membranes observed in this study would suggest that CANP may perform a specific role, in vivo, in targetting specific substrates. Observation of a ~90kDa CANP isoform is novel as the molecular weight of the purified cytosolic form and the cDNA encoded form of the large subunit for each CANP isoform from several different tissues were reported to be between 80,000 and 82,000 (Imajoh et al., 1988). However, Spalla et al. (1985) reported a molecular weight of 92,000 for uCANP purified from the hearts of hypertensive rats. It is possible that the immunoreactive 90kDa peptide observed in this study may be a novel isoform of CANP.  V. HSR calcium release The earlier observations that (a) Ca -induced C a 2+  lumen required a threshold intralumenal C a  2 +  2 +  release from the SR  (Ohnishi, 1979) and (b) tetanised  frog and rat muscle (Gonzalez-Serratos et al., 1979; Sembrowich et al., 1983) accumulated elemental C a  2 +  within the cisternal region of the SR in association  with loss of contractile function, suggested that intralumenal C a for the regulation of C a  2 +  2 +  was critical  release. In the case of the latter, it was not clear  200  whether failure of some step in excitation contraction-coupling caused cisternal Ca  2 +  accumulation, or whether the accumulation of C a  2 +  impaired it's release.  However, the observation of reduced force production concomittant with sarcoplasmic C a SR C a  2 +  2 +  imbalances ("Ca overload") suggested that the alteration of 2+  release may also be related to C a  2 +  activated CANP proteolysis of the  release channel. In light of the suggested functional association of calsequestrin with the release channel (Meszaros and Ikemoto, 1985; Meszaros et al., 1987) it was proposed that CANP mediated structural modifications of the channel may alter intralumenal C a  2 +  handling by, possibly, calsequestrin.  CANP was,  therefore, used as a physiological tool to probe the relationship between the release channel and the intralumenal C a  2 +  compartment.  In this study, HSR vesicles were chosen as an in vitro model for the study of Ca  2 +  release since the relationship between intralumenal C a  2 +  and C a  2 +  release  could be studied in isolation whilst retaining much of the structural and functional characteristics of the SR in vivo (see Ikemoto et al., 1989). Meissner (1984) has shown that HSR vesicles, passively loaded with C a , rapidly release 2 +  -75% of intralumenal C a  2 +  within 75ms upon rapid dilution into media  containing 5mM AMP-PCP and 4uM trigger C a . The observed C a 2 +  2 +  efflux  rate constant was 90s" under these conditions. In the absence of nucleotide the 1  rate constant was reduced by almost 2 orders of magnitude (~1.5s ) with only a _1  slight reduction in the amount of total C a  2 +  released. The latter conditions were  comparable to those of Figure 16 where 65-80% of vesicular C a 15 seconds when C a  2 +  2 +  contents within  loaded vesicles were rapidly diluted into iso-osmotic  media containing lOuM free C a . This assay system has been highly successful 2 +  in characterising the rapid kinetic properties of the C a  2 +  release channel in both  cardiac and skeletal SR (Meissner, 1986a; Meissner and Henderson, 1987). Meissner (1984,1986a) concluded that the rate of C a  2 +  release from HSR vesicles  201  was consistent with the rate observed, in vivo. In the present study, quantitation of the initial rapid kinetics of C a  release was precluded by the manual  2 +  methods employed. Figure 16A also shows that the amount of C a  2 +  retained by  the vesicles was non-linearly dependent upon the concentration of C a loading medium. Up to ~60nmol Ca .mg HSR" , the vesicular C a 2+  1  was steeply dependent upon extralumenal C a . 2 +  loading medium, (>2mM) vesicular C a  2 +  2 +  With elevated C a  2 +  in the  loading 2 +  in the  retention increased more gradually.  This was different from that reported earlier by Morii and Tonomura (1983) who showed that C a  retention was proportional to the C a  2 +  2 +  concentration in the  loading buffer, up to 20mM. It should be noted, however, that their reported ~20nmol Ca .mg SR" retained in the presence of lOmM C a 2+  1  than that reported here. The additional presence of 5mM M g buffer may have resulted in competition for intralumenal C a thus lower C a  2 +  2 +  dependence of C a  here, was the decrease in the fractional C a 2 +  2 +  is 5 fold lower in their loading  binding sites and  retention.  In parallel with the non-linear C a  Ca  2 +  2 +  2 +  2 +  loading, observed  release expressed as a percentage of  retained (Figure 16). This is also contrary to Morii and Tonomura (1983)  who reported that fractional C a  2 +  release was independent of intralumenal C a  2 +  retention. The basis for this discrepancy is unclear. A curious observation was a concomittant increase in the amount of C a induction of C a  2 +  2 +  remaining in the vesicles, after  release (Figure 16A), when C a  2 +  in the loading medium was  increased. This was also unclear, although it may be related to an effect of C a  2 +  toward vesicle aggregation. In the experiments described in Figure 28A (lane C), increasing the C a  2 +  concentration in the sucrose gradient to lOmM led to  formation of an undispersed, compact disc of all unfractionated HSR membranes (Gilchrist, Katz and Belcastro, unpublished observations). However, Meissner (1986a) has shown that with up to 62mM C a  2 +  in the loading medium, the rates  202  of initial C a  z +  release progressively increased.  This indicates that the C a  releasing function of vesicles is not significantly impaired by high C a  2 +  z +  loads,  both intralumenally and extralumenally. Observation of non-linear C a  2 +  loading in the present study would appear to  indicate the presence of two intralumenal C a differing C a  2 +  binding compartments with  2 +  affinities. It is proposed that a higher affinity C a  2 +  compartment  of intermediate capacity is expressed at low intralumenal C a  2 +  loading  (<60nmol.mg"l). Saturation of this compartment appears to be followed by filling of a lower affinity C a  2 +  compartment. The size of the putative higher  affinity compartment is consistent with its identity as calsequestrin. Although, sarcolumenin, a 160kDa glycoprotein, localised to the cisternae (Leberer et al., 1989) reportedly binds C a  2 +  with intermediate affinity (kd=300-600uM) and high  capacity (400nmol.mg ) the low content of this protein in these membranes (see _1  Figures 5 and 6) precludes significant C a  2 +  binding contribution from this  protein. By elimination, calsequestrin would likely represent both the relatively high and low intralumenal C a  2 +  binding compartments. This is consistent with  the observation that sepharose-immobilised calsequestrin exhibits mutiple C a  2 +  binding affinity (Charuk et al., 1990) as evidenced from non-linear scatchard analysis. An additional method for the study of C a spectroscopy of C a  2 +  2 +  release is dual-wavelength  binding metalochromic dyes. This method, first developed  by Ohnishi and Ebashi (1963), takes advantage of the fact that, at a sufficiently high intralumenal C a  2 +  sink to extralumenal C a  accumulate all extravesicular C a  2 +  with C a  2 +  load ratio, SR vesicles can  2 +  release studied through  subsequent rapid addition of various effectors to the cuvette. Scarpa et al. (1978) first showed that the metalochromic dye, Antipyrylazo III (APIII) is suitable for the study of SR function. APIII binds C a  2 +  with a 2:1 (APIII:Ca ) stoichiometry 2+  203  (Rios and Schneider, 1981) and exhibits a linear CaD2 difference absorbance over a 10-60uM C a  2 +  concentration range.  A disadvantage of this method is the substantial artifactual absorbance shift that is observed at the customary 720-790nm wavelength pair upon the addition of high ATP (>500uM) in the presence of low M g  2 +  (Rubtsov and Murphy, 1988,  Morii et al., 1985). Consequently, the initial phase of C a been poorly resolved when SR C a millimolar ATP.  2 +  2 +  accumulation has  transport is initiated by the addition of  An alternate strategy that eliminates the ATP absorbance  artifact at 720nm is the inclusion of excess M g  2 +  (lOmM) in assay media. This  has been adopted when Murexide (Inesi and Scarpa, 1972), Arsenazo HI (Herbette et al., 1981) and, Antipyrylazo III (Scarpa et al., 1978) were employed to monitor C a  2 +  transients at the respective wavelength pairs. In view of the  effects of elevated M g  upon (a) HSR C a  2 +  2 +  release (Meissner and Henderson,  1987) and (b) divalent cation:AP III difference spectra (Figures. 17 and 18) it was of interest to determine the general appropriateness of this strategy. As shown in Figure 20A, the absorbance artifact was significantly reduced when vesicles were pre-incubated in the presence of 3mM M g  2 +  prior to  addition of nucleotide and was completely eliminated at lOmM total M g  2 +  (Figure 20B). This effect is predicted by the spectral scans shown in Figures 17C and 18C. However, loss of both the secondary and tertiary phases of uptake was also observed with elevated M g . 2 +  This may reflect M g  2 +  inhibition of C a  2 +  release channel opening subsequent to the initial fast phase and probably accounts for M g  2 +  stimulation of HSR C a  1985). Additionally, Ca -induced C a 2+  Ca  2 +  (Figure 20A). C a  free C a  2 +  2 +  2 +  2 +  uptake reported earlier (Watras,  release was less sensitive to trigger  release was not triggered by 5uM free C a  was required to minimally stimulate C a  trigger C a , 25-30nmol C a . m g 2 +  2+  _1  2 +  2 +  and lOuM  release. With 20uM free  was released. M g , therefore, appears to 2 +  204  decrease the C a  2 +  sensitivity of Ca -induced C a 2+  rapid kinetic studies of ^ C a 4  2 +  2 +  release in agreement with  release from passivley loaded HSR vesicles  (Meissner et al., 1986). A 60% reduction in the rate of C a Ca  2 +  2 +  re-uptake, after 20uM  addition, was also observed when compared to the lower M g  in Figure 19 where the sum of added and released C a 50nmol Ca .mg ). At lOmM M g 2+  _1  2 +  2 +  was comparable (—40-  2 +  (Figure 20B) the initial rate of C a  was much reduced and Ca -induced C a 2+  condition  2 +  uptake  release was completely abolished  2 +  with a greatly extended re-uptake of added C a . These data indicate that M g 2 +  may also inhibit C a  2 +  2 +  channel closing subsequent to release, although the  alternative possibility, that of Ca -ATPase inhibition (see McWhirter et al., 2+  1987), was not investigated. It was also evident that M g  2 +  elevation decreased  the sensitivity of measurement. Under the conditions defined by Figure 19, at both wavelength pairs, AlOuM C a At lOmM total M g  2 +  2 +  produced a AA of 0.0025 absorbance units.  (Figure 20B) A50uM C a  2 +  resulted in a AA of only 0.0024  absorbance units. Clearly, elevation of assay M g , in order to eliminate ATP absorbance 2 +  artifacts during dual-wavelength spectroscopy of Ca :AP III AA changes, is an 2+  inappropriate general strategy for HSR C a  2 +  transport studies. Selection of new  wavelength pairs in accordance with the influence of ATP upon dyedigand difference spectra (Figure 18) facilitates study of HSR C a  2 +  uptake and release at  relatively high levels of ATP and intracellularly appropriate concentrations of Ca  2 +  and M g . 2 +  In addition, resolution of initial rapid uptake of C a  2 +  is  improved without loss of measurement sensitivity. The triphasic C a  2 +  uptake kinetic profile of HSR was first reported by Chu et  al. (1983) who observed that this phenomenon was peculiar to the presence of PIPES buffer and an ATP regeneration system in C a  2 +  transport media.  Numerous studies have since confirmed that original observation using PIPES  205  (Morii et al., 1985; Rubtsov and Murphy, 1988; Gilchrist et al, 1990), and MOPS (Plank et al., 1988). In a detailed study, Morii et al. (1985) observed that the rate of the slow phase was decreased in the presence of effectors which stimulated Ca  release (e.g. adenine nucleotides). It was suggested that the slow phase  2 +  resulted from the opening of the C a Ca  2 +  efflux.  2 +  channel and therefore an increase in net  The subsequent fast phase was attributed to channel closure  involving a multi-state transition between channel conformations. mechanism for this remains obscure.  The  Interestingly, Morii et al. (1985) also  showed that as the amount of initial C a  2 +  was increased at constant protein  concentration, the rate of the slow phase decreased. Conversely, at a fixed C a , 2 +  increased protein concentration led to diminuition of channel opening with increased rates of slow phase C a  accumulation.  2 +  These results were also  obscure and remained unexplained. In the present study, it was considered that these results reflected regulation by a saturable intralumenal C a If intralumenal C a Ca  2 +  2 +  regulated C a  sink that exceeded the C a  observed.  2 +  compartment.  channel opening, then at a sufficiently large load, C a  2 +  2 +  2 +  channel opening should not be  Furthermore, by incrementally loading the vesicles with C a  2 +  subsequent to initial uptake it should be possible to identify the size of the intralumenal pool. This hypothesis was tested and confirmed in Figure 21. Ca  2 +  induced C a  intralumenal C a  2 +  2 +  release did not occur until a finite concentration of  had been obtained (Figure 21). Furthermore, the threshold  was independent of whether C a  2 +  was added prior or subsequent to C a  uptake. Observation of triphasic kinetics at higher initial C a  2 +  2 +  loads (traces F  and G) also coincided with observation of partial stimulation of C a  2 +  release  upon accumulation of 80-90nmol Ca .mg HSR" . It should be noted that with 2+  addition of lower (5uM) C a stimulation of C a  2 +  2 +  1  pulses (control trace, Figure 26A) partial  release prior to maximal C a  2 +  release was not observed.  206  The intralumenal C a unaffected.  2 +  requirement for maximal C a  This suggests C a  2 +  induced C a  channel opening  2 +  this assay system, where a dynamic redistribution of C a is removed, intralumenal C a  slow phase will depend upon how C a and intralumenal C a  2 +  2 +  2 +  occurs (i.e. as  increases), observation of the  2 +  is distributed between extralumenal  pools at anytime during uptake.  determined by the ratio of the C a  2 +  regulated  Therefore, in  2 +  2 +  release was  may be  interdependently by both intralumenal and extralumenal C a .  extralumenal C a  2 +  sink to the C a  2 +  This, in turn, is  load. This, however, does  not account for tertiary rapid uptake. The effect of further loading of vesicles with pulse C a , upon C a 2 +  Ca  2 +  2 +  induced  release, was investigated. Figure 22 (trace A) shows that vesicles failed to  reaccumulate all extralumenal C a Eventually, spontaneous C a  2 +  2 +  subsequent to induction of release.  release occurred similar to that observed in Figure  19 (phase d of the inset). The magnitude and the extent of release was variable. However, addition of further CP, in preliminary studies, showed that the released C a  2 +  could be reaccumulated. This had been demonstrated earlier in  similar studies (Watras and Katz, 1984) and indicated that ATP had been exhausted as suggested (McWhirter et al., 1987). Supplementation of uptake media with up to 20mM CP led to sustained uptake of pulsed C a . 2 +  As  indicated in Figure 23A, this also resulted in stoichiometric accumulation, at high Ca  2 +  loads, of phosphate that, presumably, was generated from CP hydrolysis.  This phenomenon was associated with loss of C a  2 +  induced C a  2 +  release. A  possible explanation for this would be that apatite formation diminished the available pool of intralumenal releasable C a . 2 +  Ca  2 +  A similar loss of pH induced  release was also observed with Ca :oxalate loaded vesicles (Entman et al.,  1978).  2+  207  Earlier studies showed that elevated CP in the presence of a regenerating system inhibited spontaneous C a  release from both light SR (longitudinal SR)  2 +  and triad membranes (Palade et al., 1983). Palade et al. (1983) also reported (observations not shown) that the presence of CP, alone, inhibited C a  2 +  The presence of elevated CP did not inhibit the initial maximal C a  release in  2 +  release.  the present study, (Figure 22A traces D to F; and Figure 22B). Furthermore, C a  2 +  release inhibition, due to accumulation of creatine, was also not indicated since release inhibition was sustained in parallel filtration studies (Figure 24) in the absence of elevated creatine or CP. Figure 23A indicated the presence of an intralumenal phosphate free C a  compartment (~125nmol.Ca .mg HSR" )  2 +  2+  that approximated the amount of C a  2 +  1  accumulated prior to maximal C a  release (Figure 21). This suggests that during initial C a  2 +  2 +  uptake, Pi, generated  from ATP turnover was not accessible to the intralumenal C a  2 +  pool. This may  indicate that, prior to maximal release, either (a) phosphate was membrane impermeable or (b) C a  2 +  was bound with high affinity to presumably,  calsequestrin. The former possibility appears unlikely since small amounts of phosphate (10-15nmol Pi.mg HSR" ) were detected upon filters prior to release. 1  The second possibility seems more likely, therefore. This would suggest that maximal C a  2 +  induced C a  2 +  release attends conformational transition of  calsequestrin from a relatively high or intermediate C a of relatively low C a  2 +  2 +  affinity state to a state  binding affinity.  This suggestion is consistent with similar observations by Miyamoto and Kasai (1979) who observed two intralumenal low affinity C a  2 +  binding sites with  dissociation constants of 1.05xlO" M and 3.8x10" M and maximal binding 3  2  capacities of ~45 and 153nmol.Ca .mg SR" , respectively. 2+  1  This approximates  the ~160nmol Ca .mg HSR" observed close to intravesicular C a 2+  after loading in 62mM C a  1  2 +  2 +  saturation  (Meissner, 1984). The higher affinity compartment  208  was suggested by Miyamoto and Kasai (1979) to be calsequestrin while the identity of the lower affinity compartment was obscure. Time dependent loss of releasable C a competition for C a  2 +  between 2 C a  2 +  (Figure 22A, trace E) might then reflect  2 +  pools. The first pool would be Ca :Pi. 2+  The second pool would be Ca :calsequestrin. 2+  In the case of Ca :Pi, an 2+  apparent dissociation constant, assuming pH 7.0 intralumenally, of 3.018xlO M _1  can be calculated for the equilibrium complex using absolute stability constants and the computational program described by Fabiato (1979). Assuming a high affinity C a  binding site for calsequestrin during initial C a  2 +  (Kd<lmM), then C a  2 +  will be largely associated with this protein.  2 +  decreased affinity of calsequestrin for C a , during C a 2 +  would shift toward the Pi pool where-upon  2 +  uptake With  release, the equilibrium  apatite formation occurs at a  sufficiently high concentration of the Ca :Pi equilibrium complex, as evidenced 2+  in Figure 23A. With each C a  2 +  uptake and release cycle, less C a  2 +  might then be  available for subsequent release due to incomplete filling of the releasable C a pool. Unlike C a system, C a  2 +  2 +  2 +  release from passively loaded vesicles in the present assay  release is a net result of C a  2 +  efflux and C a  2 +  influx. It is not clear  what proportion of the entire releasable pool in these vesicles that the ~35nmol Ca .mg HSR" , observed in Figures 21 and 22, actually represents. In Figure 2+  1  16A ~60nmol Ca .mg HSR" was released at loading level of lOOnmol C a . In 2+  1  vivo, the exchangable SR C a  2 +  2 +  pool during muscle contraction can be calculated  at ~70nmol.mg HSR" , assuming an SR volume of lOul.mg protein" and lOOul 1  1  SR.ml muscle" (see Endo, 1977). This is close to the ~60nmol Ca .mg HSR" 1  2+  1  released at a loading level of lOOnmol Ca .mg HSR" in Figure 16A. However, 2+  Ca  2 +  could also be released at low C a  of an intralumenal C a  2 +  2 +  1  loads (Figure 16), contrary to the notion  threshold requirment for C a  2 +  induced C a  2 +  release  observed in Figure 21 and, in vivo (see Endo, 1977). The discrepancy between  209  Ca  z +  released in passively vs actively loaded vesicles may implicate a role for  the C a - ATPase in the regulation of C a 2 +  2 +  release as suggested (Meszaros and  Ikemoto, 1985a,b; Ikemoto et al., 1989). This has been suggested to be mediated by filling of calsequestrin with C a  (Ikemoto et al., 1989).  2 +  In the present study, the data from Figure 21 is consistent with regulation of Ca  2 +  release by intralumenal C a  Ca  2 +  release with elevated intralumenal Pi (Figure 22A), is consistent with the  2 +  bound to calsequestrin. Furthermore, loss of  converse property; that depletion of this pool inhibits C a  2 +  release.  It is  appropriate to comment on results published by Nelson and Nelson (1990) after completion of the experimental results reported here. These authors showed, similarly, that C a intralumenal C a  2 +  2 +  induced C a threshold.  increasing trigger C a  2 +  release, in HSR vesicles, required an  Nelson and Nelson (1990) observed that  was required to induce release, subsequent to  2 +  observation of initial maximal release. Nelson and Nelson (1990) suggested that this represented the presence of a second extralumenal C a was observed with lOuM C a  2 +  2 +  trigger site. This  pulses as employed in Figure 21. Figure 25  shows a similar apparent loss of extralumenal C a Ca  2 +  2 +  sensitivity for C a  2 +  induced  release. Although this was observed with low (luM) ryanodine this was  also commonly observed in control samples (Gilchrist, Katz and Belcastro, unpublished observations). Nelson and Nelson (1990) reported, under almost identical assay conditions to Figure 22, an intralumenal C a ~70nmol Ca .mg HSR" after 7xl0nmol C a 2+  1  release in a 1ml volume (therefore, lOuM C a were made with 0.5-2uM total C a  2 +  2 +  2 +  2 +  threshold for  additions to elicit maximal  pulses). Subsequent additions  in the cuvette until a secondary release was  observed. Nelson and Nelson (1990) suggested that this reflected the presence of a second C a  2 +  trigger site. The notion of a second extralumenal C a  is refuted in the present study on the basis that (a) less C a  2 +  2 +  trigger site  was added by  210  Nelson and Nelson (1990) to induce secondary releases (2uM) than primary releases (lOuM) and (b) the loss of C a  2 +  induced C a  2 +  release correlates with  inorganic phosphate precipitation when ATP regeneration is employed. In light of the foregoing evidence that intralumenal C a  regulates C a  2 +  2 +  release, it was of interest to determine the effect of ryanodine binding to the release channel upon intralumenal C a  2 +  loading.  In accord with earlier  observations (Jenden and Fairhurst, 1967; Fairhurst and Hasselbach, 1970; Fleischer et al., 1985; Meissner, 1986b; Lattanzio et al., 1987), ryanodine evidently maintained the open state of the channel with loss of C a  2 +  uptake (Figure 25).  Under the short pre-incubation conditions employed, elevated ryanodine (ImM) did not appear to result in channel closure and increased C a  2 +  retention as  observed (Feher et al., 1988) and was unaffected by prolonged pre-incubation. The hypothesis that ryanodine effects might depend upon C a  2 +  channel states  was tested in the following way. In Figure 25, the channel, prior to addition of Mg.ATP, was presumed to be open. This was deduced from the preliminary observation that vesicles isolated without a final lOOmM KC1 washing released ~85-90nmol C a  .mg HSR" when added to lOOmM KC1 containing APIII  2 +  1  reaction media. The source of this C a  2 +  was presumably calsequestrin and the  release was observed through a steady increase in the absorbance signal during pre-incubation. Addition of Mg.ATP without C a  2 +  addition produced absorbance traces  almost identical to that observed in Figure 19 (Gilchrist, Katz, Belcastro; unpublished observations). In order for C a  2 +  to be released, the channel must,  therefore, have been open. Addition of ryanodine after C a  2 +  uptake with the  channel, presumably, closed, led to an increased intralumenal C a Ca  2 +  induced C a  2 +  release with failure to reaccumulate C a  2 +  2 +  sensitivity of  in the presence of  5uM CP. At higher concentrations of ryanodine (2mM) spontaneous C a  2 +  211  release occurred with partial reaccumulation of C a . These results, as in Figure 2 +  25, suggested that ryanodine maintained or locked the channel in an open state. Figure 25 and Figure 26B also showed that supplementation of the uptake medium with CP enabled accumulation of released C a . Further triggering of 2 +  Ca  2 +  release was attenuated, though, despite vesicle loading with C a . This 2 +  was independent of whether CP was added prior to uptake or after release. In this instance Pi measurements of parallel filtration studies were not performed. It is unclear, therefore, whether this facilitation of uptake was due to Ca :Pi 2+  precipitation, altough Figures 22 to 24 suggest that this is likely. The curious loss of  subsequent C a  2 +  release indicates  that ryanodine  also  precludes  replenishment of the intralumenal releasable pool of C a . This may reflect the 2 +  mechanism by which ryanodine has been shown to deplete the intralumenal Ca  2 +  stores in arterial smooth muscle (Ashida et al., 1988; Ito et al., 1986; lino et  al., 1988) and cardiac purkinje fibres (Marban and Weir, 1985). In addition, these results may account for observation of ryanodine inhibition of C a (Fabiato, 1985), C a  2 +  2 +  release  oscillations (Lakatta et al., 1985) and after contractions  (Sutko and Kenyon, 1983) in cardiac cells. Depletion of intracellular cardiac Ca  2 +  pools, identified as SR, was also demonstrated, in radioisotope perfusion  studies of C a  2 +  distribution, by prior administration of ryanodine (Hunter et al.,  1983). The depletion of intralumenal SR C a  2 +  pools may, therefore represent an  event subsequent to ryanodine stimulation of C a  2 +  release as suggested by  Figures 25 and 26A. This effect of ryanodine, to inhibit the reaccumulation of C a , is not due to inhibition of the Ca -ATPase (Besch, 1985). Thus, it appears 2 +  2+  that ryanodine inhibition of C a replenish intralumenal C a  2 +  2 +  release may be due to an inability of SR to  stores for subsequent release. Inhibition of C a  2 +  release by ryanodine may therefore reflect a coupling between the functional states of the C a  2 +  channel and calsequestrin.  As indicated by Figure 26B,  212  ryanodine may serve to maintain calsequestrin in a low affinity C a  z +  binding  state via maintenance of the open channel state. The interdependence between Ca  2 +  channel states and the extent of HSR  demonstrated in Figure 27.  preloading was clearly  Ryanodine induced C a  2 +  release was always  preceded by a delay followed by a rapid (-HOOnmol Ca .mg HSR" .min" ) 2+  1  1  release as observed earlier (Palade, 1987b). As shown in Figure 27, the rate of Ca  2 +  release was not detectably different at any level of C a  discrimination between rates of C a since the rate was too fast.  2 +  2 +  loading. However,  release could not be precisely determined  Conversely, the amount of C a  dependent upon the extent of C a  2 +  release was  2 +  preloading. Furthermore, the amount of  ryanodine required to induce release decreased with elevated C a The observation of ryanodine stimulation of C a  2 +  2 +  loading.  release brings into question  the state of the channel required for ryanodine binding. Evidently, ryanodine must bind to the channel in a presumed closed state in order for ryanodine to elicit C a  2 +  release.  Observation of ryanodine induced C a  2 +  release from  preloaded vesicles always required high concentrations of ryanodine (>125uM). On the other hand, loss of C a  2 +  accumulation in C a  2 +  depleted states (i.e.  ryanodine addition prior to uptake) was observed at lower concentrations of ryanodine (lOOuM). The findings of Figures 25 to 27 are at variance with other reports, both in terms of the effect of ryanodine and the amount of ryanodine used to demonstrate these effects. In the first instance, several studies have shown that ryanodine (>300uM) closes the channel in planar bilayer studies (Nagasaki and Fleischer, 1988; Lai and Meissner, 1989) and increases C a  2 +  uptake in vesicle studies (Jones et al., 1979; Jones and Besch, 1979; Seiler et al., 1984; Hasselbach and Migala, 1987; Feher et al., 1988). In one other study (Fairhurst, 1974), 300uM ryanodine was shown to decrease C a  2 +  uptake by  skeletal SR vesicles. However, under similar assay conditions from the same  213  group Fleischer et al. (1985) and McGrew et al. (1989) showed that similar concentrations of ryanodine (400nM) was without effect upon and increased, respectively, C a  accumulation in skeletal SR vesicles. The difference between  2 +  the assays was that (i) longer pre-incubation times with ryanodine in the latter report were employed and (ii) vesicles were isolated in the absence of protease inhibitors in the earlier report. As shown by Meissner (1986b), the activatory and inhibitory effects of ryanodine were modified by the incubation times of ryanodine with HSR vesicles. Prolonged incubation (>30minutes) inhibited C a  2 +  efflux at lOOuM  ryanodine whereas short incubations (<30 minutes) activated C a same ryanodine concentration.  2 +  efflux at the  In the present study, ImM ryanodine effects  (Figure 25) were independent of incubation times.  The only case where  inhibitory effects of ryanodine upon channel opening might be indicated is after ryanodine induced C a  2 +  release (Figure 26A and 27) where partial re-uptake  was observed.  At low concentrations of ryanodine (Figure 26A) the C a  released after C a  2 +  2 +  triggering was not partially reaccumulated. This may reflect  differential effects of ryanodine at high affinity (activatory) and low affinity (inhibitory) binding sites (Figure 42).  In most cases, though, the effect of  ryanodine was to either inhibit uptake in C a ryanodine and C a  2 +  induced C a  2 +  2 +  depleted vesicles or to stimulate  release in actively loaded vesicles.  The results of the present study clearly demonstrate that ryanodine effects are significantly dependent upon the amount of HSR intralumenal C a . Below a 2 +  loading level of ~60nmol Ca .mg HSR" , addition of ryanodine 2+  1  did not  stimulate release. Above 60nmol Ca .mg HSR" , increasing intralumenal C a 2+  1  2 +  loading decreased the amount of ryanodine required to induce release. With the suggestion, earlier, of two classes of intralumenal C a  2 +  binding sites, it is  possible that subtle conformational changes may occur within the channel at  214  different levels of loading. This may occur beyond an intralumenal load of -5060nmol Ca .mg HSR" with formation of a lower affinity intralumenal C a 2+  1  2 +  site that i), "primes" the channel for opening and ii), "primes" the intralumenal releasable C a  2 +  pool for release. This remains a speculative view and must  await further studies.  VI. CANP effects upon HSR Calcium release In parallel studies to the above the functional effects of the channel selective protease, CANP (Figures 29, 30) were investigated. Prior to the recognition that the high molecular weight protein doublet visualised on SDS-PAGE gels actually represented the CANP cleaved form of the native channel, many studies had already been conducted upon the characterisation of HSR C a  2 +  uptake and  release. General agreement prevailed that HSR vesicles contained a ligand gated Ca  2 +  channel that was activated by nucleotides (Smith et al., 1985), uM C a  (Meissner, 1984) and inhibited by M g  2 +  2 +  (Meissner, 1984) and ruthenium red  (Fleischer, 1985). Indeed, much of what is now known concerning the regulation of C a  2 +  channel events (i.e. channel opening and C a  2 +  release) was established  from studies in which the channel was proteolytically modified by CANP. In preliminary spectroscopic studies, it was apparent that addition of leupeptin to isolation buffers "decreased" the rate at which C a  2 +  could be accumulated  (Gilchrist, Katz, and Belcastro; unpublished observations). It should be pointed out that these studies employed lower concentrations of protein (0.25 to 0.5mg.ml ) with similar concentrations of C a _1  2 +  to that employed in Figure 19.  At these concentrations, the slow phase of uptake (<lnmol Ca .mg HSR" .min." 2+  1  ) was greatly extended.  1  As discussed earlier, this behaviour reflected the  dependence of channel opening upon the C a  2 +  sink:Ca  2+  load ratio. These  studies were repeated in Figure 39A and with 0.5mg.HSR protein.ml" with 1  215  observation of faster accumulation of C a  z +  uptake during the secondary (slow)  and tertiary (intermediate) phases. These were small changes but were always observed when the effect of protease inhibition was evaluated. With extensive exogenous protease treatment of HSR vesicles, the slow phase C a  2 +  uptake was  elevated 2-fold (Figure 39B). Consistent with the hypothesis that intralumenal Ca  2 +  was important in the regulation of channel opening, it was proposed that  CANP proteolysis of the channel may modify the. intralumenal C a  2 +  compartment. This was tested in Figures 36, 37 and 38. In each case, u- and mCANP (i) elevated passive C a  2 +  loading and fractional C a  release in skeletal  2 +  (Figure 36 and Table 5) and cardiac HSR (Figure 37) and (ii) increased ATP dependent C a  2 +  ruthenium red.  accumulation in skeletal HSR in the presence and absence of Much of the increase in intralumenal C a  2 +  retention was  associated with limited proteolysis (ie production of 410 and 150kDa peptides). Extended proteolysis produced only minor further modification. This suggests that modified C a  2 +  handling by HSR vesicles was associated with initial  cleavage of the 550kDa peptide to 410 and 150kDa products. The associated increase in the fractional release of C a increased C a  2 +  2 +  is interesting since Figure 16 shows that  loading decreased the fractional release of C a . This suggested 2 +  that CANP increased the intralumenal releasable pool of C a . 2 +  As shown in  Figures 41 and 42, extended CANP proteolysis also resulted in small increases in low and high affinity [ H]-ryanodine binding. 3  intralumenal C a  2 +  This indicated that the  sensitivity of ryanodine- and C a -induced release may be 2 +  increased in CANP treated SR. These hypothesis were tested using the assays developed in Figures 21 and 27. Extended CANP proteolysis (a) decreased the amount of C a  2 +  loading required for C a  2 +  induced C a  increased the time for reaccumulation of released C a  2 +  2 +  release and (b)  (Figure 40). These were  reflected in a decrease in the amount of ryanodine required to stimulate  216  ryanodine induced C a  z +  release.  These results suggest that the increased  sensitivity to C a - and ryanodine-induced C a 2 +  2 +  release may be related to  elevated intralumenal binding of C a . This is a tentative conclusion, however. 2 +  Although these were consistent observations it is not clear, why CANP treated vesicles passively and actively loaded more C a  2 +  less intralumenal C a  release (Figure 40 and Table 7).  2 +  for the induction of C a  2 +  (Figures 36-38), yet required  An interesting finding is that after induction of release, the time course of C a  2 +  reaccumulation during the slow phase was longer after CANP treatment. This suggests a more sustained channel opening and may reflect loss of channel inactivation observed by Rardon et al. (1990) using vesicles incorporated into bilayers. Rardon et al. (1990) also observed elevated (single site) ryanodine binding in CANP treated skeletal HSR. In CANP treated cardiac HSR, ryanodine binding was depressed although CANP-mediated channel inactivation was observed with both HSR types.  These observed effects of CANP show  similarities to and differences from the effects of trypsin and chymotrypsin upon channel function. With limited trypsin digest of skeletal HSR vesicles, increases were observed in C a 4 5  Ca  2 +  2 +  loading, channel open time (Meissner et al., 1989) and  uptake (Chu et al., 1988; Shoshan-Barmatz and Zarka, 1988).  Concomittant loss of high affinity ryanodine binding was observed (Meissner et al., 1989; Shoshan-Barmatz and Zarka, 1988; Chu et al., 1988) subsequent to tryptic digest.  With extended trypsin digestion, Meissner et al. (1989)  demonstrated loss of channel function as determined from bilayer recordings and loss of passive 45 2+ Ca  loading. These effects of trypsin were markedly different from the effects of CANP reported in the present study and by Rardon et al. (1990). These differences appear to reflect the selectivity of cleavage sites for CANP, in contrast to trypsin. A documented feature of CANP is that it tends to cleave between functional domains rather than within (Suzuki and Ohno,  217  1990). In this regard, Suzuki and Ohno (1990) suggested that CANP fulfills a regulatory role in which the regulation is considered unidirectional. Rather than fulfilling a digestive role, CANP may act as a "biotransformer" (Suzuki and Ohno, 1990). The absence of obvious impairment of channel function after CANP treatment, may be consistent with this.  It is unlikely, therefore, that  CANP cleaves within the V8 and Achromobacter endoprotease sensitive modulator region (residues 2619-3016) of the C a  2 +  channel (see Marks et al.,  1990). A single site cleavage model, as proposed earlier, for limited CANP proteolysis, would predict cleavage at either residues -1360 or -3670 (assuming 110 daltons per residue and a 150kDa digest product) of the 5032-5037 residue protein (see Takeshima et al., 1989; Zorzato et al., 1990). In a recent study, it has been suggested (Wang et al., 1989) that CANP effects may be mediated by binding to recognition sites rich in proline (P), glutamate (E), aspartate (D), serine (S), and threonine (T) residues (PEST regions). This was based upon the earlier observation (Rogers et al., 1986), that several proteins with short half-lives contain one or more PEST sequences. It was suggested that potential phosphorylation of serine and threonine residues may generate a highly charged hydrophilic loop region (Rechsteiner, 1987) that may facilitate Ca  2 +  dependent binding of CANP (Wang et al., 1990).  The additional  observation was that many CaM binding proteins are good CANP substrates and that many CANP substrates contain one or more PEST sequences. The fact that the release channel is a CaM binding protein (Seiler et al., 1984) suggested that a search for PEST sequences might reveal potential CANP cleavage sites. Table 8 lists the predicted PEST sequences in the channel protein obtained from the "PEST-FIND" computer program (see methods). Nine "good" PEST sequences were obtained, 2 of which were located close to residue 1360. These two regions are  separated  by  Table 8. PEST regions in the calcium release channel sorted by score.  PEST  Residue #  Sequence  From  To  score  [  1865 2042 4447 3288 4582 4866 1355 1303 4231  1894 2061 4473 3310 4649 4885 1371 1323 4294  33.08 13.63 8.01 4.76 2.39 1.56 .68 .21 .13  MIEPEVFTEEEEEEEEEEEEEEEEEEDEEE CGIQLEGEEEEPEEETSLSS PEGAGGLGDMGDTTPAEPPTPEGSPIL GPEAPPPALPAGAPPPCTAVTSD VSDSPPGEDDMEGS A AGDLA /.. /FLEESTG YMEPALWCLSLLH SEDEDEPDMLCDDMMTCYLF EGTPGGTPQPGVEAQPV CTAGATPLAPPGLQPPAEDEA MEUFV AFCEDTIFEMQIA AQ/.. / EG A AG AEG AAGTVAAG ATAR  a  a. A PEST score of > 0 was indicative of a good PEST region.  ]  219  -50 residues and may represent the sites of cleavage from production of the -450 and 410kDa peptides seen clearly in Figure 2. This is a tentative proposal, though. A corollary to the apparent requirment of higher order structures for site recognition by CANP was that CANP binding and cleavage sites may be determined by tertiary structure rather than strictly primary structure (Wang et al., 1989).  VII. Contribution of this work to the existing literature. The physiological mechanism for the triggering of C a  2 +  release from the SR  cisternae of skeletal muscle in response to t-tubule depolarisation is unknown. Two hypotheses have been forwarded to account for this triggering. The first suggests that a channel gating voltage sensor within the junctional membranes may respond to a movement of charge between the t-tubules and the SR (Schneider and Chandler, 1973). The second proposes that a flux of cytosolic Ca  2 +  may elicit SR C a  associated with the C a The source of this C a  2 +  2 +  2 +  release through direct binding to a trigger site  release channel complex (see Endo, 1977; Fabiato, 1989). may be (i) L-type C a  2 +  channels located within the t-  tubules (ii), bound to specific site on the cytoplasmic leaflet of the t-tubule membrane (Frank, 1982) or (iii) derived from an initial depolarisation induced release of C a  2 +  which elicits a further regenerative C a  from the more laterally disposed C a  2 +  channels.  2 +  induced C a  Ca  2 +  Ca  2 +  2 +  release  (Ford and Podolsky, 1970;  Nesterov, 1988). In this work, the role of intralumenal C a induced C a  2 +  2 +  in regulating C a  2 +  release has been examined. It has been shown that observation of  induced C a  2 +  release is dependent upon a critical level of intralumenal  loading. This has been shown to occur under "physiologically appropriate"  conditions of free M g  2 +  (-ImM). In addition, preliminary observations have  shown that the effects of the alkaloid ryanodine upon C a  2 +  release are also  220  sensitively dependent upon the level of intralumenal C a  2 +  loading. Indirect  evidence has been presented that different levels of intralumenal C a may sensitize the SR C a  2 +  channel to extralumenal C a  Endo (1981) now acknowledges, C a  induced C a  2 +  2 +  2 +  2 +  effectors.  loading Thus, as  release is a real and  perhaps physiologically relevant observation, albeit requiring different preconditions to the depolarisation induced C a  2 +  release (see Ikemoto et al., 1989).  Little is known of the potential role of and cellular requirement for a regenerative type C a  2 +  release that is dependent upon critical intralumenal C a  loading of the cisternal region.  2 +  It could be argued, however, that such a  mechanism may confer the advantage that the release of C a  2 +  is well  coordinated with it's uptake from the cytosol. In this way, release would not occur until a finite pool of cytosolic C a  2 +  had been redistributed back into the  SR. The evidence for this is currently lacking. Taken further, such a mechanism may confer an autoregulatory character to SR C a has pointed out, the spatial distribution of C a  2 +  2 +  release. As Williams (1990)  pumps and channels within the  cell confers a kinetic property to the regulation of intracellular C a Rather that C a  2 +  handling.  signalling existing as a consequence of simply the binding  2 +  constant between C a  2 +  and its site of action, Williams (1990) has suggested that  the intracellular cycling of C a  2 +  between C a  2 +  pumps, effector sites and C a  channels establishes a homeostatic level within which C a  2 +  2 +  metabolism is  coordinated and kinetically integrated with energy metabolism. Changes in the regulation of C a  2 +  handling resets the level of homeostasis in the cell. In the  quest to understand the mechanism of SR C a overlooked that the regulation of SR C a  2 +  2 +  release it is, perhaps, somewhat  handling may also be important in the  control of protein expression and growth coupled to the metabolic demands of the cell. The possibility that the C a  2 +  release channel is important in this regard  is suggested from the enhanced expression of the ryanodine receptor in neonate  221  cardiactissue(Michalak, 1988; see also Michalak, 1985) and in dennervated fast twitch skeletal muscle (Zorzato, et al., 1989). In this regard, Martonosi (1982) has suggested that the pattern of cytosolic  transients determines the expression  of SR protein, in particular the Ca -ATPase.  This may be similar to the  2+  frequency modulated C a  2 +  signal in a variety of cells that is initiated by IP3 and  propagated by regenerative C a  2 +  induced C a  Conceivably, the requirement of intralumenal C a  release (Berridge, 1990).  2 +  2 +  saturation for C a  may represent a means by which the patterning of C a  sensitive releasable C a  2 +  2 +  2 +  in C a  2 +  and IP3  pools, in a variety of cell types are regulated by the  saturation of intralumenal C a "Quantal" release of C a  release  transients can be  2 +  regulated. This is similar to the view that intralumenal C a  2 +  2 +  compartments (Irvine, 1990). Irvine postulated a  from these structures that involved the saturation of  intralumenal C a . The quantal nature of SR C a 2 +  2 +  release was also evident in  the present study. Within this scheme, variations in the frequency and amplitude of cytosolic Ca  2 +  transients may be tolerable within limits determined, in part, by the  metabolic state of the cell. Asynchrony between C a  2 +  and Pi metabolism may  lead to apatite formation within the SR lumen. As also shown in this study, Pi accumulation by the SR either directly or, as suggested, indirectly through competition for the releasable C a  2 +  pool, leads to inhibition of C a  2 +  release.  This may represent part of the mechanism of muscle fatigue after intense muscle contraction, consistent with earlier observations (Gonzalez Serratos et al., 1978; Somylo et al., 1978, 1981). Within skeletal muscle, such an event may actually constitute a safety device when muscle activity approaches the limits of muscle capabilities. The question then is what are those limits, how are they set and can they be changed?  Nesterov (1985) has argued that the morphofunctional  characteristics of muscle across a wide variety of species are well correlated to  222  the use of either sodium or C a  2 +  electrochemical gradients across plasma  membranes, in triggering muscle contraction. It was suggested that reduction of functional activity leads to a decline in the dependence upon transsarcolemmal N a and an increased dependence upon C a +  2 +  gradients. In devervated muscle,  a similar transformation may occur with increased synthesis of a different isoform (perhaps cardiac) of the channel as tentatively suggested by Zorzato et al. (1989). These would be long term adaptations and presumably would evoke a different pattern of intracellular C a  2 +  signalling.  molecular defect in the malignant hyperthermia C a  It seems likely that the 2 +  release channel accounts  for the hypertrophied state of skeletal muscle in animal models (Pietrain pigs) possessing the defect. This hypertrophy, possibly, results from the alteration of Ca  2 +  signalling from a channel that is evidently more sensitive to Ca -induced  Ca  2 +  release (Nelson, 1983, 1984; Mickelson et al., 1985, 1986, 1988; O'Brien,  2+  1990). In the short term, could the patterning of C a  2 +  signals be modified within  the same structures? Data from the present study suggests that the effect of CANP may increase the sensitivity of the C a  2 +  release channel to C a  2 +  triggered  release. Within this scheme, activation of CANP in response to a variety of stressors may initiate limited proteolysis of the channel and possibly, sever structural connections within the "weak triads" (Brandt et al., 1990). This may lead to loss of electrogenic C a  2 +  release and increased regenerative C a  It should be stressed that these are tentative suggestions. whether, in vivo, C a  2 +  2 +  release.  It is not known  channels may adapt functionally in response to CANP or  whether, as suggested by Rardon et al. (1990), C a  2 +  channels are disfunctional  and fail to inactivate. The apparent localisation of CANP to the t-tubule/SR junction and the selective cleavage of the release channel, as indicated in the present study, would support the hypothesis that CANP may play an important  223  role in modification of excitation-contraction coupling during pathophysiological states.  VIII. Summary.  The purpose of this study was to investigate the relationship between intralumenal C a  2 +  loading by HSR vesicles and C a  2 +  release. It was anticipated  that structural and pharmacological intervention of C a would provide insight into the interaction between C a Ca  2 +  loaded states of calsequestrin.  channel operation  2 +  2 +  channel states and  Heavy sarcoplasmic reticulum (HSR)  membranes were purified from rabbit fast twitch skeletal muscle and canine hearts. Structural characterisation of these membranes revealed an abundance of 565kDa ryanodine receptor and 57kDa calsequestrin. The enrichment of both proteins indicated that these membrane preparations were derived from the terminal cisternae. Skeletal HSR membranes bound ryanodine with high capacity at both low and high affinity sites. C a  2 +  transport and Ca -ATPase activities were activated 2+  and inhibited, respectively in the presence of the C a ruthenium red.  In active C a  2 +  2 +  channel blocker  transport assays skeletal HSR membranes  released 35-40% of their intralumenal C a application of extralumenal trigger C a . 2 +  2 +  contents in response to the  A dual-wavelength spectroscopic  method was developed that permitted the study of initial ATP dependent C a  2 +  uptake by HSR vesicles without optical artifacts arising from the addition of nucleotide. This technique permitted accurate estimate of the initial rate of HSR Ca  2 +  uptake as a function of the initial C a  appearance of multiphasic C a  2 +  2 +  load. It was shown that the  uptake kinetics at elevated extralumenal C a  loads depended upon the relative distribution of C a  2 +  2 +  between intralumenal  224  Ca  z +  binding sites and the extralumenal space. These studies also showed that  HSR vesicles required a threshold filling of intralumenal C a Ca  2 +  induced C a  2 +  2 +  for observation of  release (CICR). The plant alkaloid ryanodine was then  employed to investigate the dependence of intralumenal C a Ca  2 +  release upon C a  2 +  regulation of  channel states. It was demonstrated that ryanodine  decreased the intralumenal C a release.  2 +  2 +  threshold requirement for C a  In addition, ryanodine induced C a  dependent upon the intralumenal C a  2 +  2 +  2 +  induced C a  2 +  release was shown to be  load. These studies suggested that state  transitions between the ryanodine receptor and calsequestrin were functionally linked and reciprocally interdependent. The effects of specific structural modification of the release channel by C a activated neutral protease (CANP) upon HSR C a  2 +  handling  2 +  was then  investigated. Isoforms of CANP requiring micromolar (uCANP) and millimolar (mCANP) concentrations of activating C a  2 +  were isolated from the cytosolic  fractions of rabbit skeletal muscle homogenates.  These CANP isoforms  selectively cleaved the purified and vesicular form of the 565kDa C a  2 +  release  channel (the major substrate) into 410 and 150kDa fragments during limited proteolysis. Evidence suggested that CANP proteolysis was initiated with single site cleavage. Extended proteolysis resulted in secondary fragmentation of the initial peptide products with formation of a 135kDa limiting peptide. Primary structural analysis of the C a  2 +  release channel revealed the presence of several  regions that were rich in proline (P), glutamate (E), aspartate (D), threonine (T), and serine (S) (PEST) residues. PEST sequences have been suggested to act as substrate recognition sites for CANP proteolysis (Wang et al., 1989). Two PEST rich sequences 1300 to 1350 amino acids down from the N-terminal which were tentatively assigned as possible initial CANP cleavage sites.  225  Extensive CANP mediated fragmentation of the 565kDa peptide resulted in subtle changes of HSR functional properties. Ryanodine binding at both low and high affinity binding sites was elevated after proteolysis. membranes demonstrated elevated intralumenal C a Ca  2 +  release after passive C a  increased the intralumenal C a  2 +  2 +  loading.  2 +  binding and fractional  In addition, CANP proteolysis  sensitivity of actively C a  C a -induced and ryanodine induced C a 2 +  2 +  2 +  loaded vesicles to  release. These studies suggest that  2 +  CANP cleaves close to a domain on the C a regulation of intralumenal HSR C a  CANP treated  2 +  channel that is important in the  handling.  The potential pathophysiological importance of the action of CANP upon HSR membranes and C a identify  a  2 +  channel operation was then studied through attempts to  structural  association  of  CANP  with  Immunolocalisation studies revealed that CANP  HSR membranes.  co-purified with HSR  membranes and was specifically associated with a CHAPS insoluble HSR membrane fraction. Greater immunoreactivity was, however, observed with light membrane fractions referable to the t-tubules. These studies indicated that CANP may be localised to the t-tubule/cisternal region of skeletal muscle in close association with the C a  2 +  release channel.  The function of CANP, in vivo, remains unclear. In the present study CANP was shown to elicit subtle changes in C a  2 +  handling by HSR membranes, in  vitro. The observed functional effects of CANP were discussed in relation to a potential role of regenerative HSR C a  2 +  release in mediating C a  during adaptation. One potential advantage of regenerative C a  2 +  2 +  signalling  release is that  it may confer an autoregulatory property to the coordination of HSR C a uptake and release. Given a finite intracellular pool of C a  2 +  2 +  that is cyclically  exchanged between the cytosol and the SR lumen it is conceivable that the C a  2 +  release pool must be replenished before the next release event is triggered. It  226  was proposed that such a mechanism may also govern the patterning of cytosolic transients which appear to be an important aspect of gene expression. The evidently subtle changes in the C a  2 +  adjust the patterning of intracellular C a of the C a  2 +  release mechanism.  handling brought about by CANP may 2 +  signals without significant impairment  It was suggested that CANP may sever  structural connections between "weak triads" in order to establish a greater dependence upon regenerative rather than electrogenic triggering of C a  2 +  release. Such a mechanism may constitute part of the process by which muscle cells respond to both acute and chronic changes in contractile behaviour.  IX. Conclusions.  1. Purified heavy sarcoplasmic reticulum membranes were isolated from rabbit fast twitch skeletal muscle and canine hearts. In both cardiac and skeletal SR, membranes were characterised by the presence of structural components (ryanodine receptor and calsequestrin) which indicated that these preparations were enriched in membranes derived from the terminal cisternae.  2. Functional characterisation of skeletal HSR showed that the C a  2 +  release  channel structure bound ryanodine with high capacity at both low and high affinity sites. C a  2 +  transport and Ca -ATPase activities were activated and 2+  inhibited, respectively in the presence of the C a  2 +  channel blocker ruthenium  red.  3. C a  2 +  loading studies in skeletal HSR vesicles indicated the presence of 2  intralumenal C a  2 +  binding sites with apparently different binding affinities.  227  4. A spectroscopic method for the assay of C a  2 +  transport was developed that  allowed for the improved resolution of the initial rapid phase of HSR C a  2 +  uptake in the presence of physiologically appropriate concentrations of M g . 2 +  5. The above spectroscopic techniques demonstrated (i), that HSR vesicles required a threshold filling of intralumenal C a induced C a  2 +  for observation of C a  2 +  release (ii), that ryanodine decreased the intralumenal C a  2 +  threshold requirment for C a Ca  2 +  2 +  induced C a  2 +  2 +  release and (iii) ryanodine induced  release was shown to be dependent upon the intralumenal C a  2 +  load.  6. Isoforms of calpain (CANP) requiring micromolar (uCANP) and millimolar (mCANP) concentrations of C a  2 +  were isolated from the cytosolic fractions of  rabbit skeletal muscle homogenates.  7. These CANP isoforms were found to selectively cleave the purified and vesicular form of the 550kDa C a  2 +  release channel into 410 and 150kDa  fragments during limited proteolysis.  8. A novel 88kDa CANP substrate was identified in HSR membranes and was cleaved in a C a  2 +  and protease dependent manner similar to the 550kDa C a  2 +  release channel.  9. Proteolysis of the release channel i) increased intralumenal C a fractional C a  2 +  2 +  loading and  release in passively loaded cardiac and skeletal HSR, ii)  increased the sensitivity of actively loaded skeletal HSR to Ca -induced and 2+  ryanodine induced C a  2 +  release.  228  10. Immunolocalisation studies indicated that CANP may be localised to the ttubule/cisternal region of skeletal muscle.  11. Analysis of the primary structural regions of the C a  2 +  release channel that  were rich in proline (P), glutamate (E), aspartate (D), threonine (T), and serine (S) (PEST) residues, indicated the presence of 2 PEST rich sequences 1300 to 1350 amino acids down from the N-terminal which were tentatively assigned as possible CANP cleavage sites.  12. The regulation of HSR C a  2 +  induced C a  2 +  release by intralumenal C a  2 +  and  its modification by CANP was discussed in relation to a potential role of regenerative HSR C a  2 +  release in mediating C a  2 +  signalling during adaptation.  229  REFERENCES Abramcheck, C.W., and Best, P.M. (1989) Physiological role and selectivity of the in situ potassium channel of the sarcoplasmic reticulum in skinned frog skeletal muscle fibers. J. Gen. Physiol. 93,1-21. Abramson, J.J., and Salama, G. (1989) Critical sulfhydryls regulate calcium release from sarcoplasmic reticulum. J. Bioenerg. Biomemb. 21, 283294. Abramson, J.J., Cronin, J.R., and Salama, G. (1988) Oxidation induced by phthalocyanine dyes causes rapid calcium release from sarcoplasmic reticulum vesicles. Arch. Biochem. Biophys. 263,245-255. Adams, B.A„ and Beam, K.G. (1990) Muscular dysgenesis in mice: a model system for studying excitation-contraction coupling. FASEB J. 4, 28092816. Allen, G. and Bottomley, R.C., and Trinnaman, B.J. (1980) Primary structure of the calcium ion-transporting adenosine triphosphate from rabbit thermolytic, tryptic and staphyloccal-proteinase peptides. Biochem J. 187,577-589. Anderson, K., Lai, F.A., Liu, Q., Rousseau, E., Erickson, H.P., and Meissner, G. (1989) Structural and functional characterization of the purified cardiac ryanodine receptor-Ca release channel complex. J. Biol. Chem. 264, 1329-1335. 2+  Andersson-Cedergren, E. (1959) Ultrastructure of motor-end plate and sarcoplasmic components of mouse skeletal muscle fiber as revealed by three-dimensional reconstructions from serial sections. J. Ultrastruct. Res. 2 (Suppl. 1), 1-191. Antoniu, B., Kim, D., Morii, M., and Ikemoto, N. (1985) Inhibitors of C a release from the isolated sarcoplasmic reticulum. Biochim. Biophys. Acta. 816, 9-17. 2 +  Appelt, D., Buenviaje, B., Champ, C , and Franzini-Armstrong, C. (1989) Quantitation of 'junctional feet* content in two types of muscle fiber from hind limb muscles of the rat. Tissue and Cell. 21, (5) 783-794. Appelt, D., Buenviaje, B., Champ, C , and Franzini-Armstrong, C. (1989) Quantitation of 'junctional feet' content in two types of muscle fiber from hind limb muscles of the rat. TISSUE & CELL 25,783-794. Argaman, A., and Shoshan-Barmatz, V. (1988) Dicyclohexylcarbodiimide interaction with sarcoplasmic reticulum - Inhibition of C a efflux. J. Biol. Chem. 263,6315-6321. 2 +  Ashida, T., Schaeffer, J., Goldman, W.R, Wade, J.B., and Blaustein, M.P. (1988) Role of sarcoplasmic reticulum in arterial contraction: comparison of ryanodine's effect in a conduit and a muscular artery. Circ. Res. 62, 854-863.  230  Badalamente, M.A., Hurst, L.C., and Stracher, Al. (1989) Neuromuscular recovery using calcium protease inhibition after median nerve repair in primates. Proc. Natl. Acad. Sci. USA. 86,5983-5987. Barcenas-Ruiz L, and Wier, W.G. (1987) Voltage dependence of intracellular Car transients in guinea pig ventricular myovytes. Circ. Res. 61,148154. +  Barrett, A.J. (1986) An introduction to the proteinases In: Proteinase Inhibitors (Barrett, A.J. and Salvesen, G., eds) pp. 3-22, Elsevier, Amsterdam. Baskin, R.J. (1971) Ultrastructure and calcium transport in crustacean muscle microsomes. J. Cell Biol. 48,49-60. Bayerdorffer, E., Streb, H., Eckhardt, L., Haase, W., and Schulz, I. (1984) J. Membr. Biol. 81,69-82. Baylor, S.M., Chandler, W.K., and Marshall, M.W. (1982) Optical measurements of intracellular pH and magnesium in frog skeletal muscle fibres. J. Physiol. (1982) 331,105-137. Beam, K.G., Tanabe, T., and Numa, S. (1989) Structure, function, and regulation of the skeletal muscle dihydropyridine receptor. Ann. NY. Acad. Sci. 560,127-137. Bean, B.P. (1985) Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology. J. Gen. Physiol. 86,1-30. Bean, B.P. (1989) Multiple types of calcium channels in heart muscle and neurons. Ann. NY. Acad. Sci. 560,334-345. Belles, B., Hescheler, J., Trautwein, W., Blomgren, K., and Karlsson, J.O. (1988) A possible physiological role of the Ca-dependent protease calpain and its inhibitor calpastatin on the Ca current in guinea pig myocytes. Pflugers Arch. 412,554-556. Berridge, M.J. (1987) Inositol trisphosphate and diacylglycerol: two interacting second messengers Ann Rev. Biochem. 56,159-93. 1  Berridge, M.J. (1990) Calcium oscillations. J. Biol. Chem. 265,9583-9586. Berridge, M.J., and Galione, A. (1988) Cytosolic calcium oscillators. FASEB J. 2, 3074-3082. Berridge, M.J., and Irvine, R.F. (1989) Inositol phosphates and cell signalling. Nature 341,197-205. Besch, H.R. (1985) Effects of ryanodine on cardiac subcellular membrane fractions. Fed. Proc. 44,2960-2963.  231  Beuckelmann, D.J., and Wier, W.G. (1988) Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. J. Physiol. 405,233255. Beukelmann, D.J., and Wier, W.G. (1988) Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. Physiol. (Lond.) 405,233-255. Bindoli, A., and Fleischer, S. (1983) Induced C a release in skeletal muscle sarcoplasmic reticulum by sulfhydryl reagents and chlorpromazine. Arch. Biochem. Biophys. 221,458-466. 2 +  Bishop, J.E., Al-Shawi, M.K., and Inesi, G. (1987) Relationship of the regulatory nucleotide site to the catalytic site of the sarcoplasmic reticulum Ca ATPase. J. Biol. Chem. 262,4658-4663. Bond, J.S., and Butler, P.E. (1987) Intracellular proteases. Ann. Rev. Biochem. 56, 333-64. Brandl, C.J., Green, N.M., Korczak, B., and MacLennan, D.H. (1986) Two C a ATPase genes: Homologies and mechanistic implications of deduced amino acid sequences. Cell. 44,597-607. 2 +  Brandt, N.R., Caswell, A.H., Wen, S., and Talvenheimo, J.A. (1990) Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads. J. Membrane Biol. 113,237-251. Brooks, B.A., Goll, D.E., Peng, Y.S., Greweling, J.A., and Hennecke, G. (1983) Effect of alloxan diabetes on a Ca-activated proteinase in rat skeletal muscle. American Physiological Society. Brown, A.M., and Birnbaumer, L. (1988) Direct G. protein gating of ion channels. Am. J. Physiol. 254 (Heart Circ. Physiol. 23), H401-H410. Brunder, D.G., Dettbarn, C , and Palade, P. (1988) Heavy metal-induced C a release from sarcoplasmic reticulum. J. Biol. Chem. 263,18785-18792.  2 +  Burgoyne, R.D., Cheek, T.R., Morgan, A., O'Sullivan, A.J., Moreton, R.B., Berridge, M.J., Mata, A.M., Colyec J., Lee, A.G., and East, J.M. (1989) Distribution of two distinct C a -ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells. Nature 342,72-74. 2  Cala, S.E., and Jones, L.R. (1983) Rapid purification of calsequestrin from cardiac and skeletal muscle sarcoplasmic reticulum vesicles by c-dependent elution from phenyl-sepharose. J. Biol. Chem. 258,11932-11936. Callewaert G., Cleemann, L., and Morad, M. (1988) Epinephrine enhances C a current-regulated C a release and C a reuptake in rat ventricular myocytes. Proc. Natl. Acad. Sci. USA. 85,2009-2013.  2 +  2 +  2 +  Campbell, K.P., Franzini-Armstrong, C , and Shamoo, A.E. (1980) Further characterisation of light and heavy sarcoplasmic reticulum vesicles. Identification of the "sarcoplasmic reticulum feet" associated with  232  heavy sarcoplasmic reticulum vesicles. Biochim. Biophys. Acta. 602, 97-116. Campbell, K.P., MacLennan, D.H., and Jorgensen, A.O. (1983) Staining of C a binding proteins, calsequestrin, calmodulin, troponin C , and S-100, with the cationic carbocyanine dye "stains-all". J. Biol. Chem. 258, 11267-11273. 2 +  Campbell, K.P., MacLennan, D.H., Jorgensen, A.O., and Mintzer, M.C. (1983) Purification and characterization of calsequestrin from canine cardiac sarcoplasmic reticulum and identification of the 53,000 dalton glycoprotein. J. Biol. Chem. 258,1197-1204. Cannell, M.B., Vaughan-Jones, R.D., and Lederer, W.J. (1985) Ryanodine block of calcium oscillations in heart muscle and the sodium-tension relationship. Fed. Proc. 44,2964-2969. Cannell, M.M., Berlin, J.R., and Lederer, W.J. (1987) Effect of membrane potential changes on the calcium transient in single rat cardiac muscle cells. Science. 238,1419-1423 Carafoli, E. (1979) The calcium cycle of mitochondria. BEBS Lett. 104,1-5. Carafoli, E. (1987) Intracellular calcium homeostasis. Ann. Rev. Biochem. 56, 395433. Carafoli, E., Rossi, C.S., and Lehninger, A.L. (1965) J. Biol. Chem. 240,2254-61. Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256,3263-70. Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256,9371-73. Caroni, P., and Carafoli, E. (1983) The regulation of the Na-Ca exchanger of heart sarcolemma. Eur. J. Biochem. 132,451-460. Caswell, A.H., and Brandt, N.R. (1989) Triadic proteins of skeletal muscle. J. of Bioenergetics and Biomembranes. 21,149-162. Catterall, W.A., Seagar, M.J. Takahashi, M., and Nunoki, K. (1989) Molecular properties of dihydropyridine-sensitive calcium channels Ann. Ny. Acad. Sci. 560,1-13 Catterall, W.A., Seagar, M.J., and Takahashi, M. (1988) Molecular properties of dihydropyridine-sensitive calcium channels in skeletal muscle. J. Biol. Chem. 263,3535-3538. Chadwick, C.C., Inui, M., and Fleischer, S. (1988) Identification and purification of a transverse tubule coupling protein which binds to the ryanodine receptor of terminal cisternae at the triad junction in skeletal muscle. J. Biol. Chem. 263,10872-10877. Chamberlain, B.K., Volpe, P., and Fleischer, S. (1984) Calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles - General characteristics. J. Biol. Chem. 259,7540-7546.  233  Chamberlain, B.K., Volpe, P., and Fleischer, S. (1984) Inhibition of calciuminduced calcium release from purified cardiac sarcoplasmic reticulum vesicles. J. Biol. Chem. 259,7547-7553. Chandler, W.K., Rakowski, R.F., and Schneider, M.F. (1976) A non-linear voltage dependent charge movement in frog skeletal muscle. J. Physiol. 254, 245-283. Charuk, J.H.M., Pirraglia, C.A., and Reithmeier, R.A.F. (1990) Interaction of rethenium red with Ca -binding proteins. Analytical Biochem. 188, 123-131. Cheon, J., and Reeves, J.P. (1988) Site density of the sodium-calcium exchange carrier in reconstituted vesicles from bovine cardiac sarcolemma. J. Biol. Chem. 263, ??. Chiu, V.C.K., and Haynes, D.H. (1977) High and low affinity C a binding to the sarcoplasmic reticulum. Use or a high affinity fluorescent indicator. Biophys. J. 18,3-22. 2 +  Chiu, V.C.K., and Haynes, D.H. (1980) Rapid kinetic studies of active C a transport in sarcoplasmic reticulum. J. Memb. Biol. 56,219-239.  2 +  Chu, A., Diaz-Munoz, M., Hawkes, M., Brush, K., and Hamilton, S.L. (1990) Ryanodine as a probe for the functional state of the skeletal muscle sarcoplasmic reticulum calcium release channel. Am. Soc. Pharm. Exp. Ther. 37, 735-741. Chu, A., Sumbilla, C , Inesi, G., Jay, S.D., and Campbell, K.P. (1990) Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle. Biochemistry. 29,5899-5905. Chu, A., Sumbilla, C , Scales, D., Piazza, A., and Inesi, G. (1988) Trypsin digestion of junctional sarcoplasmic reticulum vesicles. Biochemistry. 27,2827-2833. Chu, A., Tate, C.A., Bick, R.J., Van Winkle, W.B., and Entman, M.L. (1983) Anion effects on in vitro sarcoplasmic reticulum function. J. Biol. Chem. 258, 1656-1664. Chu,  A., Volpe, P., Costello, B., and Fleischer, S. (1986) Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum. Biochemistry 25,8315-8324.  Chyn, T., and Martonosi, A.N. (1977) Chemical modification of sarcoplasmic reticulum membranes. Biochim. Biophys. Acta. 468,114-126. Cifuentes, M*E., Ronjat, M., and Ikemoto, N. (1989) Polylysine induces a rapid C a release from sarcoplasmic reticulum vesicles by mediation of its binding to the foot 2 +  234  Clarke, D.M., Loo, T.W., and MacLennan, D.H. (1990) Functional consequences of alterations to j?olar amino acids located in the transmembrane domain of the Ca -ATPase of sarcoplasmic reticulum. J. Biol. Chem. 265,6262-6267. 2+  Clarke, D.M., Loo, T.W., Inesi, G., and MacLennan, D.H. (1989) Location of high affinity Ca -binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum C a -ATPase. Nature 339, 476478. 2+  2  Collins, J.H., Tarcsafalvi, A., and Ikemoto, N. (1990) Identification of a region of calsequestrin that binds to the junctional face membrane of sarcoplasmic reticulum. Biochemical and Biophysical Research Communications. 167,189-193. Cong, J., Goll, D.E., Peterson, A.M., and Kapprell, H.P. (1989) The role of autolysis in activity of the Ca -depenaent proteinases (u-calpain and m-calpain). J. Biol. Chem. 264,10096-10103. 2+  Corkey, B.E., Tornheim, K., Deeney, J.T., Glennon, M.C., Parker, J.C., Matschinsky, F.M., Ruderman, N.B., and Prentki, M. (1988) Linked oscillations of free C a and the ATP/ADP ratio in permeabilized FJNm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract*. J. Biol. Chem. 263,4254-4268. 2 +  Corkey, B.E., Tornheim, K., Deeney, J.T., Glennon, M.C., Parker, J.C., Matschinsky, F.M., Ruderman, N.B., and Prentki, M. (1988) Linked oscillations of free C a and the ATP/ADP ratio in permeabilized PJNm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract. J. Biol. Chem. 263,4254-4258. 2 +  Costello, B., Chadwick, C , Saito, A., Chu, A., Maurer, A., and Heischer, S. (1986) Characterization of the junctional face membrane from terminal cisternae of sarcoplasmic reticulum. J Cell Biol. 103,741-753. Croall, D.E. (1989) Proteolytic modification of calcium-dependent protease 1 in erythrocytes treated with ionomycin and calcium. American Chemical Society. Croall, D.E., and DeMartino, G.N. (1984) Comparison of two calcium-dependent proteinases from bovine heart. Bichim. Biophys. Acta. 788,348-355. Dahlmann, B., Rutschmann, M., and Reinauer, H. (1986) Effect of starvation or treatment with corticosterone on the amount of easily releasable myofilaments in rat skeletal muscles. Biochem. J. 234,659-664. Danko, S., Kim, D.H., Sreter, F.A., and Ikemoto, N. (1985) Inhibitors of C a release from the isolated sarcoplasmic reticulum. II. The effects of dantrolene on C a release induced by caffeine, C a and depolarization. Biochimica et Biophysica Acta. 816,18-24.  2 +  2 +  2 +  Davy, H.K. (1808) Electro-chemical researches, on the decomposition of the earths; with observations on the metals obtained from the alkaline earths, and on the amalgam from amonium. Philos Trans. R. Soc.  235  Lond. 98, 333. Reported in Campbell, A.K. Intracellular Calcium. Its Universal Role as Regulator. New York: John Wiley, 1983. Dayton, W.R., Schollmeyer, J.V., Chan, A.C., and Allen, C E . (1979) Biochim. Biophys. Acta. 584,216-230. Deamer, D.W., and Baskin, R.J. (1969) Ultrastructure of sarcoplasmic reticulum preparations. J. Cell. Biol. 42,296-307. DeMartino, G.N., Huff, C.A., and Croall, D. (1986) Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity. J. Biol. Chem. 261,12047-12062. Devine, C.E., and Raynes, D.G. (1975) Freeze fracture studies of membrane in vertebrate muscle, n. Smooth muscle. J. Ultrastruct. Res. 51,293-306. Diaz-Munoz, M., and Hamilton, S.L. (1990) Modulation of C a release channel activity from sarcoplasmic reticulum by annexim VI (67-kDa calcimedin). J. Biol. Chem. 265,15894-15899. 2 +  Donaldson, S.K. (1989) Mechanisms of excitation-contraction coupling in skinned muscle fibers. Medicine and Science in Sports and Exercise. 21, 411417. Donaldson, S.K., Goldberg, N.D., Walseth, T.F., and Huetteman, D.A. (1987) Iositol trisphosphate stimulates calcium release from peeled skeletal muscle fibers. Biochim. Biophys. Acta. 927,92-99. Dulhunty, A., and Valois, A. (1983) Mentations in the terminal cisternae of amphibian and mammalian skeletal muscle fibers. J. Ultrastruct. Res. 84,34-49. Dulhunty, A., Gage, P., and Valois, A. (1983) Indentations in the terminal cisternae of slow-and fast-twitch muscle fibers from normal and paraplegic rats. J. of Ultrastructure Research. 84,50-59. Dulhunty, A.F. (1989) Feet, bridges, and pillars in triad junctions of mammalian skeletal muscle: their possible relationship to calcium buffers in terminal cisternae and T-tubules and to excitation-contraction coupling. J. Membrane Biol. 109,73-83. Dunnett, J., and Nayler, W.G. (1979) Effect of pH on calcium accumulation and release by isolated fragments of cardiac and skeletal muscle sarcoplasmic reticulum. Arch. Biochem. Biophys. 198,434-438. Dupont, Y. (1977) Kinetics and regulation of sarcoplasmic reticulum ATP ase. Eur. J. Biochem. 72,185-190. Earnshaw, W.C. (1987) J. Cell Biol. 105,1479-1482. Ebashi, S. (1961) Calcium binding activity of vesicular relaxing factor. J. Biol. Chem. 50,236-244.  236  Ebashi, S., and Endo, M. (1968) Ca ion and muscle contraction. Progr. Biophys. Mol. Biol. 18,123-183. Ebashi, S., and Lipman, F. (1962) Adenosine triphosphate-linked concentration of calcium ions in a particular fraction of rabbit muscle. J. Cell Biol. 14, 389-400. Ebashi, S., and Lipmann, F. (1962) Adenosine triphosphate-linked concentration of calcium ions in a particulate fraction of rabbit muscle. J.Cell Biol. 14, 389-400. Ebashi, S., Endo, M., and Ohtsuki, I. (1969) Control of muscle contraction. Quarterly Reviews of Biophysics 2.4,351-384. Ebashi,S. (1961) Calcium binding activity of vesicular relaxin factor. J. Biochem. (Tokyo). 50,236-244. Eisenberg, B.R., and Eisenberg, R.S. (1982) The T-SR junction in contracting single skeletal muscle fibers. J. Gen. Physiol. 79,1-19. Eisenberg, D., Schwartz, E., Komaromy, M. and Wall, R. (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J. Mol. Biol. 179,125-142. Eisenberg, R.S., McCarthy, R.T., and Milton, R.L. (1983) J. Physiol. Lond. 341,495595. Endo, M. (1977) Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57,71-103. Endo, M. (1975) Conditions required for calcium-induced release of calcium from the sarcoplasmic reticulum. Proc. Japan Acad., 51,467-472. Endo, M. (1977) Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57,71-108. Endo, M. (1981) Mechanism of calcium induced calcium release in the SR membrane, In:Ohnishi, S.T. and Endo M. (Eds). The Mechanism of Gated Calcium Transport Across Biological Membranes. Academic, New York. 1-8 Endo, M., and Nakajima, Y., (1973) Release of calcium induced by "depolarisation" of the sarcoplasmic eticulum membrane. Nature New Biol. 246,216-218. Endo, M., Kakutz, Y., and Kitazawa, T. (1981) A further study of the Ca-induced Ca release mechanism. The regulation of muscle contraction: Excitation-contraction coupling. Endo, M., Tanaka, M., and Ogawa, Y. (1970) Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres. Nature 228,34-36.  237  Entman, M.L., Snow, T.R., Freed, D., and Schwartz, A. (1973) Analysis of calcium binding and release by canine cardiac relaxing system (sarcoplasmic reticulum). J. Biol. Chem. 248,7762-7772. Entman, M.L., Van Winkle, W.B., Bornet, E., and Tate, C. (1978) Spontaneous calcium releases from sarcoplasmic reticulum. Biochimica et Biophysica Acta. 551,382-388. Evans, S., and Dean, W.L. (1986) Effects of sulfhydryl reagents and other inhibitors on C a transport and inositol triphosphate-induced C a release from human platelet membranes. J. Biol. Chem. 261, 1307113075. 2 +  2 +  Exton, J.H. (1988) Mechanisms of action of calcium-mobilizing agonists: some variations on a young theme. FASEB J. 2,2670-2676. Fabiato, A. (1981) Mechanism of calcium-induced release of calcium from the sarcoplasmic reticlum of skinned cardiac cells studied with potential sensitive dyes. In: Ohnishi S.T. and Endo, M. (Eds). The Mechanism of Gated Calcium Transport Across Biological Membranes. Academic, New York. Fabiato, A. (1981) Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle. J. Gen. Physiol. 78, 457-497. Fabiato, A. (1982) Fluorescence and eifferential light absorption recordings with calcium probes and potential-sensitive dyes in skinned cardiac cells. Can. J. Physiol. Pharmacol. 60,556-567. Fabiato, A. (1982) Fluoresence and differential light absorption recordings with calcium probes and potential-sensitive dyes in skinned cardiac cells. Can. J. Phys. Pharm. 60,556-567. Fabiato, A. (1983) Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am. J. Physiol. 245, C1-C14. Fabiato, A. (1983) Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am. J. Physiol. 245, C1-C4. Fabiato, A. (1989) App[raisal of the physiological relevance of two hypotheses for the mechanism of calcium release from the mammalian cardiac sarcoplasmic reticulum: calcium-induced release versus chargecoupled release. Molecular and Cellular Biochemistry 89,135-140. Fabiato, A. and Fabiato, F. (1979) Calculator programs for multiple metals and ligands. J. Pysiol. (Paris) 75,463-505. Fabiato, A. Calcium release in skinned cardiac cells: variations with species, tissues and development. Fed. Proc. 41,2238-2244.  238  Fabiato, A., and Fabiato, F. (1975) Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells. J. physiol. 249,469-495. Fain, J.N., Wallace, M.A., and Wojcikiewicz, R.J.H. (1988) Evidence for involvement of guanine nucleotide-binding regulatory proteins in the activation of phospholipases by hormones. FASEB J. 2,2569-2574. Fairhurst, A.S., and Hasselbach, W.H. (1970) Calcium efflux from a heavy sarcotubular fraction: effects of ryanodine, caffeine and magnesium. Eur. J. Biochem. 13,504-509. Feher, J.J., Manson, N.H. and Poland, J.L. (1988) The rate and capacity of calcium uptake by sarcoplasmic reticulum in fast, slow, ana cardiac muscle: Effects of ryanodine and ruthenium red. Arch. Biochem. Biophys. 265, 171-182. Ferris, C D . , Huganir, R.L., Supattapone, S., and Snyder, S.H. (1989) Purified inositol 1,4,5-triphosphate receptor mediates calcium flux in reconstituted lipid vesicles. Nature 342,87-89. Fill, M., Ma, J., Knudson, C M . , Imagawa, T., Campbell, K.P., and Coronado, R. (1989) Role of the ryanodine receptor of skeletal muscle in excitationcontraction coupling. Ann. N.Y. Academy of Sci. 560,155-161. Fitts, R.H., Courtright, J.B., Kim, D.H., and Witzmann, F.A. (1982) Muscle fatigue with prolonged exercise: contractile and bioch4emical alterations. Am. J. Physiol. 242, C65-C73. Fleischer, S., Ogunbunmi, E.M., Dixon, M C , and Fleer, E.A.M. (1985) Localization of C a release channels with ryanodine in junctional terminal cisternae of sarcoplasmic reticulum of fast skeletal muscle. Proc. Natl. Acad. Sci. 82,7256-7259. 2 +  Fliegel, I., Ohnishi, M., Carpenter, M.R., Khanna, V.K., Reithmeier, R.A.F., and MacLennan, D.H. (1987) Proc. Natl. Acad. Sci. U.S.A. 84,1167-1171. Fliegel, L., Burns, K., MacLennan, D.H., Reithmeier, R.A.F., and Michalak, M. (1989) Molecular Cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 264,21522-21528. Fliegel, L., Burns, K., Opas, M., and Michalak, M. (1989) The high-affinity calcium binding protein of sarcoplasmic reticulum. Tissue distribution, and homology with calregulin. Biochimica et Biophysica Acta. 982,1-8. Ford, L.E., and Podolsky, R.J. (1970) Regenerative calcium release within muscle cells. Science. 167,58-59. Frank, G.B. (1982) Canadian journal of physiology and pharmacology. Roles of Extracellular and "trigger" calcium ions in excitation-contraction coupling in skeletal muscle. 60,427-439.  239  Franzini-Armstrong, C. (1975) Membrane particles and transmission at the triad. Fed. Proc. 34,1382-1389. Franzini-Armstrong, C. (1980) Structure of Sarcoplasmic reticulum. Fed. Proc. 39, 2403-2409. Franzini-Armstrong, C , and Nunzi, G. (1983) Junctional feet and particles in the triads of a fast-twitch muscle fibre. J. of Muscle Research and Cell Motility. 4,233-252. Froelich, J.P., and Taylor, E.W. (1976) Transient state kinetic effects of calcium ion on sarcoplasmic reticulum adenosine triphosphatase. J. Biol. Chem. 251,2307-2315. Fujimori, T., and Jencks, W.P. (1990) Lanthanum inhibits steady-state turnover of the sarcoplasmic reticulum calcium ATPase by replacing magnesium as the catalytic ion. J. Biol. Chem. 265,16262-16270. Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N., and Mikoshiba, K. (1989) Primary structure and functional expression of the inositol 1,4,5-triphosphate-binding protein P400 Nature 342,32-38. Gainullin, R.Z., and Saxon, M.E. (1989) Positive inotropic effect of ryanodine on rabbit ventricular muscle: dependence on the Intracellular calcium load. Gen. Physiol. Biophys. 8,555-568. Ghosh, T.K., Mullaney, J.M., Tarazi, F.I., and Gill. (1989) GTP-activated communication between distinct inositol 1,4,5-triphosphate-sensitive and -insensitive calcium pools. Nature 340,236-239. Gilchrist, J.S.C., Katz, S., and Belcastro, A.N. (1990) Improved resolution of the initial fast phase of heavy sarcoplasmic reticulum C a uptake by C a . Antipyrylazo III dual-wavelength spectroscopy. Biochem. and Biophys. Res. Comm. 168,364-371. 2 +  Gilman, A.G. (1984) G. proteins and dual control of adenylate cyclase. Cel. 36, 577-579. Gilman, A.G. (1987) G proteins: transducers of receptor-generated signals. Ann Rev. Biochem. 56,615-49. Gingold, M.P., Rigaud, J.L., and Champeil, P. (1981) Fluorescence energy transfer between ATPase monomers in sarcoplasmic reticulum reconstituted vesicles. Biochimie. 63,923-925. Goll, D.E., Kleese, W.C, Kumamoto, T., Cong, J., and Szpacenko. A. (1989) In search of the regulation and function of the Ca -dependent proteinases (calpains) . in: "Intracellular Proteolysis, Mechanisms and Regulaiton" (Katunuma, N., and Kominami, E., eds.), Japanese Scientific Societies Press, Tokyo, 82-91. 2+  1  Gonzales-Serratos, H.A., Somylo, A., MacLellan, G., Schuman, H., Borrero, L., and Somylo, A. (1978) Composition of vacuoles and sarcoplasmic  240  reticulum in fatigued muscle: electron probe analysis. Proc. Natl. Acad. Sci. 75,1329-1333. Gopalakrishna, R., and Barsky, S.H. (1986) Hydrophobic association of calpains with subcellular organelles. J. Biol. Chem. 261,13936-13942. Gopalakrishna, R., and Barsky, S.H. (1986) Hydrophobic association of calpains with subcellular organelles. J. Biol. Chem. 261,13936-13942. Gould, G.W., McWhirter, J.M., Easi, J.M., and Lee, A.G. (1987) A model for the uptake and release of C a by sarcoplasmic reticulum. Biochem. J. 245, 739-749. 2 +  Hamilton, S.L., Alvarez, R.M., FilL M., Hawkes, M.J., Brush, K.L., Schilling, W.P., and Stefani, E. (1989) [ H]PN200-110 and [ H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes. Anal. Biochem. 183,31-41. 3  3  Hashimoto, I., Sembrowich, W., and Gollnick, P.D. (1978) Calcium uptake by isolated sarcoplasmic reticulum and homogenates in different fibre types following exercise. Med Sci. Sports. 10,42. Hasselbach, W. (1979) The sarcoplasmic calcium pump. A model of energy transduction in biological membranes. Top Curr. Chem. 78,1-56. Hasselbach, W., and Makinose, M. (1961) Die calciumpumpe der 'Erschlaffungsgrana' des muskels und ihre abhangigkeit von der ATPSpaltung. Biochem. Z. 333,518-528. Hasselbach, W., and Makinose, M. (1961) Die Calciumpumpe der "Erschlaffungsgrana" des Muskels und ihre Abbangigkeit von der ATP-Spaltung. Biochem. Z. 833,518-528. Hasselbach, W., and Migala, A. (1987) Activation and inhibition of the calcium ate of sarcoplasmic reticulum by high-affinity ryanodine binding, ed. Eur. Biochem. Soc. 221,119-123.  P  Hasselbach, W., and Oetliker, H. (1983) Energetics and electrogenecity of the sarcoplasmic reticulum calcium pump. Ann. Rev. Physiol. 45,325-339. Hasselbach, W., and Waas, W. (1982) The Ca -ATPase of sarcoplasmic reticulum: IV. Energy coupling in sarcoplasmic reticulum C a transport: An overview. An N.Y. Acad. Sci. 402,459-469. 2+  2 +  Hasselbach, W., Fassold, E., Migala, A., and Rauch, B. (1981) Magnesium dependence of sarcoplasmic reticulum calcium transport. Fed. Proc. 40, 2657-2662. Hathaway, D.R., Werth, D.K., and Haeberle, J.R. (1982) J. Biol. Chem. 257, 90729077. Haynes, D.E. (1983) Mechanism of C a by C a , Mg -ATPase pump: analysis of major states and pathways. Am. J. Physiol. 244, G3-G12. 2 +  2 +  2+  241  Heegaard, C W Le Maire, M , Gulik-Krzywicki, T., and Moller, J.V. (1990) J. Biol. Chem. 265,12020-12028, V  Heilbrunn, L.V., and Wiercinski, F.J. (1947) The action of various cations on muscle protoplasm. J. Cell Comp. Physiol. 29,15-32. Herbette, L., Scarpa, A., and Blasie, J.K. (19??????) Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio. Biophys. J. 36,27-46. Herland, J.S., Julian, F.J.^and Stephenson, D.G. (1990) Halothane increases C a efflux via C a channels of sarcoplasmic reticulum in chemically skinned rat myocardium. J. Physiol. 426,1-18.  2 +  2 +  Hidalgo,  C , and Ikemoto, N. (1977) Disposition of proteins and aminophospholipids in the sarcoplasmic reticulum membranes. J. Biol. Chem. 252,8446-8454.  Hidalgo, C , Gonzalez, M.E., and Garcia, A.M. (1986) Calcium transport in transverse tubules isolated from rabbit skeletal muscle. Biochim. Biophys. Acta. 865,279-286. Hill, A.V. (1949) The abrupt transition from rest to activity in muscle. Proc. R. Soc. 136,399-420. Hill, A.V. (1949) The heat of activation and the heat of shortening in a muscle twitch. Proc. Roy. Soc. 136,195-210. Hofmann, S.L., Brown, M.S., Lee, E., Pathak, R.K., Anderson, R.G.W., and Goldstein, J.L (1989) Purification of a sarcoplasmic reticulum protein that binds C a and plasma lipoproteins. J. Biol. Chem. 264,8260-8270. 2 +  Hosey, M.M., Chang, F.C., O'Callahan, C M . , and Ptasienski, J. (1989) L-type calcium channels in cardiac and skeletal muscle. Ann. NY Acad. Sci. 560,27-38. Hui, C.S., Milton, R.L., and Eisenberg, R.S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2582-2585. Hymel, L., Schindler, H., Inui, M., Heischer, S., Streissnig, J., and Glossmann, H. (1989) A molecular model of excitation-contraction coupling at the skeletal muscle triad junction via coassociated oligomeric calcium channels. Ann. N.Y. Acad. Sci. 560,185-188. Hymel,L., Inui, M., Heischer, S., and Schindler, H. (1988) Purified ryanodine receptor of skeletal muscle sarcoplasmic reticulum forms C a activated oligomeric C a channels in planar bilayers. Proc. Natl. Acad. Sci. 85,441-445. 2 +  2 +  lino, M., Kobayashi, T., and Endo, M. (1988) Use of ryanodine for functional removal of the calcium store in smooth muscle cells of the guinea-pig. Biochim. et Biophys. Acta. 152,417-422.  242  Ikemoto, N. (1974) The calcium binding sites involved in the regulation of the urified adenosine triphosphatase of the sarcoplasmic reticulum. J. iol.Chem. 249,649-651. Ikemoto, N. (1975) Transport and inhibitory C a binding sites on the ATPase enzyme isolated from the sarcoplasmic reticulum. J. Biol. Chem. 250, 7219-7224. 2 +  Ikemoto, N., Antoniu, B., and Kim, D. (1984) Rapid calcium release from the isolated sarcoplasmic reticulum is triggered via the attached transverse tubular system. J. Biol. Chem. 259,13151-13158. Ikemoto, N., Ronjat, M , and Meszaros, L.G. (1989) Kinetic analysis of excitationcontraction coupling. J. of Bioenerg. Biomemb. 21,247-266. Imagawa, T., Smith, J.S., Coronado, R, and Campbell, K.P. (1987) Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca -permeable pore of the calcium release channel. J. Biol. Chem. 262,16636-16643. 2+  Imagawa, T., Takasago, T., and Shigekawa, M. (1989) Cardiac ryanodine receptor is absent in type I slow skeletal muscle fibers: Immunochemical and Ryanodine binding studies. J. Biochem. 106,342-348. Imajoh, S., Aoki, K., Ohno, S., Emori, Y., Kawasaki, H., Sugihara, H., and Suzuki, K. (1988) Molecular cloning of the cDNA for the large subunit of HighCa -activated neutral protease. Biochemistry. 27,8122-8128. 2+  Imajoh, S., Kawasaki, H. and Suzuki, K. (1986) The amino-terminal hydrophobic region of the small subunit of calcium-activated neutral protease (CANP) is essential for its activation by phosphatidylinositol. J. Biochem. 99,1281-1284. Imajoh, S., Suzuki, K. (1985) Reversible interaction between Ca -activated neutral protease (CANP) and its endogenous inhibitor. FEBS lett. 187, 47-50. 2+  Inesi, G. (1981) The sacrcoplasmic reticulum of skeletal and cardiac muscle. In: Dowben R.M. Dowben and Shay, J.W. (Eds.). Cell and Muscle Motil. Plenum: New York. Inesi, G., and Scales, D. (1974) Tryptic cleavage of sarcoplasmic reticulum protein. Biochemistry. 13,3298-3306. Inesi, G., Kurzmack, M., Coan, C , and Lewis, D.E. (1980) Cooperative calcium binding and ATPase activation in sarcoplasmic reticulum vesicles. J. Biol. Chem. 255,3025-3031. Inesi, G., Watanabe, T., Coan, C , and Murphy, A. (1982) The mechanism of sarcoplasmic reticulum ATPase. Ann. N.Y. Acad. Sci. 402,515-534. Inui, M., Sai to, A., and Fleischer, S. (1987) Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures. J. Biol. Chem. 262,15637-15642.  243  Inui, M., Saito, A., and Fleischer, S. (1987) Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle. J. Biol. Chem. 262, 1740-1747. Irvine, R.F. (1990) 'Quantal' C a release and the control of C a entry by inositol phosphates-a possible mechanism. FEBS Lett. 263,5-9. 2 +  2 +  Ito, K., Kohichi, T., and Sutko, J.L. (1986) Ryanodine inhibits the release of calcium from intracellular stores in guinea pig aortic smooth muscle. Circ. Res. 58,730-734. Ito, K., Takakura, S., Sato, K., and Sutko, J.L. (1986) Ryanodine inhibits the release of calcium from intracellular stores in guinea pig aortic smooth muscle. Circ. Res. 58,730-734. James, P., Inui, M., Tada, M., Chiesi, M., and Carafoli (1989) Nature and site of phospholamban regulation of the C a pump of sarcoplasmic reticulum. Nature. 342,90-92. 2 +  James, P., Maeda, M.; Fischer, R., Verma, A.K., Krebs, J., Penniston, J.T., and Carafoli, E. (1988) Identification and primary structure of a calmodulin binding domain of the C a pump of human erythrocytes. J. Biol. Chem. 263,2905-2910. 2 +  Jay, S.D., Ellis, S.B., McCue, A.F., Williams, M.E., Vedvick, T.S., Harpold, M.M., and Campbell, K.P. (1990) Primary structure of the A subunit of the DHP-sensitive calcium channel from skeletal muscle. Science 248,490492. Jenden, D.J., and Fairhurst, A.S. (1969) The pharmacology of ryanodine Pharmacol. Revs. 21,1. Jewett, P.H., Sommer, J.R., and Johnson, E.A. (1971) Cardiac muscle. Its ultrastructure in the finch and hummingbird with special reference to the sarcoplasmic reticulum. J. Cell Biol. 49,50-65. Jones, L.R., and Cala, S.E. (1981) Biochemical evidence for functional heterogeneity of cardiac sarcoplasmic reticulum vesicles. J. Biol. Chem. 256,11809-11818. Jones, L.R., Besch, H.R., Jr., Sutko, J.L. and Willerson, J.T. (1979) Ryanodine induced stimulation of net Ca2+ uptake by cardiac sarcoplasmic reticulum vesicles. J. Pharmacol. Exp. Ther. 209,48-55. Jorgensen, A.O., and Campbell, K.P. (1984) Evidence for the presence of calsequestrin in two structurally different regions of myocardial sarcoplasmic reticulum. J. Cell Biol. 98,1597-1602. Jorgensen, A.O., Shen, A.C.Y., Campbell, K.P., and MacLennan, D.H. (1983) Ultrastructure localisation of calsequestrin in rat skelletal muscle by immunoferritin labeling of ultrathin frozen sections. J. Cell Biol. 97, 1573-1581.  244  Kanazawa, T., and Boyer, P.D. (1973) Occurrence and characteristics of a rapid exchange of phosphate oxygens catalysed by sarcoplasmic reticulum vesicles. J. Biol. Chem. 248,3163-3172. Kanazawa, T., Yamada, S., Yamamoto, T., and Tonomura, Y. (1971) Reaction mechanism of the Ca -dependent ATPase of sarcoplasmic reticulum from skeletal muscle. V. Vectorial requirements for calcium and magnesium ions of three partical reactions of ATPase: formation and decomposition of a phosphorylated intermediate and ATP-formation from ADP and the intermediate. J. Biochem. 70,95-123. 2+  Kapprell, H.P., and Goll, D.E. (1989) Effect of C a on binding of the calpains to calpastatin. J. Biol. Chem. 264,17888-17896. 2 +  Katz, A.M., Louis, C.F., Repke, D.I., Fudyma, G., Nash-Adler, P.A., Kupsaw, R., and Shigekawa, M. (1980) Time-dependent changes of calcium influx and efflux rates in rabbit skeletal muscle sarcoplasmic reticulum. Biochim. Biophys. Acta. 596,94-107. Katz, A.M., Repke, D.I., and Hasselbach, W. (1977) Dependence of ionophoreand caffeine-induced calcium release from sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations. J. Biol. Chem. 252,1938-1949. Kawamoto, R.M., Brunschwig, J.-P., Kim, K.C., and Caswell, A.H. (1986) Isolation, characterization, and localization of the spanning protein from skeletal muscle triads. J Cell Biol. 103,1405-1414. Kendrick, N.C., Ratzlaff, R.W., and Blaustein, M.P. (1977) Arsenazo III as an indicator for ionized calcium in physiological salt solutions: Its use for determination of the C a A T P dissociation constant. Anal. Biochem. 83,433-450. 2+  Kenessey, A., Banay-Schwartz, B., DeGuzman, T., and Lajtha (1989) Regional distribution of brain calpastatin and of calpain II. activity with casein and with endogenous brain protein substrates. Neurocnem. Int. 15, 307-314. Kentish, J.C., Barsotti, R.J., Lea, T.J., Mulligan, LP., Patel, J.R., and Ferenczi, M.A. Calcium release from cardiac sarcoplasmic reticulum induced by photorelease of calcium or Ins(l,4,5)P3. Am. J. Physiol. 258 (Heart Circ. Physiol 27): H610-H615. Kielley, W.W., and Meyerhof, O. (1948) Studies on adenosine triphosphatase of muscle. II. A new magnesium-activated adenosine triphosphatase. J. Biol. Chem. 176,591-601. Kim, D., Ohnishi, ST., and Ikemoto, N. (1983) Kinetic studies of calcium release from sarcoplasmic reticulum. J. Biol. Chem. 258,9662-9668. Kim, D., Sreter, F.A., Ohnishi, ST., Ryan, J.F., Roberts, J., Allen, P.D., Meszaros, L.G., Antoniu, B., and Ikemoto, N. (1984) Kinetic studies of C a release from sarcoplasmic reticulum of normal and malignant 2 +  245  hyperthermia susceptible pig muscles. Biochim. et Biophys. Acta. 775, 320-327. Kim, K.C., Caswell, A.H., Brunschwig, J.P., and Brandt, N.R. (1990) Identification of a new subpopulation of triad junctions isolated from skeletal muscle; morphological correlations with intact muscle. J. Membrane Biol. 113,221-235. Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S., Tominaga, M., Kuroda, T., and Nishizuka, Y. (1989) Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (Calpain). J. Biol. Chem. 264,4088-4092. Kishimoto, A., Mikawa, K., Hashimoto, Keisuke, Yasuda, I., Tanka, S., Tominaga, M., Kuroda, T., and Nishizuka, Y. (1989) Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (Calpain). J. Biol. Chem. 264,4088-4092. Klein, P., Kanehisa, M., and DeLisi, C. (1985) The detection and classification of membrane-spanning proteins. Biochim. Biophs. Acta. 815,468-476. Klempner, M. (1985) J. Clin. Invest. 76,303-10. Knudson, C M . , Mickelson, J.R., Louis, C.F., and Campbell, K.P. (1990) Distinct immunopeptide maps of the sarcoplasmic reticulum C a release channel in malignant hyperthermia. J. Biol. Chem. 265,2421-2424. 2 +  Kouzaridea, T., and Ziff, E. (1988) The role of the leucine zipper in the fos-jun interaction. Nature. 336,646-651. Kovacs, L. Rios, E., and Schneider, M.F. (1983) Measurement and modification of free calcium transients in frog skeletal muscle fibres by a metallochromic indicator dye. J. Physiol. 343,161-196. Kraus-Friedmann, N., Carafoli, E., Biber, J., and Murer, H. (1982) Ann. N.Y. Acad. Sci. 402,440-442. Kretsinger R.H., and Barry, C D . (1975) The predicted structure of the calcium binding component of troponin. Biochim. Biophys. Acta. 405,40-52. Kumagai, H., Ebashi, S., and Takeda, F. (1955) Essential relaxing factor in muscle other than myokinase and creatine phosphokinase. Nature. 176,166. Kyte, J., and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157,105-132. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature,. 227,680-685. Lai, F.A., and Meissner, G. (1989) The muscle ryanodine receptor and its intrinsic C a channel activity. J. Bioenerg. Biomemb. 21,227-246. 2 +  Lai, F.A., Andersoru K., Rousseau, E., Liu, Q., and Meissner, G. (1988) Evidence for a C a channel within the ryanodine receptor complex from 2 +  246  cardiac sarcoplasmic reticulum. Biochem. Biophys. Res. Commun. 151, 441-449. Lai, F.A., Erickson, H., Block, B.A., and Meissner, G. (1987) Evidence for a junctional feet-ryanodine receptor complex from sarcoplasmic reticulum. Biochem. Biophys. Res. Commun. 143,704-709. Lai, F.A., Erickson, H.P., Rousseau, E., Liu, Q., and Meissner, G. (1988) Purification and reconstitution of the calcium release channel from skeletal muscle. Nature 331,315-319. Lai, F.A., Misras, M., Xu, L., Smith, A., and Meissner, G. (1989) The ryanodine receptor-Ca release channel complex of skeletal muscle sarcoplasmic reticulum: Evidence for a cooperatively coupled, negatively charged homotetramer. J. Biol. Chem. 264,16676-16785. 2+  Lakatta, E.G., Capogrossi, M . C , Kort, A.A., and Stern, M.D. (1985) Spontaneous myocardial calcium oscillations: overview with emphasis on ryanodine and caffeine. Fed. Proc. 44,2977-2983. Landschulz, W.H., Johnson, P.F., and McKnight, S.L. (1988) The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science. 240,1759-1764. Lattanzio, F.A., Schlatterer, R.G., Nicar, M., Campbell, K.P., and Sutko, J.L. (1987) The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes. J. Biol. Chem. 262,2711-2718. Lea, T.J., Griffiths, P.J., Tregear, R.T., and Ashley, C.C. (1986) An examination of the ability of inositol 1,4,5,-trisphosphate to induce calcium release and tension development in skinned skeletal muscle fibres of frog and Crustacea. FEBS Lett. 207,153-161. Leavis, P.C. and Gergely, J. (1984) Thin filament proteins and thin filament linked regulation of vertebrate muscle contraction. CRC crit. Rev. Biochem. 16,235-305. Leberer, E., Charuk, J.H.M., Clarke, D.M., Green, N.M., Zubrzycka-Gaarn, E., and MacLennan, D.H. (1989a) Molecular cloning and expression of cDNA encoding the 53,000-dalton glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 264. 3484-3493. Leberer, E., Charuk, J.H.M., Green, N.M., and MacLennan, D.H. (1989b) Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum. Proc. Natl. Acad. Sci. 86,6047-6051. Leberer, E., Timms, B.G., Campbell, K.P., and MacLennan, D.H. (1990) Purification, Calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (sarcalumenin)-Dalton Glycoproteins of the sarcoplasmic reticulum. J. Biol. Chem. 265,1011810124. Leblanc, N., and Hume, J.R. (1990) Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum. Science 248,372-376.  247  Lederer, W.J., Cannell, M.B., Cohen, N.M., and Berlin, J.R. (1989) Excitationcontraction coupling in heart muscle. Mol. Cell. Biochem. 89,115-119. LeMaire, M.J., Moller, J.V., and Tanford, C. (1976) Retention of enzymatic activity by detergent-solubilised sarcoplasmic Ca -ATPase. Biochemistry. 15, 2336-2342. 2+  Lentz, B.R., Clubb, K.W., Barrow, D.A., and Meissner, G. (1983) Ordered and disordered phospholipid domains coexist in membranes containing the calcium pump protein of sarcoplasmic reticulum. Proc. Natl. Acad. Sci. 80,2917-2921. LePeuch, C.J., and Demaille, J.G. (1989) Covalent regulation of the cardiac sarcoplasmic reticulum calcium pump. Cell Calcium 10,397-400. Leung, A.T., Imagawa, T., Block, B., Franzini-Armstrong, C , and Campbell, K.P. (1988) Biochemical and ultrastructural characterization of the 1,4dihydropyridine receptor from rabbit skeletal muscle. J. Biol. Chem. 263,994-1001. Litosch, I., and Fain, J.N. (1986) Regulation of phosphoinositide breakdown by guanine nucleotides. Life Sci. 39,187-194. Liu, Q.Y., Lai, F.A., Rousseau, E., Jones, R.V., and Meissner, G. (1989) Multiple conductance states of the purified calcium release channel complex from skeletal sarcoplasmic reticulum. Biophys. J. 55,415-424. Llinas, R, and Yarom. Y. (1981) J. Physiol. 315,549-567. Llinas, R., Sugimori, M., Lin, J.W., and Cherksey, B. (1989) Proc. Natl. Acad. Sci. USA. 86,1689-1693. Llinas, R.R., Sugimori, M., and Cherksey, B. (1989) Voltage-dependent calcium conductances in mammaliam neurons. Ann. N.Y. Acad. Sci. 560, 103111. Long, C. and Mouat, B. (1973) The binding of calcium ions by erythrocytes and •ghost'-cell membranes. Biochem. J. 123,829-836. Lowry, O.H., Rosebrough, N., Farr, A., and Randell, R, (1951) Protein measurement with folin-phenol reagent. J. Biol. Chem. 193,265-275. Ma, J., Fill, M., Knudson, C M . , Campbell, K.P. and Coronado, R. (1988) Ryanodine receptor of skeletal muscle is a gap junction-type channel. Science 242,99-102. MacLennan, D.H., and Wong, P.T.S. (1971) Isolation of a calcium sequestering rotein from sarcoplasmic reticulum. Proc. Natl. Acad. Sci. 68, 1231235.  ?  MacLennan, D.H., Brandl, C.J., Korzak, G., and Green, N.M. (1985) Amino-acid sequence of a C a ^ + Mg " " dependent ATPase from rabbit muscle 2  2  1  248  sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature. 316,696-700. MacLennan, D.H., Duff, C , Zorzato, R, Fujii, J., Phillips, M., Korneluk, R.G., Frodis, W., Britt, B.A., and Worton, R.G. (1990) Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia. Nature. 343,559-561. Maki, M., Takano, E., Mori, H., Sato, A., Murachi, T. and Hatanaka, M. (1987) All four internally repetitive domain of pig calpastatin possess inhibitory activities against calpain I and II. FEBS lett. 223,174-180. Makinose, M., and Boll, W. (1979) The role of magnesium on the sarcoplasmic calcium pump. In: Mukata, Y., and Packer, L. (Eds.). Cation Flux Across Biomembranes. Academic: New York. 89-100. Makinose, M., and Hasselbach, W. (1971) ATP synthesis by the reverse of the sarcoplasmic calcium pump. FEBS Lett. 12,271-272. Marban, E., Wier, W.G. (1985) Ryanodine as a tool to determine the contributions of calcium entry and calcium release to the calcium transient and contraction of cardiac purkinje fibers. Circ. Res. 56,133-138. Marks, A.R., Fleischer, S., Nadal-Girnard, B., and Tempst, P. (1990) Mapping protease sensitive regions of the ryanodine receptor from skeletal muscle. Biophys. J. 57,176a. Marks, A.R., Tempst, P., Hwang, K.S., Taubman, M.B., Inui, M., Chadwick, C , Fleischer, S., and Nadal-Ginard, B. (1989) Molecular cloning and characterisation of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle sarcoplasmic reticulum. Proc. Natl. Acad. Sci. 86,8683-8687. Marsh, B.B. (1951) A factor modifying muscle fibre synaeresis. Nature 167,10651066. Marsh, B.B. (1952) The effects of adenosine triphosphate on the fibre volume of a muscle homogenate. Biochim. Biophys. Acta. 9,243-260. Martonosi, A. (1968) Sarcoplasmic reticulum V. The structure of sarcoplasmic reticulum membranes. Biochim. Biophys. Acta. 150,694-704. Martonosi, A.N. (1980) Calcium pumps (Introduction) Fed. Proc. 39,2401-2402. Martonosi, A.N. (1982) The development of the sarcoplasmic reticulum membranes. Ann Rev. Physiol. 44,337-372. Martonosi, A.N. (1984) Mechanisms of C a release from sarcoplasmic reticulum of skeletal muscle. Physiol. Rev. 64,1240-1320. 2 +  Martonosi, A.N., and Beeler, T.V. (1983) Mechanisms of C a transport by sarcoplasmic reticulum. In: Peachey, L.D., and Adrian, R.H. (Eds). Handbook of Physilogy. X. Skeletal Muscle. Am. Physiol. Soc: Bethesda. 417-485. 2 +  249  Maruyama, K Clarke, D.M., Fujii, J., Inesi, G., Loo, T.W., and MacLennan, D.H. (1989) Functional consequences of alterations to amino acids located in the catalytic center (Isoleucine 348 to Threonine 357) and Nucleotidebinding domain of the Ca -ATPase of sarcoplasmic reticulum. J. Biol. Chem. 264,13038-13042. v  2+  Masuda, H., and DeMeis, L. (1973) Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate. Inhibition by calcium ions. Biochemistry. 12,4581-4585. Mathias, R.T., Levis, R.A. and Eisenberg, R.S. (1980) Electrical models of excitation-contraction coupling and charge movement in skeletal muscle. J. Gen. Physiol. 76,1-31. McClellan, P., Lash, J.A., and Hathaway, D.R. (1989) Identification of major autolytic cleavage sites in the regulatory subunit of vascular calpain n. J. Biol. Chem. 264,17428-17431. McCormack, J.G., and Denton, R.M. (1989) The role of C a ions in the regulation of intramitochondrial metabolism and energy production in rat heart. Mol. Cell. Biochem. 89,121-125. 2 +  McGrew, S.G., Wolleben, C , Siegl, P., Inui, M., and Fleischer, S. (1989) Positive cooperativity of ryanodine binding to the calcium release channel of sarcoplasmic reticulum from heart and skeletal muscle. Biochemistry 28,1686-1691. Mcintosh, D.B., and Boyer, P.D. (1983) Adenosine 5'-Triphosphate modulation of catalytic intermediates of calcium ion activated adenosine triphosphatase of sarcoplasmic reticulum subsequent to enzyme phosphorylation. Biochemistry. 22,2867-2875. McMillin, J.B., and Pauly, D.F. (1988) Control of mitochondrial respiration in muscle. Mol. Cell. Biochem. 81,121-129. McPherson, P.S., and Campbell, K.P. (1990) Solubilization and biochemical characterization of the high affinity rH] ryanodine receptor from rabbit brain membranes. J. Biol. Chem. 265,18454-18460. McWhirter, JA1, Gould, G.W., East, J.M. and Lee, A.G. (1987) Characterisation of C a uptake and release by vesicles of skeletal-muscle sarcoplasmic reticulum. Biochem. J. 245,731-738. 2 +  Meissner, G. (1973) ATP and C a binding by the C a protein of sarcoplasmic reticulum. Biochim. Biophys. Acta. 298,906-926. 2 +  2 +  Meissner, G. (1975) Isolation and characterisation of two types of sarcoplasmic reticulum vesicles. Biochim. Biophys. Acta. 389,51-68. Meissner, G. (1984) Adenine nucleotide stimulation of Ca -induced C a release in sarcoplasmic reticulum. J. Biol. Chem. 259,2365-2374. 2+  2 +  250 Meissner, G. (1986) Ryanodine activation and inhibition of the C a release channel of sarcoplasmic reticulum. J. Biol. Chem. 261,6300-6306. z +  Meissner, G. (1986a) Evidence of a role for calmodulin in the regulation of calcium release from skeletal muscle sarcoplasmic reticulum. Biochemistry. 25,244-251. Meissner, G., and Henderson, J.S. (1987) Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent o n C a and is modulated by M g , adenine nucleotide, and calmodulin. J. Biol. Chem. 262,3065-3073. 2 +  Meissner, G., Darling, E., and Eveleth, J. (1986) Kinetics of rapid C a release by sarcoplasmic reticulum. Effects of C a , M g , and Adenine nucleotides. Biochemistry 25,236-244. 2 +  Meissner, G., Rousseau, E., Lai, F.A., L i u , Q-Yy and Anderson K.A. (1988) Biochemical characterization of the C a release channel of skeletal and cardiac sarcoplasmic reticulum. M o l . Cell. Biochem. 82,59-65. 2 +  Meissner, G., Rousseau, Eric, and Lai F.A. (19S9) Structural and functional correlation of the trypsin-digested C a release channel of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 264,1715-1722. 2 +  Mellgren, R.., Lane, R.D., and Kakar, S.S. (1987a) A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity. Biochem. Biophys. Acta. 930,370-377. Mellgren, R.L. (1987) Calcium-dependent proteases: an anzyme system active at cellular membranes? FASEB J. 1,110-115. Mellgren, R.L. (1987) Calcium-dependent proteases: an enzyme system active at cellular membranes? F A S E B J. 1,110-115. Mellgren, R.L. (1988) O n the mechanism of binding of calpastatin, the protein inhibitor of calpains, to biologic membranes. Biochem. Biophys. Res. C o m m u n . 150,170-176. Mellgren, R.L., and Rozanov, C B . (1990)Calpain Il-dependent solubilization of a nuclear protein kinase at micromolar calcium concentrations. Biochem. Biophys. Res. C o m m . 168,589-595. Mellgren, R.L., Lane, R.D., and Kakar, S.S. (1987b) Isolated bovine myocardial sarcolemma and sarcoplasmic reticulum vesicles contain tightly bound calcium-dependent protease inhibitor. Biochem. Biophys. Res. C o m m . 142,1025-1031. Mellgren, R.L., Repetti, A., M u c k , T . C and Easly, J. (1982) Rabbit skeletal muscle calcium-dependent protease requiring millimolar C a . J. Biol. Chem. 257,7203-7209. Mellgren, R.L., Repetti, A., Muck, T . C , and Easly, J. (1982) J. Biol. Chem. 257, 7203-7209.  251  Melzer, W., Rios, E., and Schneider, M.R (1984) Time course of calcium release and removal in skeletal muscle fibers. Biophys. J. 45,637-641. Mermier, P., and Hasselbach, W. (1975) The biphasic active transport of calcium by the fragmented sarcoplasmic reticulum as revealed by the flow dialysis method. Eur. J. Biochem. 64,613-620. Meszaros, L.G., and Ikemoto, N. (1955a) Conformational changes of C a ATPase as early events of C a release from sarcoplasmic reticulum. J. Biol. Chem. 260,16076-16079. 2 +  2 +  Meszaros, L.G., and Ikemoto, N., (1985b) Ruthenium red and caffeine affect the Ca -ATPase of the sarcoplasmic reticulum. Biochem. Biophys. Res. Commun. 127,836-842. 2+  Meszaros, L.G., Brown, K.L., and Ikemoto, N. (1987) 4',6-Diamidino-2phenylindole^ a novel conformational probe of the sarcoplasmic reticulum C a pump, and its effect on C a release. J. Biol. Chem. 262,11553-11558. 2 +  2 +  Meyer, R.A., Sweeney, H.L., and Kushmerick, M.J. (1984) A simple analysis of the "phosphocreatine shuttle". Am. J. Physiol. 246, C365-C377. Michalak, M. (1985) The sarcoplasmic reticulum membrane, in: (ed. Martonosi, A.N.) The Enzymes of Biological Membranes. Plenum Press, New York. 3,115-155. Michalak, M. (1988) Identification of the Ca2+ release activity and ryanodine receptor in sarcoplasmic reticulum membranes during cardiac myogenesis. Biochem. J. 253,631-636. Michalak, M., Campbell, K.P., and MacLennan, D.H. (1980) Localisation of the high-affinity calcium binding protein and an intrinsic glycoprotein in sarcoplasmic reticulum membranes. J. Biol. Chem. 255,1317-1326. Michalak, M., Famulski, K., and Carafoli, E. (1984) J. Biol. Chem. 259,15540-47. Michalak, M., Famulski, K., and Carafoli, E. (1984) J. Biol. Chem. 259, 1554015547. Michalak, M., Famulski, K., and Carafoli, E. (1984) The Ca -pumping ATPase in skeletal muscle sarcolemma. Calmodulin dependence, regulation by cAMP-dependent phosphorylation, and purification. J. Biol. Chem. 259,15540-15547. 2+  Mickelson, J.R., Beaudry, T.M., and Louis, C.F. (1985) Arch. Biochem. Biophys. 242,127-145. Mickelson, J.R., Gallant, E.M., Litterer, L.A., Johnson, K.M., Rempel, W.E., and Louis, K.M. (1988) Abnormal sarcoplasmic reticulum ryanodine receptor in malignant hyperthermia. J. Biol. Chem. 263,9310-9315. Mickelson, J.R., Litterer, L.A..Jacobson, B.A., and Louis, C.F. (1990) Stimulation and inhibition of [ H] ryanodine binding to sarcoplasmic reticulum 3  252  from malignant hyperthermia susceptible pigs. Biophys. 278,251-257.  Arch. Biochem.  Mickelson, J.R., Ross, J.A., Reed, B.K., and Louis, C.F. (1986) Enhanced C a induced release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle. Biochim. et Biophys. Acta. 862,318-328. 2 +  Mignery, G.A., Sudhof, T.C, Takei, K., and Camilli, T.C. (1989) Putative receptor for inositol 1,4,5-triphosphate similar to ryanodine receptor. 342, 192195. Mikos, G.J., and Snow, TLR. (1987) Failure of inositol 1,4,5-triphosphate to elicit or potentiate C a release from isolated skeletal muscle sarcoplasmic reticulum. Biochim. et Biophys. Acta 927,256-260. 2 +  Mitchell, R.D., Simmerman, H.K.B., and Jones, L. (1988) C a Binding effects on protein conformation and protein interactions of canine cardiac calsequestrin. J. Biol. Chem. 263,1276-1381. 2 +  Mitra, R., and Morad, M. (1986) Two types of calcium channels in guinea-pig ventricular myocytes. Proc. Natl. Acad. Sci. USA 83,5340-5344. Miyamoto, H and Kasai, M. (1979) Asymmetric distribution of calcium binding sites of sarcoplasmic reticulum fragments. J. Biochem. 85,765-773 Moews, P.G., and Kretsinger, R.H. (1975) Refinement of the structure of carp  Moller, J.V., Lind, K.E., and Andersen, J.P. (1980) Enzyme kinetics and substrate stabilisation of detergent-solubilized and membraneous (Ca +Mg ) activated ATPase from sarcoplasmic reticulum. Effects of proteinprotein interactions. J. Biol. Chem. 255,1912-1920. 2+  2+  Moncrief, N.D., Kretsinger, R.H., and Goodman, M. (1990) Evolution of EF-hand calcium-modulated proteins. I. relationships based on amino acid sequences. J. Mol. Evol. 30,522-562. Mooibroek, M.J., and Wang, J.H. (1988) Integration of signal-transduction processes. Biochem. Cell Biol. 66,557-566. Morad, M., Goldman, Y.E., and Trentham, D.R. (1983) Nature. 304,635-638. Morii, H., and Tonomura, Y. (1983) The gating behavior of C a channel for Ca -induced C a release in fragmented sarcoplasmic reticulum. J. Biochem. 93,1271-1285. 2 +  2+  2 +  Morii, H., Takisawa, H., and Yamamoto, T. (1985) Inactivation of a Ca -induced Ca release channel from skeletal muscle sarcoplasmic reticulum during active C a transport. J. Biol. Chem. 260,11536-11541. 2+  2 +  2 +  253  Morii, M., Danko, S., Kim, D., and Ikemoto, N. (1986) Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum. J. Biol. Chem. 261,2343-2348. Morton, M.E., Caffrey, J.M., Brown, A.M., and Froehner, S.C. (1988) Monoclonal antibody to the ??? Subunit of the dihydropyridine-binding complex inhibits calcium currents in BC3H1 myocytes. J. Biol. Chem. 263, 613616. Moutin, M., Abramson, J.J., Salama, G., and Dupont, Y. (1989) Rapid A g induced release of C a from sarcoplasmic reticulum vesicles of skeletal muscle: a rapid filtration study. Biochim. et Biophys. Acta 984, 289-292. +  2 +  Moutin, M., and Dupont, Y. (1988) Rapid filtration studies of Ca -induced C a release from skeletal sarcoplasmic reticulum. J. Biol. Chem. 263, 42284235. 2+  2 +  Movsesian, M.A., Nishikawa, M., and Adelstein, R.S. (1984) J. Biol. Chem. 259, 8029-32. Movsesian, M.A., Thomas, A.P., Selak, M., and Williamson, J.R. (1985) Inositol trisphosphate does not release C a from permeabilized cardiac myocytes and sarcoplasmic reticulum. FEBS Lett. 185,328-332. 2 +  Murachi, T. (1983) Calpain and Calpastatin. Trends Biochem. Sci. 8,167-169. Murachi, T. (1988) Intracellular regulatory system involving calpain and calpastatin. Biochem. Int. 18, (2) 263-294. Murachi, T., Tanaka, K., Hatanaka, M. and Murakami, T. (1979) Intracellular Ca -dependent protease (calpain) and its high-molecular-weight endogenous inhibitor (calpastatin) In: Advances in Enzyme Regulation (Wiker, G., ed.) Vol. 19,407-423. Pergammon Press, Oxford. 2+  Nabauer M., Callewaert, G., Cleeman L., and Moran M. (1989) C a current, but not gating charge, regulates C a release in mammalian cardiac myocytes. Science (in press). 2 +  2 +  Nagainis, P.A., Wolfe, F.H., Sathe, S., and Goll, D.E. (1988) Autolysis of the millimolar Ca-requiring form of the Ca-dependent proteinase from chicken skeletal muscle. Biochem. Cell Biol. 66,1023-1031. Nagasaki, K., and Fleischer, S. (1988) Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum. Cell Calcium 9,1-7. Nagasaki, K., and Kasai, M. (1983) Fast release of calcium from sarcoplasmic reticulum vesicles monitored by chlortetracycline fluorescence. J. Biochem. 94,1101-1109. Nagasaki, K., Fleischer, S. (1988) Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum. Cell Calcium 9,1-7.  254  Nagura, S., Kawasaki, T., Taguchi, T., and Kasai, M. (1988) Calcium release from isolated sarcoplasmic reticulum due to 4,4'-dithiodipyridine. J. Biochem. 104,461-465. Nayler, W.G., and Dresel, P.E. (1984) C a Mol. Cell. Cardiol. 10,165-174.  2 +  and the sarcoplasmic reticulum. J.  Nelson, T.E. (1983) Abnormality in calcium release from skeletal sarcoplasmic reticulum or pigs susceptible to malignant hyperthermia. J. Clin. Invest. 72,862-870. Nelson, T.E. (1984) Dantrolene does not block calcium pulse-induced calcium release from a putative calcium channel in sarcoplasmic reticulum from malignant hyperthermia and normal pig muscle. FEBS LETT. 167,123-126. Nelson, T.E., and Nelson, K.E. (1990) Intra-and extraluminal sarcoplasmic reticulum membrane regulatory sites for Ca -induced C a release. FEBS. Lett. 263, 292-294. 2 +  Nesterov, V.P. (1985) Interaction between parameters of sodium and potassium ion distribution and the contractile properties of muscles. J. Evol. Biochem. Physiol. 20, (5). Nesterov, V.P. (1988) Possible mechanisms of Na -induced release of calcium ions from the sarcoplasmic reticulum of skeletal muscle fibres of vertebrates. Fisiol ZH. 34,60-66. +  Neyses, J., Locher, R., Stimpel, M., Streuli, R., and Vetter, W. (1985) Biochem. J. 227,105-12. Neyses, L., Reinlib, L., and Carafoli, E. (1985) J. Biol. Chem. 260,10283-87. Nicoll, D.A., Longoni, S., and Philipson, K.D. (1990) Molecular cloning and functional expression of the cardiac sarcoplemmal N a - C a exchanger. Science 250,563-568. +  2 +  Nosek, T.M., Williams, M.F., Zeigler, ST., and Godt, R.E. (1986) Inositol trisphosphate enhances calcium release in skinned cardiac and skeletal muscle. Am. J. Physiol. 250 (Cell Physiol. 19), C807-C811. Nowycky, M. C , Fox, A.P., and Tsien, R.W. (1985) Three types of neuronal calcium channel with different calcium sensitivity. Nature. 316,440-43. O'Brien, P.J. (1990) Microassay for malignant hyperthermia susceptibility: hypersensitive ligand-gating of the C a channel in muscle sarcoplasmic reticulum causes increased amounts and rates of Carelease. Molec. Cell. Biochem. 93,53-59. 2 +  O'Callahan, C M . , and Hosey, M.M. (1988) Multiple phosphorylation sites in the 165-kilodalton peptide associated with dihydropyridine-sensitive calcium channels. Biochemistry. 27,6071-6077.  255  O'Callahan, C M . , Ptasienski, J., and Hosey, M.M. (1988) Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C.J. Biol. Chem. 263,17342-17349. O'Shea, E.K., Rutkowski, R., and Kim, P.S. (1989) Evidence that the leucine zipper is a coiled coil. Science. 243,538-542. Ogawa, Y.. and Harafuji, H. (1990) Osmolarity-dependent characteristics of [ H]ryanodine binding to sarcoplasmic reticulum. J. Biochem. 107, 894-898. 3  Ohnishi, S.T. (1979) Calcium-induced calcium release from fragmented sarcoplasmic reticulum. J. Biochem. 86,1147-1150. Ohnishi, S.T. (1979) Interaction of metallochromic indicators with calcium sequestering organelles. Biochim. Biophys. Acta. 585,315-319. Ohnishi, S.T., Taylor, S., and Gronert, G.A., (1983) Calcium-induced C a release from sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia: The effects of halothane and dantrolene. FEBS LETT. 161,103-107. 2 +  Ohnishi, T., and Ebashi, S. (1963a) Spectrophotometrical measurement of instantaneous calcium binding of the relaxing factor of muscle. J. Biol. Chem. 54,506-511. Ohnishi, T., and Ebashi, S. (1963b) The velocity of calcium binding of isolated sarcoplasmic reticulum. J. Biol. Chem. 55, No. 6. Okita, J.R., Frojmovic, M.M., Kristopeit, S., Wong, T., and Kunicki, T.J. (1989) Montreal platelet syndrome: A defect in calcium-activated neutral proteinase (Calpain). Blood. 74,715-721. Osguthorpe, D.J., and Robson, B. (1978) Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120,97-120. Otsu, K., Willard, H.F., Khanna, V.K., Zorzato, F., Green, N.M., and MacLennan, D.H. (1990) Molecular cloning of cDNA encoding the C a release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. J. Biol. Chem. 265,13472-13483. 2 +  Otsuka, Y., and Goll, D.E. (1987) Purification of the Ca -dependent proteinase inhibitor from bovine cardiac muscle and its interaction with the millimolar Ca -dependent proteinase. J. Biol. Chem. 262,5839-5851. 2+  2+  Palade, P. (1987a) Drug-induced C a release from isolated sarcoplasmic reticulum. I. Use of pyrophosphate to study caffeine-induced C a release. J. Biol. Chem. 262,6135-6141. 2 +  2 +  Palade, P. (1987b) Drug-induced C a release from isolated sarcoplasmic reticulum. II. Releases involving a Ca -induced C a release channel. J. Biol. Chem. 262,6142-6148. 2 +  2 +  256  Palade, P. (1987c) Drug-induced Ca "t release from isolated sarcoplasmic reticulum. III. Block of Ca -induced C a release by organic polyamines. J. Biol. Chem. 262,6149-6154. z  2+  2 +  Palade, P., Mitchell, R.D., and Fleischer, S. (1983) Spontaneous calcium release from sarcoplasmic reticulum. J. Biol. Chem. 258,8098-8107. Peachey, L.D., and Franzini-Armstrong, C. (1983) Structure and function on membrane systems of skeletal muscle cells. In: Peachey, L.D., and Adrian, R.H. (Eds). Handbook of Physiology. X. Skeletal Muscle. Am. Physiol. Soc: Bethesda. 23-71. Pelzer, D., Grant, A.O., Cavalie, A., Pelzer, S., Sieber, M., Hofmann, F., and Trautwein, W. (1989) Calcium channels reconstituted from the skeletal muscle dihydropyridine receptor protein complex and its peptide subunit in lipid bilayers. Ann. NY Acad Sci 560,138-153. Penniston, J.T. (1982) Ann. N.Y. Acad. Sci. 402,296-303. Pessah, I.N., and Schiedt, M.J. (1990) Early over-expression of low-affinity [ H] ryanodine receptor sites in heavy sarcoplasmic reticulum fraction from dystrophic chicken pectoralis major. Biochim. Biophys. Acta. 1023, 98106. 3  Pessah, I.N., Francini, A.O., Scales, D.J., Waterhouse, A.L., and Casida, J.E. (1986) Calcium-ryanodine receptor complex; Solubilization and partial characterization from skeletal muscle junctional sarcoplasmic reticulum vesicles. J. Biol. Chem. 261,8643-8648. Pessah, I.N., Stambuk, R.A., and Casida, J.E. (1987) Ca -activated ryanodine binding: Mechanisms of sensitivity and intensity modulation by M g , caffeine and adenine nucleotides. Molec. Pharm. 31,232-238. 2+  2 +  Pessah, I.N., Waterhouse, A.L., and Casida, J.E. (1985) The calcium-ryanodine receptor complex of skeletal and cardiac muscle. Biochem. Biophys. Res. Commun. 128,449-456. Philipson, K.D. and, Nishimoto, A.Y. (1980) J. Biol. Chem. 255,6880-82. Philipson, K.D., and Nishimoto, A.Y. (1983) ATP-dependent N a transport in cardiac sarcolemmal vesicles. Biochim. Biophys. Acta. 733,133-141. +  Pick, U., and Karlish, S.J.D. (1982) Regulation of the conformational transition in the Ca-ATPase from sarcoplasmic reticulum by pH, temperature, and calcium ions. J. Biol. Chem. 257,6120-6126. Plank, B., Wyskovsky, W., Hohenegger, Hellmann, G., and Suko, J. (1988) Inhibition of calcium release from skeletal muscle sarcoplasmic reticulum by calmodulin. Biochim. Biophys. Acta. 938,79-88. Pontremoli, S., and Melloni, E. (1986) Extralysosomal protein degradation. Ann. Rev. Biochem. 55,455-81.  257  Porter, KR., and Palade, G.E. (1957) Studies on the endoplasmic reticulum. HI. Its form and distribution in striated muscle cells. J. Biol. Chem. 3,269-300. Prabhu, S.D., and Salama, G. (1990) The heavy metal ions A g and H g trigger calcium release from cardiac sarcoplasmic reticulum. Arch. Biochem. Biophys. 277,47-55. 2 +  2 +  Rao, J.K.M., and Argos, P. (1986) A conformational preference parameter to redict helices in integral membrane proteins. Biochim. Biophys. Acta. 59,197-214.  g  Rardon, D.P., Cefali, D.C., Mitchell, R.D., Seiler, S.M., Hathaway, D.R., and Jones, L.R. (1990) Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II effects on the C a release channel. Circ. Res. 67,84-96. 2 +  Raynes, D.G., Devine, C.E., and Sutherland, C.L. (1975) Freeze-fracture studies of membrane systems in vertebrate muscle. I. Striated muscle. J. Ultrastruct. Res. 50,306-320. Rechsteiner, M. (1987) Regulation of enzyme levels by proteolysis: the role of PEST regions. Adv. Rev. Cell. Biol. 3,1-30. Reeves, J., and Sutko, J.L. (1983) Competitive interactions of sodium and calcium with the sodium-calcium exchange system of cardiac sarcolemmal vesicles. J. Biol. Chem. 258,3178-3182. Reeves, J.P. (1985) The sarcolemmal sodium-calcium exchange system. Curr. Top. Membr.. 25,77-127. Reeves, J.P., and Sutko, J.L. (1980) Sodium-calcium exchange activity generates a current in cardiac membrane vesicles. Science. 208,1461-1464. Reithmeier, R.A.F., Ohnishi, M., Carpenter, M.R., Slupsky, J.R., Gounden, K., Fliegel, L., Khanna, V.K., ana MacLennan, D.H. (1987) Calsequestrin in Calcium-binding proteins in health and disease (Norman, A.W., Vanaman, T.C, and Means, A.R., Eds.). Harcourt Brace Jovanovich, San Diego, 62-71. Reuter, H., and Seitz, N. (1968) The dependence of calcium efflux from cardiac muscle on temperature ana external ion compostion. J. Physiol. (Lond.) 195,451-470. Ringer, S.A. (1883) A further contribution regarding the influence of difference constituents of the blood for the contractions of the heart. J. Physiol. (Lond.) 4,29-42. Rios, E., and Brum G. (1987) Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle. Nature. 325, 717720. Rios, E., and Schneider, M.F. (1981) Stoichiometry of the reactions of calcium with the metallochromic indicator dyes antipyrylazo III and arsenazo III. Biophys. J. 36,607-621.  258  Rodgers, G.M., Cong, J., Goll, D.E., and Kane, W.H. (1987) Activation of coagulation factor V by calcium-dependen proteinase. Biochim. Biophys. Acta. 929,263-270. Rogers, S., Wells, R. and Reichsteiner, M (1986) Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science. 234, 364368. Rohrkasten, A., Meyer, H.E., Nastainczyk, W., Sieber, Manfred, and Hofmann, F. (11988) cAMP-dependent protein kinase rapidly phosphorylates serine-687 of the skeletal muscle receptor for calcium channel blockers. J. Biol. Chem. 263,15325-15329. Ross, C.A., Meldolesi, J., Milner, T.A., Satoh, T., Supattapone, S., and Snyder, S.H. (1989) Inositol 1,4,5-triphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons. Nature 339, 468-470. Rousseau, E., LaDine, J., Liu, Q., and Meissner, G. (1988) Activation of the c release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds. Arch. Biochem. Biophys. 267,75-86. Rousseau, E., Smith, T.S., Henderson, J.S., and Meissner,G. (1986) Single channel and ^ Ca " flux measurements of the cardiac sarcoplasmic reticulum calcium channel. Biophys. J. 50,1009-1014. 4  24  Rubin, R.P. (1970) The role of calcium in the release of neurotransmitter substances and hormones. Pharmacol. Rev. 22,389-428. Rubtsov, A.M., and Murphy, A.J. (1988) Caffeine interaction with the c-release channels of heavy sarcoplasmic reticulum. Evidence that 170 kD cbinding protein is a caffeine receptor of the c-channels. Biochem. Biophys. Res. Commun. 154,462-468. Rubtsov, A.M., Smirnova, M.B., and Boldyrev, A.A. (1988) Interaction of different nucleotides with c-release channels from heavy sarcoplasmic reticulum. Biochemistry International 17,629-636. Ruoslahti, E., and Pierschbacher, M.D. (1986) Arg-Gly-Asp: A versatile cell recognition signal. Cell. 44,517-518. Saito, A., Seiler, S., Chu, A., and Fleischer, S., (1984) Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle. J Cell Biol. 99,875-885. Saito, A., Wang, C.T., and Fleischer, S. (1978) Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid. J. Cell. Biol. 79,601-616. Salama, G., and Abramson, J. (1984) Silver ions trigger C a release by acting at the apparent physiological release site in sarcoplasmic reticulum. J. Biol. Chem. 259,13363-13369. 2 +  259  Salviati, G., and Volpe, P. (1988) C a release from sarcoplasmic reticulum of skinned fast- and slow-twitch muscle fibers. Am. J. Physiol. 254 (Cell Physiol. 23): C459-465. 2 +  Samis, J.A., and Elce, J.S. (1989) Immunogold electron-microscopic localization of calpain I in human erythrocytes. Thromb. Haem. 61,250-253. Scarpa, A., Brinley, FJ. Jr., and Dubyak, G. (1978) Antipyrylazo III, a "Middle Range" C a * metallochromic indicator. Biochemistry. 17,1378-1386. 2  Schneider, M.F., and Simon, B.J. (1988) Inactivation of calcium release from the sarcoplasmic reticulum in frog skeletal muscle J. Physiol. 405,727-745. Schwartz, I.M., McCleskey, E.W., and Aimers, W. (1985) Nature. 314,747-751. Schwartz, L.M., McClesky, E.W., and Aimers, W. (1985) Dihydropyridine receptors in muscle are voltage-dependent but most are not functional calcium channels. Nature. 314,747-751. Scott, B.R., Simmerman, H.K.B., Collins, J.H., Ginard, B.N. and Jones, L.R. (1988) Complete amino acid sequence of canine cardiac calsequestrin deduced by cDNA cloning. J. Biol. Chem. 263,8958-8964. Seiler, S., Wegeners, A.D., Whang, D.D., Hathaway, D.R., and Jones, L.R. (1984) "eh molecular weight proteins in cardiac and skeletal muscle High  Sembrowich, W.L., Johnson, D., Wang, E., and Hutchinson, T.E. (1983) Electron microprobe analysis of fatigued fast- and slow-twitch muscle. Int. Series Sport Sci.: Biochem. Exercise. 13,571-576. Shigekawa, M., and Dougherty, J.P. (1978) Reaction mechanism of C a dependent ATP hydrolysis by skeletal muscle sarcoplasmic reticulum in the absence of added alkali metal salts. HI. Sequential occurrence of ADP-sensitive and ADP-insensitive phosphoenzymes. J. Biol. Chem. 253,1458-1464. 2 +  Shigekawa, M., Wakabayashi, S., and Nakamura, H. (1983) Reaction mechanism of Ca -dependent adenosine triphosphatase of sarcoplasmic reticulum. J. Biol. Chem. 258,8698-8707. 2+  Shoshan-Barmatz, V. (1986) Chemical modification of sarcoplasmic reticulum. Biochem. J. 240,509-517. Shoshan-Barmatz, V. (1988) Activation of C a reticulum. J. Memb. Biol. 103,67-77.  2 +  release in isolated sarcoplasmic  Shoshan-Barmatz, V., and Zarka, A. (1988) Trypsin destruction of the high affinity ryanodine binding sites of the junctional sarcoplasmic reticulum. J. Biol. Chem. 263,16772-16779.  260  Shoshan-Barmatz, V., Ouziel, N., and Chipman, D.M. (1987) Tryptic digestion of sarcoplasmic reticulum inhibits Car accumulation oy action on a membrane component other than the Ca - ATPase. J. Biol. Chem. 262, 11559-11564. +  Smallwood, I.I., Gugi, B., and Rasmussen, H. (1988) Regulation of Erythrocyte C a pump activity by protein kinase C. J. Biol. Chem. 263,2195-2202. 2 +  Smith, j.S., Coronado, R., and Meissner, G. (1985) Sarcoplasmic reticulum contains adenine nucleotide-activated calcium channels. Nature. 316, 446-449. Smith, J.S., Imagawa, T., Ma, J., Fill, M., Campbell, K.P., and Coronado, R. (1988) Purified ryanodine receptor from rabbit skeletal muscle is the calciumrelease channel of sarcoplasmic reticulum. J. Gen. Physiol. 92,1-26. Smith, J.S., Rousseau, E., and Meissner, G. (1989) Calmodulin modulation of single sarcoplasmic reticulum Ca -release channels from cardiac and skeletal muscle. Circ. Res. 64,352-359. 2+  Smith, O.L.K., Wong, C.Y., and Gelfand, R.A. (1989) Skeletal muscle proteolysis in rats with acute streptozocin-induced diabetes. Diabetes. 38, 11171122. Soler,  R, Fernandez-Belda, F., and Gomez-Fernandez, J.C. (1989) Characterization of the tetraphenylboron-induced calcium release from sarcoplasmic reticulum. Eur. J. Biochem.181,513-518.  Somlyo, A.V. (1979) Bridging structures spanning the functional gap at the triad of skeletal muscle. J. Cell. Biol. 80,743-750. Somlyo, A.V., Gonzales-Serratos, H.G., Shuman, H., McClellan, G., and Somlyo, A.P. (1981) Calcium release and ionic changes in the sarcoplasmic reticulum of tetanised muscle: an electron probe study. J. Cell. Biol. 90, 577-594. Somlyo, A.V., Gonzalez-Serratos, H., McClellan, G., Shuman, H., Borrero, L.M., and Somlyo, A.P. (1978) Electron microprobe analysis of the sarcoplasmic reticulum and vacuolated t-tubule system of fatigued frog muscles. Ann. N.Y. Acad. Sci. 307,232-234. Spalla, M., Tsang, W., Kuo, T.H., Giacomelli, F., and Wiener, J. (1985) Purification and characterization of two distinct Ca -activated proteinases from hearts of hypertensive rats. Biochim. Biophys. Acta. 830,258-266. 2+  Sperelakis, N., and Wahler, G.M. (1988) Regulation of C a influx in myocardial cells by beta adrenergic receptors, cyclic nucleotides, and phosphorylation. Mol. Cell. Biochem. 82,19-28. 2 +  Squier, T.C, Bigelow, D.J., Fernandez-Belda, F , deMeis, L., and Inesi, G. (1990) J. Biol. Chem. 265,13713-13720.  261  Stewart, P.S., MacLennan, D.H., and Shamoo, A.E. (1976) Isolation and characterisation of tryptic fragments of adenosine triphosphatase of sarcoplasmic reticulum. J. Biol. Chem. 251,712-719. Stuart, J., and Abramson, J.J. (1988) Adenine nucleotides stimulate oxidationinduced calcium efflux from sarcoplasmic reticulum vesicles. Arch. Biochem. Biophys. 264,125-134. Su, J.Y., and Hasselbach. W. (1984) Caffeine-induced calcium release from isolated sarcoplasmic reticulum or rabbit skeletal muscle. Pflugers Arch. 400,14-21. Suarez-Isla, B.A., Irribarra, V., Oberhauser, A., Larralde, L., Bull, R., Hidalgo, C , and Jaimovich, E. (1988) Inositol (l,4,5)-trisphosphate activites a calcium channel in isolated sarcoplasmic reticulum membranes. Biophys. J. 54, 737-741. Sumbilla, C , and Inesi, G. (1987) Rapid filtration measurements of C a from cisternal sarcoplasmic reticulum vesicles. 210,31-36.  2 +  release  Sutko, J.L., and Kenyon, J.L. (1983) Ryanodine modification of cardiac muscle responses to potassium-free solutions - Evidence for inhibition of sarcoplasmic reticulum calcium release. J. Gen. Physiol. 82,385-404. Sutko, J.L., Ito, K., and Kenyon, J.L. (1985) Ryanodine: a modifier of sarcoplasmic reticulum calcium release in striated muscle. Fed. Proc. 44,2984-2988. Suzuki, K., and Ohno, S. (1990) Calcium activated neutral protease-structurefunction relationship and functional implications. Cell structure and function. 15,1-6. Suzuki, K., Imajoh, S., Emori, Y., Kawasaki, H., Minami, Y. and Ohno, S. (1987) Calcium-activated neutral protease and its endogenous inhibitor. FEBS lett. 220,271-277. Suzuki, K., Imajoh, S., Emori, Y., Kawasaki, H., Minami, Y., and Ohno, S. (1987) Calcium-activated neutral protease and its endogenous inhibitor. FEBS. Lett. 220,271-277. Suzuki, K., Imajoh, S., Emori, Y., Kawasaki, H., Minami, Y., and Ohno, S. (1988) Regulation of activity of calcium activated neutral protease, in (ed. Weber, G.) Advances in enzyme regulation. Pergamon Press, Oxford 27,153-167. Suzuki, K., Kawashima, S., and Imahori, K. (1984) Structure and function of Ca -activated protease, pp. 213-226 in Calcium Regulation in Biological Systems (S. Ebashi, M. Endo, K. Kakiuchi and Y. Nishizuka, eds.), Academic Press, New York. 2+  Suzuki, K., Ohno, S., Emori, Y., Imajoh, S., and Kawasaki, H. (1987) Calciumactivated neutral protease (CANP) and its biological and medical implications. Prog. Clin. Biochem. Med. 5  262  Suzuki, K., Tsuji, S., Kubota, S., Kimura, Y., and Imahori, K. (1981) J. Biochem. (Tokyo). 90,275-278. Tada, M., Kirchberger, M.A., Li, H.C., and Katz, A.M. (1975) J. Biol. Chem. 250, 2640-46. Tada, M., Yamamoto, T , and Tonomura, Y. (1978) Moecular mechanism of active calcium transport by sarcoplasmic reticulum. Physiol. Rev. 58,1-79. Takakuwa, Y., and Kanazawa, T. (1982) Role of Mg + in the C a - C a exchange mediated by the membrane bound ( C a , Mg )-ATPase of sarcoplasmic reticulum vesicles. J. Biol. Chem. 257,10770-10775. 2  2 +  2 +  2+  Takamatsu, T., and Wier, W.G. (1990) Calcium waves in mammalian heart: quantification of origin, magnitude, waveform, and velocity. FASEB. 4, 1519-1525. Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., and Numa, S. (1989) Primary structure and expression from complimentary DNA of skeletal muscle ryanodine receptor. Nature 339,439-445. Takisawa, H., Makinose, M. (1983) Occlusion of calcium in the ADP-sensitive phosphoenzyme of the adenosine triphosphatase of sarcoplasmic reticulum. J. Biol. Chem. 258,2986-2992. Tan, F.C., Goll, D.E., and Otsuka, Y. (1988) Some properties of the millimolar Ca-dependent proteinase from bovine cardiac muscle. J. Mol Cell Cardiol. 20,983-997. Tanabe, T., Beam, K.G., Adams, B.A. Niidome, Tetsuhiro, and Numa, S. (1990a) Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling. Nature. 346,567-569. Tanabe, T., Mikami, A., Numa, S., and Beam, K.G. (1990b) Cardiac-type excitation-contraction coupling in dysgenic skeletal muscle injected with cardiac dihydropyridine receptor cDNA. Nature. 344,451-453. Tanaka, M., Ozawa, T., Maurer, A. (1986) Apparent cooperativity of C a binding associated with crystallization of Ca-binding protein from sarcoplasmic reticulum. Arch. Biochem. Biophys. 251,369-378. 2 +  Tate, C.A., and Taffet, G.E. (1989) The regulatory role of calcium in striated muscle. Med. Sci. Sp. Exer. 21,393-398. Tate, C.A., Van Winkle, W.B., and Entman, M.L. (1980) Time-dependent resistence to alkaline pH of oxalate-supported calcium uptake by sarcoplasmic reticulum. Life Sciences 27,1453-1464. Tatsumi, S., Suzuno, M., Taguchi, T., and Kasai, M. (1988) Effects of silver ion on the calcium-induced calcium release channel in isolated sarcoplasmic reticulum. J. Biochem.104,279-284.  263  Taylor, C.W., and Merritt, J.E. (1986) Trends Pharmacol. Sci. 7,238-42. Teruel, J.A. and Inesi, G. (1988) Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adeosine triphosphatase. Biochemistry. 27,5885-5890. Thorley-Lawson, D.A., and Green, N.M. (1973) Studies on the location and orientation of proteins in the sarcoplasmic reticulum. Eur. J. Biochem. 40,403-414. Tornheim, K. (1988) Fructose 2, 6-bisphosphate and glycolytic oscillations in skeletal muscle extracts. J. Biol. Chem. 263,2619-2624. Trimm, J.L., Salama, G., and Abramson, J.J (1986) Sulfhydryl oxidation induces rapid calcium release from sarcoplasmic reticulum vesicles. J. Biol. Chem. 261,16092-16098. Trimm, J.L., Salama, G., and Abramson, J.J. (1988) Limited tryptic modification stimulates activation of C a release from isolated sarcoplasmic reticulum vesicles. J. Biol. Chem. 263,17443-17451. 2 +  Vanderkooi, J.M., Ierokomos, A., Nakamura, H., ancL Martonosi, A. (1977) Fluorescence energy transfer between C a transport ATPase molecules in artificial membranes. Biochemistry. 16,1262-1267. 2 +  Vasington, F.D., and Murphy, J. (1961) Active binding of calcium by mitochondria. Fed. Proc. 20,146. Vasington, F.D., and Murphy, J.V. C a uptake by rat kidney mitochondria and its dependence on respiration and phosphorylation. J. Biol. Chem. 237, 2670-2677. + +  Vergara, J., Tsien, R.Y., and Delay, M. (1985) Inositol 1,4,5,-trisphosphate: a ossible chemical link in excitation-contraction coupling in muscle, roc. Natl. Acad. Sci. U.S.A. 82,6352-6356.  P  Vianna, A.L. (1975) Interaction of calcium and magnesium in activiting and inhibiting the nucleoside triphosphatase of sarcoplasmic reticulum vesicles. Biochim. Biophys. Acta. 410,389-406. Virk. S.S., Kirk, C.J., and Shears, S.B. (1985) Biochem. J. 226,741-48. Vittone, L., Mundina, C , Chiappe de Cingolani, G., and Mattiazzi, A. (1990) Am. J. Physiol. 258 (Heart Circ. Physiol. 27), H318-H325. Volpe, P., Salviati, G., De Virgilio, R, and Pozzan, T. (1985) Inositol 1,4,5,trisphosphate induces calcium release from sarcoplasmic reticulum of skeletal muscle. Nature. 316,347-249. Wagenknecht, T., Grassucci, R., Frank, J., Saito, A., Inui, M., and Heischer, S. (1989) Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum. Nature 338,167-170.  264  Waisman, D.m., Salimah, B.p., and Anderson, M.J. (1985) J. Biol. Chem. 260,16521660. Wakabyashi, S., and Shigekawa, M. (1984) Role of divalent cation bound to phosphenzyme intermediate of sarcoplasmic reticulum ATPase. J. Biol. Chem. 259,4427-4436. Walker, J.W., Somlyo, A.B., Goldman, Y.E., Somlyo, A.V., and Trentham, D.R. (1987) Kinetics of smooth and skeletal muscle avtivation by laser pulse photolysis of caged inositol 1,4,5,-trisphosphate. Nature. 327,249-252. Walker, J.W., Somlyo, A.V., Goldman, Y.E., Somlyo, A.P., and Trentham. D.R. (1987) Kinetics of smooth and skeletal muscle activation by laser pulse photolysis of caged inositol 1,4,5-trisphosphate. Nature (Lond.) 327, 249-252. Wang, K.K.W., Roufogalis, B.D., and Villalobo, A. (1988) Further characterization of calpain-mexliated proteolysis of the human erythrocyte plasma membrane Ca -ATPase. Arch. Biochem. and Biophys. 267,317-327. 2+  Wang, K.K.W., Villalobo, A., and Roufogalis, B.D. (1988) Activation of the C a ATPase of human erythrocyte membrane by an endogenous C a dependent neutral protease. Arch. Biochem. Biophy. 260,696-704. 2 +  2 +  Wang, K.K.W., Villalobo, A., and Roufogalis, B.D. (1989) Review Article Calmodulin-binding proteins as calpain substrates. Biochem. J. 262, 693-706. Watanabe, N., Vande Woude, G.F., Ikawa, Y., and Sagata, N. (1989) Specific proteolysis of the c=mos proto-oncogene product by calpain on fertilization of Xenopus eggs. Nature. 342,505-517. Watras, J. (1985) Effects of M g on calcium accumulation by two fractions of sarcoplasmic reticulum from rabbit skeletal muscle. Biochim. et Biophys. Acta 812,333-344. 2 +  Weber, A. (1976) Symp. Soc. Exp. Biol. 30,445-000. Weber, A., Herz, R., and Reiss, I. (1963) On the mechanism of the relaxing effect of fragmented sarcoplasmic reticulum. J. Gen. Physiol. 46,679-702. Weber, A., Herz, R., and Reiss, I. (1966) Study of the kinetics of calcium transport by isolated fragmented sarcoplasmic reticulum. Biochem. Z. 345, 329369. Weber, A., Herz, R., and Reiss, I. (1966) Study of the kinetics of calcium transport by isolated fragmented sarcoplasmic reticulum. Biochem. Z. 345, 329369. Weber, K., and Osborn, M. (1969) The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244,4406-4412.  265  Wier, W.G., Yue, D.T., and Marban, E. (1985) Effects of ryanodine on intracellular C a transients in mammalian cardiac muscle. Fed. Proc. 44, 29892993. 2 +  Williams, R.J.P (1990) Calcium and cell steady states. In: (eds. Pochet, R., Lawson, D.E.M., and Heizmann, C.W.) Calcium Binding Proteins in Normal and Transformed Cells. Plenum Press, London. Williams, R.J.P. (1976) Symp. Soc. Exp. Biol. 30,1-17. Williams, R.W., and Beeler, T.J. (1986) Secondary structure of calsequestrin in solutions and in crystals as determined by raman spectroscopy. J. Biol. Chem. 261,12408-12413. Wolfe, F.H., Sathe, S.K., Goll, D.E., Kleese, W.C, Edmunds, T., and Duperret, S.M. (1989) Chicken skeletal muscle has three Ca -dependent proteinases. Biochim. Biophys. Acta. 998,236-250. 2+  Yamada, S., and Tonomura, Y. (1972) Phosphorylation of the C a - M g ) dependent ATPase of the sarcoplasmic reticulum coupled with cation translocation. J. Biochem. 71,1101-1104. 2+  2+  Yamada, S., Sumida, M., and Tonomura, Y. (1972) Reaction mechanism of the Ca -dependent ATPase of sarcoplasmic reticulum from skeletal muscle.Vin. Molecular mechanism of the conversion of osmotic energy to chemical energy in the sarcoplasmic reticulum. J. Biochem. 72,153/1548. 2+  Yamamoto, T., Takisawa, H., and Tonomura, Y. (1979) Reaction mechanisms for ATP hydrolysis and synthesis in the sarcoplasmic reticulum. Curr. Top. Bioenerg. 9,179-236. Yatani, A., Codina, J., Imoto, Y., Reeves, J.P., Birnabaumer, L., and Brown, A.M. (1987) Direct regulation of mammalian cardiac calcium channels by a G. protein. Science Wash. DC. 238,1288-1292. Yatani, A., Imoto, Y., Codina, J., Hamilton, S.L., Brown, A.M., and Birnbaumer, L. (1988) The stimulatory G protein of adenyl cyclase, Go, also stimulates dihydropyridine-sensitive C a channels. J. Biol. Chem. 263, 98879895. 2 +  Yazaki, P.J., Salvatori, S., Sabbadini, R.A., and Dahms, A.S. (1990) Calsequestrin, an intracellular calcium-binding protein of skeletal muscle sarcoplasmic reticulum, is homologous to aspartactin, a putative laminin-binding protein of the extracellular matrix. Biochem. Biophys. Res. Commun. 166,898-903. Yoo, S.H., and Albanesi, J.P. (1990) Inositol 1, 4, 5-trisphosphate-triggered C a release from bovine adrenal medullary secretory vesicles. J. Biol. Chem. 265,12446-12448.  2 +  Yoshihara, Y., Ueda, H., Imajoh, S., Takagi, H., and Satoh, M. (1988) Calciumactivated neutral protease (CANP). a putative processing enzyme of  266  the neuropeptide, kyotorphin, in the brain. Bioch. Biophys. Res. Comm. 155,546-553. Zaidi, N.R, Lagenaur, C.F., Abramson^ J.J., Pessah, I., and Salama, G. (1989) Reactive disulfides trigger C a * release from sarcoplasmic reticulum via oxidation reaction. J. Biol. Chem. 264,21725-21736. 2  Zaidi, S.I.M., and Narahara, H.T. (1989) Degradation of skeletal muscle plasma membrane proteins by calpain. J. Memb. Biol. 110,209-216. Zarain-Herzberg, A., Fliegel, L. and MacLennan, D.H. (1988) Structure of the rabbit fast-twitch skeletal muscle calsequestrin gene. J. Biol. Chem. 263, 4807-4812.  Zorato, F., Salviati, G., Facchinetti, T., and Volpe, P. (1985) Doxorubicin induces calcium release from terminal cisternae of skeletal muscle - A study on isolated sarcoplasmic reticulum and chemically skinned fibers. J. Biol. Chem. 260,7349-7355. Zorzato, R, Chu, A., and Volpe, P. (1989) Antibodies to junctional sarcoplasmic reticulum proteins: probes for the Ca -release channel. Biochem. J. 261,863-870. 2+  Zorzato, R, Fujii, J., Otsu, K., Phillips, M., Green, N.M., Lai, F.A., Meissner, G., and MacLennan, D.H. (1990) Molecular ila cloning of cDNA encoding human and rabbit forms of the C a release channel lannei (ryanodine tryc jcept receptor) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 265, 2244-2256. 2 +  Zorzato, R, Volpe, P., Damiani, E., Quaglino, D. Jr., and Margreth, A. (1989) Terminal cisternae of deneryated rabbit skeletal muscle: alterations of functional properties of C a release channels. Am. J. Physiol. 257 (Cell Physiol. 26): C504-C511. 2 +  

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
United States 60 0
China 6 9
Canada 3 0
India 3 0
Czech Republic 2 0
Japan 2 0
City Views Downloads
Washington 37 0
Ashburn 10 0
Unknown 6 19
Shenzhen 5 9
Phoenix 3 0
Seattle 3 0
Tokyo 2 0
Mountain View 2 0
Wilmington 2 0
Jalalpur 1 0
Bhandup 1 0
Etobicoke 1 0
Redmond 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0076804/manifest

Comment

Related Items