Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Characterization of voltage-dependent calcium channels in T lymphocytes Kotturi, Maya Fay 2005

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2005-994937.pdf [ 19.01MB ]
Metadata
JSON: 831-1.0092362.json
JSON-LD: 831-1.0092362-ld.json
RDF/XML (Pretty): 831-1.0092362-rdf.xml
RDF/JSON: 831-1.0092362-rdf.json
Turtle: 831-1.0092362-turtle.txt
N-Triples: 831-1.0092362-rdf-ntriples.txt
Original Record: 831-1.0092362-source.json
Full Text
831-1.0092362-fulltext.txt
Citation
831-1.0092362.ris

Full Text

\  CHARACTERIZATION OF VOLTAGE-DEPENDENT CALCIUM CHANNELS INJT_ LYMPHOCYTES by Maya Fay Kotturi B.Sc, University of Victoria, 1996 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology; The Biomedical Research Centre; Biotechnology Laboratory) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 2004 © Maya Fay Kotturi, 2004  ABSTRACT  In T lymphocytes, sustained calcium (Ca ) influx through Ca  channels  localized in the plasma membrane is critical for T cell activation and proliferation. Previous studies intimated that L-type voltage-dependent C a role in C a  2 +  2 +  channels (VDCCs) play a  mobilization during T lymphocyte activation. However, the function of  VDCCs in these non-excitable cells is still poorly understood. In order to explore the role of L-type VDCCs in T lymphocytes, molecular and pharmacological analyses were employed to identify L-type VDCCs, and to define their contribution to C a  2 +  influx  pathways in T lymphocytes. In human T lymphocytes, two novel splice isoforms of the channel-forming  OCIF-  subunit of retinal L-type V D C C were identified. It was found that both of the ociF-subunit splice isoforms contain unique structural features, distinct from the 0Ci F-subunit originally isolated from human retina that may render these channel variants insensitive to changes in membrane depolarization. Through cDNA cloning with a human spleen library, the complete cDNA sequence of one of the 0Ci F-subunit splice variants was isolated for future functional studies. The mRNA expression of the aiF-subunit splice isoforms appeared to be regulated by T cell receptor (TCR)-induced activation in the human Jurkat T cell leukemia line, and to a lesser extent in human peripheral blood T lymphocytes (PBTs). In addition, the 0CiF-subunit protein was detected in Jurkat T cells and human PBTs. To further investigate the contribution of C a  2+  influx through L-type VDCCs, the  effects of the 1,4-dihydropyridine (DHP) L-type Ca channel agonist, (+/-) Bay K 8644, 2+  and antagonist, nifedipine, on Jurkat T cells, human PBTs and mouse splenocytes were  ii  assessed. It was found that treatment of T lymphocytes with (+/-) Bay K 8644 increased intracellular C a  2 +  and induced the activation of phospho-extracellular regulated kinase 1/2  (Erkl/2), whereas nifedipine blocked C a  2+  influx, the activity of Erkl/2 and nuclear  factor of activated T cells, interleukin-2 (IL-2) production and IL-2 receptor expression. Nifedipine also significantly suppressed splenocyte proliferation in an in vitro mixed lymphocyte reaction. Since patients receive nifedipine for the treatment of cardiovascular diseases and other  clinical  disorders,  it  was  important  to  further  examine  the  potential  immunosuppressive effects associated with nifedipine administration. It was found that nifedipine inhibited the proliferation of male antigen (H-Y)-specific TCR-transgenic C D 8 T cells in transplanted male mice in vivo. Finally, a study exploring the effects of +  nifedipine administration on circulating T lymphocytes in renal disease patients showed T cells from renal patients secreted less IL-2 compared to T cells isolated from a healthy individual. This suggested that nifedipine therapy may act as an immunosuppressant. The results demonstrate that alternative splicing of the human retina otiF-subunit has led to the expression  of structurally unique aiF-subunits in T lymphocytes. 2_|_  Furthermore, the pharmacological studies indicate that L-type Ca significant role in the C a  2+  channels play a  influx pathways mediating T lymphocyte activation and  proliferation in vitro and in vivo.  in  T A B L E OF CONTENTS  ABSTRACT ii T A B L E OF CONTENTS iv LIST OF FIGURES vii LIST OF TABLES x LIST OF ABBREVIATIONS xi ACKNOWLEDGEMENTS xiv DEDICATION xv CHAPTER 1: GENERAL INTRODUCTION 1 1.1 Role of Calcium in Regulating Distinct Cellular Processes 1 1.2 T Cell Activation 4 1.2.1 Early Signaling Events Proximal to the T Cell Receptor 4 1.2.2 Immunological Synapse Formation and Sustained T Cell Signaling 9 1.2.3 Calcium's Role as an Intracellular Secondary Messenger 12 1.2.4 Calcium-Dependent Transcriptional Regulation 14 1.3 Intracellular Calcium Release and Regulation in T Cells 19 1.3.1 IP3 Receptor Calcium Channels 19 1.3.2 Ryanodine Receptor Calcium Channels 23 1.4 Communication between Calcium Store Release and Calcium Influx Pathways 26 1.4.1 Diffusible Messenger Model 27 1.4.2 Conformational Coupling Model 29 1.4.3 Secretion Model 31 1.5 Calcium Influx Pathways in T Cells 32 1.5.1 IP3 Receptor Calcium Channels 33 1.5.2 Transient Receptor Potential Calcium Channels 34 1.5.3 Voltage-Dependent Calcium Channels 39 CHAPTER 2: MATERIALS AND METHODS 45 2.1 Cell Lines and Culture Conditions 45 2.2 Isolation and Culture of Human Peripheral Blood T Lymphocytes 45 2.3 Cell Separations 46 2.4 Nested RT-PCR of Odp-Subunit and DNA Sequencing 48 2.5 Cloning of (XiF-Subunit cDNA from Human Retina and Spleen Libraries 50 2.6 Production of Carboxyl-Terminal FLAG-tagged (XiF-Subunit 52 2.7 Immunofluorescence Staining 54 2.8 Immunoprecipitation Analysis of (XiF-Subunit 54 2.9 Flow Cytometry Analysis of (Xip-Subunit 56 2.10 Real-Time PCR 56 2.11 Construction of Plasmids for Stable siRNA Expression 58 2.12 Generation of Stable Cell Lines Expressing siRNA 61 2.13 Immunoprecipitation Analysis of p53 62 2.14 Measurement of Intracellular Calcium Levels 63 2.15 Immunoblot Analysis of Phospho-p44/p42 MAP Kinase 64  iv  2.16 NFAT-Luciferase Assay 65 2.17 IL-2 Assay 65 2.18 Flow Cytometry Analysis of IL-2R and CD69 68 2.19 Mice 68 2.20 Mixed Lymphocyte Reaction 68 2.21 In Vivo Proliferation Assay 69 2.22 Statistical Analysis 71 CHAPTER 3: MOLECULAR IDENTIFICATION OF L-TYPE V O L T A G E DEPENDENT CALCIUM CHANNELS IN T LYMPHOCYTES 72 3.1 Introduction 72 3.2 Results 76 3.2.1 L-Type CtiF-Subunit mRNA Transcript is Expressed in T Cells 76 3.2.2 Identification of Novel Alternative Splice Variants of the an-Subunit in T Cells 78 3.2.3 Carboxyl-Terminus of the Retina (XiF-Subunit Resides in the Cytoplasm 84 3.2.4 Alternative Splice Variants of CtiF-Subunit are Differentially Expressed in Human Leukocytes 87 3.2.5 0CiF-Subunit Protein is Expressed in Human T Cells 88 3.2.6 Comparison of GCiF-Subunit Alternative Splice Variant mRNA Expression in Resting and Activated T Cells 93 3.2.7 Comparison of CtiF-Subunit Protein Expression in Resting and Activated Jurkat T Cells 100 3.3 Discussion 102 CHAPTER 4: DEFINING T H E CONTRIBUTION OF L-TYPE CALCIUM CHANNELS TO CALCIUM INFLUX DURING T L Y M P H O C Y T E ACTIVATION IN VITRO  113  4.1 Introduction 113 4.2 Results 119 4.2.1 Examination of the Effects of siRNA on (XiF-Subunit Expression in Jurkat T Cells 119 4.2.2 Induction of Calcium Influx in Jurkat T Cells and Human PBTs by (+/-) Bay K 8644 121 4.2.3 Nifedipine Inhibits Anti-CD3 Induced Calcium Influx in Jurkat T Cells and Human PBTs 124 4.2.4 (+/-) Bay K 8644 and Nifedipine Modulate Phospho-p44/p42 MAP Kinase Activation in Jurkat T Cells and Human PBTs 127 4.2.5 Nifedipine Blocks NFAT-Transcriptional Activity in Jurkat T Cells 129 4.2.6 IL-2 Production and IL-2R Expression is Inhibited by Nifedipine in Jurkat T Cells and Human PBTs 131 4.2.7 Nifedipine Suppresses Splenocyte Proliferation 138 4.3 Discussion 138 CHAPTER 5: DETERMINING T H E ROLE OF L-TYPE CALCIUM CHANNELS IN T LYMPHOCYTES IN VIVO  5.1 Introduction 5.2 Results 5.2.1 Nifedipine Inhibits T Cell Proliferation in Mice  146  146 151 151  v  5.2.2 Increasing the Number of Nifedipine Doses Augments Anti-Proliferative Effect 154 5.2.3 Anti-Proliferative Effect of Nifedipine is Dependent on the Timing of Administration 156 5.2.4 Nifedipine Specifically Blocks T Cell Proliferation 157 5.2.5 Nifedipine Reduces T Cell Recovery Through an Antigen-Dependent Mechanism 159 5.2.6 Examination of Human PBT Function from Renal Disease Patients Following DHP Administration 162 5.2.7 Uremic Serum from Renal Disease Patients Inhibits IL-2 Secretion from Normal Human PBTs 166 5.3 Discussion 168 CHAPTER 6: DISCUSSION 172 6.1 General Conclusions 172 6.2 Future Directions 179 REFERENCES 182 APPENDIX A 196 APPENDIX B 205  vi  LIST O F F I G U R E S  Figure 1-1: T cell signaling events proximal to the TCR  6  Figure 1-2: Ca -mediated signaling events during T cell activation  15  Figure 1-3: Three proposed models linking ER C a activation  28  2+  2 +  store-depletion to C R A C channel  Figure 1-4: Phylogenic tree of the three mammalian TRP subfamilies  35  Figure 1-5: Membrane topology and structure of the three mammalian TRP subfamilies. 37 Figure 1-6: Nomenclature, chromosome location and tissue expression of the different channel-forming cci -subunits 41 Figure 1-7: Model of the subunit structure and composition for VDCCs  42  Figure 2-1: PCR cloning strategy used to isolate oti F-subunit cDNA sequences from human retina and spleen cDNA libraries  51  Figure 2-2: The pIRES-hrGFP-la mammalian expression vector  53  Figure 2-3: The mammalian expression pSUPER directs stable synthesis of siRNA transcripts  59  Figure 2-4: The pNFAT-TA-Luc vector monitors NFAT-mediated signaling transduction pathways in mammalian cells 66 Figure 2-5: Experimental design for the in vivo proliferation assay Figure 3-1: The channel-forming oti F-subunit of L-type V D C C s is expressed in T lymphocytes  70  77  Figure 3-2: Schematic representation of mRNA splice sites and putative protein topology of the voltage negative splice variant of 0Ci F-subunit isolated from human spleen 80 Figure 3-3: Schematic representation of mRNA splice sites and putative protein topology of the voltage positive splice variant of ctiF-subunit isolated from human spleen 83 Figure 3-4: FLAG-tagged retina OC]F-subunit is expressed in HeLa cells  86  vn  Figure 3-5: Expression of alternatively, spliced isoforms of ociF-subunit mRNA in human leukocytes 89 Figure 3-6: Detection of OtiF-subunit protein in Jurkat T cells and human PBTs  91  Figure 3-7: Anti-CD3 stimulation alters mRNA expression levels of OtiF-subunit splice variants and LTRPC2 channel in Jurkat T cells and human PBTs 94 Figure 3-8: Activation-induced splicing may control the mRNA expression of the voltage negative splice variant of the aiF-subunit in Jurkat T cells 96 Figure 3-9: mRNA expression of different TRP subfamily members in Jurkat T cells, human PBTs and human spleen Figure 4-1: Chemical Structures of L-Type C a  2 +  Channel Modulators  99 115  Figure 4-2: Stable expression of siRNAs targeted to the otiF-subunit in Jurkat T cells did not knock-down aiF-subunit protein expression  120  Figure 4-3: (+/-) Bay K 8644 induces C a influx in a dose-dependent manner in the human Jurkat T cell leukemia line and human PBTs  123  2 +  Figure 4-4: Nifedipine blocks C a human PBTs :  2 +  influx in the human Jurkat T cell leukemia line and 125  Figure 4-5: (+/-) Bay K 8644 modulates phospho-p44/42 M A P kinase activation in Jurkat T cells and human PBTs 128 Figure 4-6: Nifedipine modulates phospho-p44/42 MAP kinase activation in Jurkat T cells 130 Figure 4-7: Inhibition of N F A T transcription by nifedipine in the human Jurkat T cell leukemia line 132 Figure 4-8: Nifedipine prevents IL-2 secretion from Jurkat T cells and human PBTs... 134 Figure 4-9: Decreased IL-2R expression in Jurkat T cells and human PBTs after treatment with nifedipine  137  Figure 4-10: Nifedipine suppresses splenocyte proliferation  139  Figure 5-1: T cell proliferative response to H-Y male antigen is decreased in mice following repeated nifedipine treatment  153  Figure 5-2: Anti-proliferative effect of nifedipine is dependent on the number of doses administered to mice 155  vni  Figure 5-3: One later dose of nifedipine suppresses T cell proliferation in response to the H-Y male antigen in mice 158 Figure 5-4: Anti-proliferative effect of nifedipine is not due to non-specific drug toxicity. 160 Figure 5-5: Nifedipine treatment does not suppress antigen-independent T cell recovery in female mice 161 Figure 5-6: DHP administration may decrease IL-2 secretion from PBTs of renal disease patients 165 Figure 5-7: Uremic serum from renal disease patients reduces IL-2 secretion from human PBTs 167 Figure 6-1: Proposed model for C a signaling in T lymphocytes  2+  channels involved in distinct phases of C a  2+  178  ix  LIST OF TABLES  Table 2-1: pSUPER constructs used for stable expression of siRNA in Jurkat T cells.... 60 Table 5-1: Summary of pharmacokinetic parameters of nifedipine and amlodipine in humans 148 Table 5-2: Summary of the DHP dosage, creatinine levels and T cell characteristics ofthe human subjects used in the study 163  LIST O F A B B R E V I A T I O N S Arp2/3 ANOVA AP-1 2-APB APC APCs ATCC bp BSA Ca [Ca ], cADPR CaMK CCB cDNA CFSE CIF CMV Con A CRAC CREB CsA c-SMAC CSNB DAG DHP DMEM DMSO dT EDTA EGTA ELISA ER Erkl/2 ExPASy EYFP FACS FITC FBS HEPES hrGFP H-Y antigen GAPDH 2+  2+  actin related proteins 2 and 3 Analysis of Variance activating protein-1 2-aminoethoxyborane allophycocyanin antigen presenting cells American Type Culture Collection base pair bovine serum albumin calcium intracellular Ca concentration cyclic adenosine diphosphate ribose Ca /calmodulin-dependent kinase C a channel blocker complementary deoxyribonucleic acid 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester Ca -influx factor cytomegalovirus concanavalin A Ca -release activated Ca cAMP response element binding protein cyclosporin A central S M A C congenital stationary night blindness diacylglycerol 1,4-dihydropyridine Dubelco's Modified Eagle Medium dimethyl sulfoxide 2'-deoxy-thymidine ethylenediaminetetraacetic acid ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid Enzyme-Linked Immunosorbent Assay endoplasmic reticulum extracellular regulated kinase 1/2 Expert Protein Analysis System enhanced yellow fluorescent protein fluorescent-activated cell sorter fluorescein isothiocyanate fetal bovine serum 4-(2-hydroxyethyl)piperazine-l -ethanesulfonic acid humanized recombinant green fluorescent protein histocompatibility antigen encoded on Y chromosome glyceraldehyde-3-phosphate dehydrogenase 2+  2+  2+  XI  GSK 1CAM 7 RAC  glycogen synthase kinase intracellular adhesion molecule Ca -release activated C a current immunoglobulin interleukin-2 IL-2 receptor intraperitoneal inositol 1,4,5-trisphosphate inositol 1,4,5-trisphosphate receptor internal ribosomal entry site intravenous immunoreceptor tyrosine-based activation motif inducible T cell kinase potassium kilobase kilo Dalton voltage-dependent potassium channel linker for activation of T cells lymphocyte-specific PTK p56 lymphocyte function-associated antigen long-TRP channel monoclonal antibody magnetic cell sorting mitogen-activated protein myocyte enhancer factor murine erythroleukemia cells minimum essential medium major histocompatibility complex messenger RNA mixed lymphocyte reaction nicotinamide adenine dinucleotide nuclear factor of activated T cells cytoplasmic component of N F A T nuclear subunit of N F A T nuclear factor K B no treatment polyacrylamide gel electrophoresis p21-activated kinase peripheral blood mononuclear cell phosphate buffered saline peripheral blood T lymphocyte phycoerythrin pleckstrin homology phytohemagglutinin propidium iodide phosphoinositol 3-kinase 2+  C  Ig IL-2 IL-2R i.p. IP IP3R IRES i.v. ITAM ltk K kb kDa K channel LAT Lck LFA LTRPC mAb MACS MAP MEF MELC MEM MHC mRNA MLR NAD NFAT NFATc NFATn NFKB NT PAGE Pak PBMC PBS PBT PE PH PHA PI PI3K 3  +  v  +  2 +  Lck  xii  PIP PIP PKC PLC PMSF pS p-SMAC pSUPER PTEN TPEN PTK PVDF RACE rhIL-2 RNA RPMI RT-PCR RT RyR SD SDS SE SH siRNA SLP-76 SMAC SNAP-25 SOCs SR TCR 2  3  Tg Th TNF TPA TRP TRPC TRPM TRPV VDCC WASP ZAP-70  phosphoinositol 4,5-bisphosphate phosphatidylinositol 3,4,5-trisphosphate protein kinase C phospholipase C phenylriiethanesulfonyl fluoride picoSiemens peripheral S M A C suppression of endogenous R N A phosphatase and tensin homologue deleted on chromosome ten N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene diamine protein tyrosine kinase polyvinylidene fluoride rapid amplification of cDNA ends recombinant human IL-2 ribonucleic acid Roswell Park Memorial Institute reverse transcriptase-polymerase chain reaction room temperature ryanodine receptor standard deviation sodium dodecyl sulfate Sort Enhanced Src-homology small interfering RNA SH2-containing leukocyte protein 76 supramolecular activation cluster synaptosome-associated protein of MW25 store-operated C a channels sarcoplasmic reticulum T cell receptor transgenic T helper tumor necrosis factor 12-O-tetradecanoylphorbol 13-acetate transient receptor potential TRP canonical TRP melastatin TRP vanilloid voltage-dependent Ca channel Wiskott-Aldrich syndrome protein zeta-associated protein-70 2 +  ACKNOWLEDGEMENTS  I am indebted to Dr. Wilfred Jefferies for his scientific guidance and expert training that were essential for the development of my skills as a scientist. I am also grateful for Dr. Jefferies' enthusiasm for research and his unending support and kindness. I thank Jason Grant for his assistance with the immunofluorescence experiments, Dr. Douglas Carlow for his technical expertise with the mouse studies, and Lindsey Turner for her assistance with the statistical analysis. I am grateful to Geoffery Osborne and Andrew Johnson for their expert technical assistance provided for the intracellular C a  2+  measurements. I thank the undergraduate directed studies students, Adam Mott and Junella Lee for their dedicated and enthusiastic work ethic on various aspects of my project. I thank the members of the Jefferies' lab that generously donated blood so that I could perform the studies with human T lymphocytes. 1 am grateful to many individuals for their valuable comments on my manuscripts and thesis, including my committee members; Dr. Kenneth Baimbridge, Dr. Pauline Johnson, and Dr. Fumio Takei, as well as Dr. Cheryl Pfeifer, Dr. Maki Ujiie, Dr. Douglas Carlow and Dara Dickstein. I also would like to thank all of the individuals of the Jefferies' lab, past and present, for creating a warm and friendly work environment. Finally, 1 am grateful to the Department of Microbiology and Immunology, The Biomedical Research Centre, and Biotechnology Laboratory for creating a stimulating and scientifically diverse environment to perform my graduate degree. This work was partially funded by the Graduate Engineering and Technology Scholarship (G.R.E.A.T.) from the Science Council of British Columbia.  DEDICATION  I would like to dedicate this work to my loving parents, Susan Mary Kotturi and Murthi Sreerama Kotturi, and my dear brother, Gopaul Kotturi. Without their unending love and support I would not have been able to accomplish my goal of becoming a successful scientist.  XV  CHAPTER 1: GENERAL INTRODUCTION  1.1 Role of Calcium in Regulating Distinct Cellular Processes  2~F  The Ca  ion is a highly versatile signaling agent that regulates many complex  cellular processes in the human body involved in life and death (1). For instance, C a signaling  initiates  fertilization,  controls  muscle  contraction  2 +  and secretion of  neurotransmitters, and mediates cellular proliferation, necrosis and apoptosis (1). The "on" and "off switches of these Ca -dependent processes are coordinated at one level 2+  by alterations in the intracellular C a [Ca ]i 2+  2+  concentration  [Ca ]i 2+  of cells. In resting cells, the  is approximately 10-100 nM, which is 20,000 fold-less than the [Ca ] found 2+  extracellularly (2). Upon cellular stimulation, intracellular C a 2 (iM from the release of C a  membrane C a  2+  2 +  2 +  2+  from the extracellular compartment through plasma  channels (2). Since  surrounding intracellular C a  levels rapidly rise to 0.5-  through channels located in intracellular membrane C a  2 +  stores and the influx of C a  2+  [Ca ]j 2+  is critical for cell function, the events  release and removal are tightly regulated through  numerous ion channels, buffers, exchangers and pumps. Further control of Ca -dependent processes is achieved by modifying spatial and 2+  temporal patterning of the C a  2 +  signal (2). The spatial aspects of C a  categorized as either elementary C a waves (1). Elementary C a with C a  2+  2 +  2+  2+  signaling have been  events, or intracellular and intercellular global C a  events and C a  2 +  waves are visualized by imaging cells loaded  indicator dyes through confocal microscopy (3). An elementary C a  defined as a localized release of C a  2 +  2 +  from second-messenger operated C a  2 +  2+  event is channels 1  found in intracellular Ca  stores, such as the endoplasmic reticulum (ER) and the  sarcoplasmic reticulum (SR) in muscle (4). In response to low second-messenger concentrations, elementary C a 1,4,5-trisphosphate  2 +  events include single-channel opening of either inositol  receptor (IP3R) or ryanodine receptor (RyR) C a  2+  channels in  intracellular stores, which are visualized as blips or quarks, respectively (2). Higher concentrations of second-messengers induce the coordinated opening of a group of  IP3RS 2_|_  or RyRs that are visualized as puffs and sparks, respectively (2). Since elementary Ca signals are localized intracellular events, the result is a specific physiological outcome near the vicinity of C a elementary C a  2 +  2 +  release. An example of a cellular process that is regulated by  events is neuronal excitability. In the neuron cell body, transient, highly  localized puffs of C a  2 +  released from IP3R C a  2+  channels located in the ER activate C a 2+  activated potassium (K ) channels in the plasma membrane (5). The activation of the K +  +  channels induces K ion efflux across the plasma membrane, which in turn directly alters +  the membrane potential, preventing further electrical activity (1, 5). Elementary C a  2 +  release from IP3RS may also augment other neuronal processes, such as secretion of synaptic vesicles containing neurotransmitters. There is accumulating evidence that elementary C a  2+  release from IP3RS enhances localized [ C a ^ that is necessary to trigger 2  neurotransmitter exocytosis (5). Long-lasting elementary C a  2 +  events that produce puffs and sparks from either the  ER or SR lead to an intracellular global C a  2+  wave, that moves swiftly through the  cytoplasm of an entire cell (4). During fertilization in mammals, sperm fusing with the egg plasma membrane generates repetitive increases in cytosolic C a 2_|_  approximately two hours (2). Each increase in intracellular Ca  2 +  that last for  from IP Rs is a global 3  2  Ca  wave that begins at the sperm attachment site and spreads throughout the egg (6). As  a result of the C a  2+  wave, exocytosis of enzyme-containing cortical granules that are  responsible for "hardening" the structure of the fertilized egg is initiated, preventing additional sperm fusion (6). Another example of a cellular process regulated by intracellular global C a  2 +  waves is the excitation-contraction coupling of cardiac muscle.  When a motor nerve triggers an action potential in a muscle cell, voltage-operated plasma membrane C a Ca  2 +  2+  channels open in response to membrane depolarization, initiating a small  influx, that stimulates the release of a C a  2+  spark from four to six RyRs located in  the SR (7). This response is amplified following the release of more C a RyRs. A global C a  2+  2 +  sparks from  wave is then generated, inducing the contraction of each myofibril  (7). When cells are connected to one another through gap junctions, intracellular C a  2 +  waves can spread to many neighboring cells, leading to the generation of intercellular global C a  2+  waves (2). The first recorded intercellular Ca  waves were in hepatocytes  from intact liver tissue after stimulation with the agonist, vasopressin (8). Intercellular Ca  2+  waves through coordinated release of C a  2 +  from IP3RS in the liver are thought to  directly effect hepatic functions, including the movement and secretion of bile (8). 2__ |  The temporal characteristics of Ca  signaling, such as speed, amplitude and  frequency modulation, generate additional diversity of the C a elementary and global C a  2+  2+  signal. Furthermore, both  events can have distinct oscillation frequencies and durations  depending on the cellular process. For instance, altering the C a  2+  oscillation frequency in  lymphocytes can directly result in differential activation of proinflammatory transcription factors (9). High frequency oscillations are necessary to activate the transcription factor, nuclear factor of activated T cells (NFAT), whereas infrequent oscillations only stimulate  3  the transcription factor nuclear factor K B ( N F K B ) in T lymphocytes (9). The prolonged activation and nuclear retention of N F A T for up to two hours is required to promote lymphocyte proliferation (10). However, very high concentrations of Ca in C a  2 +  or disruptions  homeostasis can be deleterious to cells, inducing cell death by either necrosis or  apoptosis (11). Together, both spatial and temporal patterning of the Ca Ca  2 +  signal produce complex  signaling networks with durations of seconds to many hours depending on the  cellular process. In order to generate distinct physiological outputs, the intricate Ca signals are "decoded" by various intracellular signaling molecules (1). The intracellular "decoders" provide a final level of control over the physiological outcome of C a 2+  dependent processes. As an example of the complexity of Ca type, the precise nature and regulation of C a  2+  signals in a single cell  signals during T lymphocyte activation  will be discussed.  1.2 T Cell Activation  1.2.1 Early Signaling Events Proximal to the T Cell Receptor T lymphocyte activation is initiated through the TCR. The TCR complex is multisubunit transmembrane receptor, consisting of the disulfide-linked, clonotypic TCR-cq3 or TCR-y5 heterodimer, non-covalently associated with the TCR invariant chains; the TCR-^ homodimer, and the CD3-5, -e, and -y chains (12). Assembly of the TCR-ap7CD3 complex occurs in the ER through a series of tightly coordinated protein interactions (13,  4  14). Once assembled on the cell surface, it is believed that the minimal TCR/CD3 complex contains one TCR-ap or TCR-yS heterodimer associated with a CD3-e8 and a CD3-ey dimer, since the stoichiometry is one 8-, one y- and two e-chains per CD3 complex (15) (Figure 1-1). However, when quantitative flow cytometry was used to study subunit stoichiometry, it was demonstrated that there may be three CD3-e chains for every two TCR heterodimers (16). Since the crystal structure of the entire TCR/CD3 complex has not been completed, the exact stoichiometry of the subunits is still under debate. It is well established that the TCR/CD3 complex is coupled to intracellular signaling machinery by the large cytoplasmic domains of the CD3-8, -£, and -y chains and the TCR-^ homodimer. T lymphocytes expressing TCR-aP become activated when the TCR/CD3 complex recognizes antigen peptide bound to the major histocompatibility complexes (MHC) on antigen presenting cells (APCs), such as B lymphocytes, dendritic cells and macrophages. The extracellular domains of CD8 expressed on cytotoxic T cells, and CD4 found on T helper (Th) cells bind to membrane-proximal regions of M H C Class I and II, respectively (17). CD28 is a T cell surface receptor that binds to the costimulatory molecules, CD80 (B7.1) and CD86 (B7.2) expressed on APCs (17). Engagement ofthe coreceptors, CD4 and CD8, and the costimulatory receptor, CD28 present on aP T lymphocytes by their appropriate ligands on APCs is also necessary to achieve full activation responses (17). In the absence of the CD28 costimulation signal, T C R engagement induces a state of T cell anergy or unresponsiveness (18). Following ligation of the TCR/CD3 by a peptide-MHC complex, nonreceptor protein tyrosine kinases (PTKs) associated with the TCR/CD3 complex are activated  5  ZAP-70  Figure 1-1: T cell signaling events proximal to the T C R . Following peptide-MHC recognition by the TCR, ITAM sequences in the CD3-e5 and CD3-ey dimers, and the TCR-^ invariant chains are phosphorylated (designated as P) by Lck. ZAP-70 is activated by Lck phosphorylation and subsequently phosphorylates the adapter protein SLP-76. SLP-76 in conjunction with L A T recruits PI3K to the plasma membrane, which converts the membrane phospholipid PIP2 to PIP . Newly synthesized PIP3 interacts with the Tec family kinases, ltk and Rtk, which in turn phosphorylate PLCyl. The intracellular messengers, IP3 and D A G , are released into the cytoplasm when activated PLC-yl hydrolyzes PIP2. This T cell signal transduction diagram was adapted from Winslow et al. (19). 3  (Figure 1-1). Since the TCR/CD3 complex does not contain enzymatic activity in the cytoplasmic domains, the association and recruitment of PTKs is necessary for the activation of signaling cascades downstream of the antigen receptor. Three classes of PTKs implicated in T cell activation are the Src, zeta-associated protein (ZAP)-70 and Tec family kinases (12). The Src family members, lymphocyte-specific P T K p 5 6  Lck  (Lck)  and Fyn, noncovalently associated with the cytoplasmic tails of both CD4 and CD8 coreceptors, are activated through dephosphorylation of their negative regulatory tyrosine residue by the transmembrane receptor, protein tyrosine phosphatase, CD45 (20). In addition, CD4 and CD28 induce autophosphorylation of Lck by recruiting Lck to the TCR and sustaining Lck activation (21). Once activated, Lck and Fyn specifically phosphorylate tyrosine residues on CD3-5, -e, and -y, and TCR-^ chains at a motif known as the immunoreceptor tyrosine-based activation motif (ITAM) (22). The specific consensus sequence for an ITAM motif is YxxI/L(x)6-8YxxI/L, where Y denotes a tyrosine, I is isoleucine, L is leucine, and x is any amino acid (22). There is one ITAM motif present on each CD3 chain and three on a TCR-^ chain, therefore a minimal TCR/CD3 complex contains ten ITAMs. ITAM sequences on TCR-£ can either be phosphorylated once (TCR-^ p21) or dual phosphorylated (TCR-^ p23), generating two distinct ITAM isoforms (12). The ITAMs are predominantly phosphorylated by Lck and to a lesser extent by Fyn (12). Dual-phosphorylated ITAMs recruit ZAP-70 from the cytoplasm, causing ZAP70 to bind to ITAMs in the TCR-^ chain through its tandem, phosphotyrosine recognition domains,  the  Src-homology  (SH2)  domains  (22).  ZAP-70  becomes  tyrosine  phosphorylated and activated as a result of autophosphorylation and phosphorylation by  7  Lck (23). Phosphorylation of ZAP-70 generates docking sites for the recruitment of the SH2-containing adapter or linker proteins that lack enzymatic activity, but contain numerous binding domains, which allow the formation of multiprotein signaling complexes (22). Proteins that bind to phosphotyrosine residues of ZAP-70 include; the transmembrane adapter protein, linker for activation of T cells (LAT); the scaffolding adapter protein, SH2-containing leukocyte protein 76 (SLP-76); and the Grb2 family of adapter proteins (12). The Grb2 family consists of Grb2, Grap, and Gads that are small linker proteins, containing both SH2 and SH3 domains (22). SLP-76 interacts with L A T through binding to Gads (22). When tyrosine phosphorylated predominantly by ZAP-70, L A T and SLP-76 recruit additional proteins to the plasma membrane, including; phosphoinositol 3-kinase (PI3K); the guanine nucleotide exchange factor for the Rho-family of small GTPases, Vav; and the adapter protein, Nek (12). PI3K converts phosphoinositol 4,5-bisphosphate (P1P ) to phosphatidylinositol 3,4,5-trisphosphate 2  (PIP ) (24). The amount of PIP 3  3  synthesized at the plasma membrane is tightly regulated by PI3K and the lipid phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome ten), which converts PIP3 to PIP2 (19). The Tec family kinase, inducible T cell kinase (ltk), associates with P1P in the membrane via an amino-terminal pleckstrin homology (PH) 3  domain (12). An additional Tec family kinase that lacks a PH domain, Rlk, is also recruited to the plasma membrane through palmitoylation of a cysteine-string motif (12). Once ltk and Rlk are phosphorylated and activated by Src family kinases, the Tec kinases in turn directly phosphorylate phospholipase C (PLC)-yl. Activated PLC-yl catalyzes the hydrolysis of the plasma membrane PIP2, generating two cytoplasmic second messengers,  8  inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (19). While IP3 stimulates the release of C a  2+  from intracellular C a  2 +  stores in the ER, D A G activates protein kinase C  (PKC), particularly PKC0, and the downstream Ras signal transduction pathway (19). TCR engagement also induces polarized actin cytoskeleton rearrangement in T lymphocytes. The trimolecular complex of SLP-76, Vav and Nek is critical for this process. Through the SH3 domains of Nek, the serine/threonine kinase Pak (p21activated kinase) and the actin-binding protein, WASP (Wiskott-Aldrich syndrome protein) bind to the trimolecular complex (12). Vav and Nek also recruit GTP-bound Rac and Cdc42, which subsequently activate Pak and WASP (25). Once in its active state, WASP recruits and activates the actin related proteins 2 and 3 (Arp2/3) complex, which binds actin-ATP and initiates actin polymerization (25).  1.2.2 Immunological Synapse Formation and Sustained T Cell Signaling The tyrosine phosphorylation cascade initiated through the TCR/CD3 complex occurs within seconds following  T C R engagement by peptide-MHC  complexes.  However, sustained T cell signaling through the TCR/CD3 complex for many hours is necessary for the transcriptional activation of cytokine genes required for T cell proliferation and clonal expansion. In fact, naive T cells require approximately 20 h of sustained signaling through the TCR to be committed to proliferation (26). In order to establish and maintain long-lasting T cell signaling, the contact zone between T cells and APCs must overcome many physical obstacles. First of all, large glycoproteins on the T cell surface, such as CD43 and CD45, impose a steric hindrance to the TCR and peptideM H C interaction due to the smaller size of both the T C R and M H C molecules (27).  9  Second, there is a low-affinity interaction with a dissociation constant of 0.2 to 200 U.M between the TCR and peptide-MHC (28). The continual movement of T cells is also preventative in maintaining a sustained signal (27). Furthermore, T cells must search for a minimum of 10 antigenic peptides amongst many thousands of peptides presented by M H C molecules (29). Remarkably, only 10 peptide-MHC complexes are necessary to sustain T cell signaling (28). It is thought that the mechanism used to overcome all of these barriers is the formation of the immunological synapse. The immunological synapse is the specialized contact area between T cells and the interacting APCs, which contains highly organized molecular structures called supramolecular activation clusters (SMACs) (30). SMACs are micrometer scale domains, consisting of lymphocyte antigen receptors, costimulatory receptors, adapter proteins, cytoskeletal proteins, intracellular signaling molecules, and unique membrane domains (29). Using peptide-MHC and the adhesion ligand, intracellular adhesion molecule (ICAM)-l, integrated into a glass-supported planar bilayer, it was established that the formation of SMACs and the immunological synapse is a multiple step process (27). During the first 30 seconds, the junction between T cells and APCs is formed with peptide-MHC and T C R engagement in an outer ring, and the interacting T cell pV integrin, lymphocyte function-associated antigen (LFA)-l with ICAM-1 on APCs in the center of the ring. CD4 is also involved in the first stage of S M A C formation since CD4 deficient cells are less capable of stopping and forming immunological synapses (27). In the next 5 minutes, TCRs and peptide-MHC complexes translocate to the central (c)S M A C , while adhesion molecules move to the outer ring or peripheral (p)-SMAC through an actin-mediated process. Translocation of cell surface receptors relies on actin  10  cytoskeletal reorganization through the previously described trimolecular SLP-76, Vav and Nek complex (25). Finally, the third stage involves the stabilization of the immunological synapse for several hours by maintaining c-SMAC and p-SMAC structures. Since immune synapse  stabilization occurs 5 minutes after the T C R  engagement, synapse formation is not necessary for inducing tyrosine kinase activation, but is important for sustaining signaling events. Sustained signaling through the TCR is maintained by a process termed the serial triggering mechanism. Once TCR engagement has initiated tyrosine phosphorylation, the peptide-MHC can disengage and then become available to a new TCR/CD3 complex (26). This explains how only 10 peptide-MHC complexes can serially trigger a T cell through hundreds of TCRs (27). Further investigations on the T cell synapse have revealed that the c-SMAC contains not only the TCR/CD3 complex, but also CD28, protein kinases Lck, Fyn and PKC0, and the adapter protein CD2 associated protein (30). The p-SMAC consists of LFA-1, the cytoskeletal protein talin, and CD45 (30). Interestingly, although there are 10 different PKC isoforms in T lymphocytes, only PKC0 translocates to the c-SMAC upon TCR engagement (30). Additional support for a role for PKC9 in the c-SMAC came when mature T cells from PKC0 knock-out mice failed to respond to T C R induced activation (31). CD45 is also recruited to the early c-SMAC prior to synapse stabilization in order to dephosphorylate and activate Lck (32). Smaller, alternatively spliced isoforms of CD45 may also be present in the c-SMAC of the mature immune synapse, potentially sustaining Lck activity (25). Immunological synapse formation also involves the reorganization of plasma membrane structures into specialized "membrane domains" termed lipid rafts at the T cell  11  and  A P C interface.  structures  that  are  Lipid  rafts are detergent-insoluble,  highly  enriched  in  cholesterol,  liquid-ordered membrane GM1  gangliosides  and  glycosylphosphatidylinositol anchored proteins (25). In resting T cells, lipid rafts contain very few proteins and are randomly distributed in the plasma membrane with a diameter of less than 70 nm (33). Upon TCR engagement, proteins such as the TCR/CD3 complex, acylated Lck and L A T selectively associate with aggregated lipid rafts, whereas CD45 is generally considered to be excluded from rafts at the immunological synapse (33). Lipid raft coalescence is thought to bring signaling molecules such as Lck and L A T into close proximity with one another to enable more efficient signaling (33). Therefore, the aggregation of lipid rafts is likely an integral component of the T cell signaling process.  1.2.3 Calcium's Role as an Intracellular Secondary Messenger In T lymphocytes, a critical event during immunological synapse formation is the sustained increase in [Ca ]i. Intracellular C a 2+  involving an immediate, transient release of C a  2+  2 +  mobilization is a binary response through second messenger-gated C a  channels located in the ER, followed by prolonged Ca milieu for up to two hours, through C a Sustained C a  2+  2+  2 +  influx from the extracellular  channels in the plasma membrane (34).  mobilization has many physiological consequences within T lymphocytes,  including enzymatic activation, granule exocytosis and gene transcription (35). For instance, the Ca -dependent protease, calpain, is activated following binding of this 2+  enzyme to C a . Once in its active form, calpain releases LFA-1 from its cytoskeletal 2+  anchor, allowing LFA-1 to move into the contact area between T cells and APCs (35). Increases in [Ca ]j are also thought to induce cytolytic granule exocytosis from cytotoxic 2+  12  T cells since sustained Ca  gradients are present in cytotoxic T cells during lysis of  target cells (35). In addition, a rise in intracellular C a  2 +  is necessary for the long-term  activation of cytokine genes in Th cells, which will be discussed in more detail below (36) . Elevated cytoplasmic  Ca  2 +  levels also promote the binding of C a  2 +  to a  ubiquitous, 17 kilo Dalton (kDa) Ca -dependent regulatory protein called calmodulin 2+  (37) . In turn, Ca /calmodulin complexes regulate the activity of several enzymes 2+  required  for  transcription  factor  activation,  including  the  serine/threonine  Ca /calmodulin-dependent kinase (CaMK) family members, CaMKII and CaMKIV, and 2+  the serine/threonine protein phosphatase, calcineurin, also known as protein phosphatase 2B (19). Although the role of CaMKII in T cells is not well defined, CaMKIV may indirectly mediate the transcriptional upregulation of fos, which associates with jun forming the heterodimeric activating protein (AP)-l transcription factor (37). CaMKIV is suggested to activate fos transcription through phosphorylation and subsequent activation of cAMP response element binding protein, CREB that binds to a cAMP responsive element upstream of fos (37). The calcineurin isoforms functioning in T cells are calcineurin A a , and A(3 (38). A predominant role for calcineurin Ap has been suggested since calcineurin AP~'~ mice show a more severe defect in T cell maturation and function compared to calcineurin Aa" '' mice (39, 40). The activity of calcineurin is tightly regulated through Ca /calmodulin 2+  and endogenous calcineurin inhibitors. Calcineurin is activated by interacting with Ca /calmodulin complex through its catalytic domain, and direct binding of C a 2+  regulatory subunit (38).  2 +  to its  In contrast, the activity of calcineurin is attenuated by  13  upregulation of the calcineurin inhibitor, calcipressin 1, following calcineurin signaling (38). Calcipressin 1 exhibits its inhibitory action by directly binding between the catalytic and regulatory domains of calcineurin, inhibiting calcineurin phosphatase activity (38). The calcineurin binding protein, Cabinl/Cain-1, also binds to calcineurin and inhibits its phosphatase activity potentially through a negative-regulatory involving C a  2+  feedback  mechanism  (35).  Pharmacological inhibitors, such as the fungal metabolites cyclosporin A (CsA) and tacrolimus (FK506) are widely used to inhibit the phosphatase action of calcineurin in human patients (41). Both CsA and FK506 serve as potent immunosuppressive agents that are used largely to prevent immune responses in transplant therapy. CsA and FK506 do not bind to calcineurin directly. Instead, CsA and FK506 bind to the intracellular immunophilin receptors, cyclophilin and FK506-binding protein, respectively (41). The drug/immunophilin complexes then interact with the regulatory domain of calcineurin, abolishing its catalytic activity and any downstream dephosphorylation events mediated by this phosphatase (38).  1.2.4 Calcium-Dependent Transcriptional Regulation Through the regulation of calcineurin activity, C a  2 +  in turn, mediates the  activation of many transcription factors, including NFAT, N F K B and myocyte enhancer factor (MEF)-2 in T lymphocytes (Figure 1-2). When calcineurin is activated through elevated [Ca ]i, the cytoplasmic component (NFATc) of NFAT transcription complexes 2+  is dephosphorylated by calcineurin at multiple phosphoserine residues in the aminoterminus (38, 42). Calcineurin binds to the regulatory domain of NFATc through a  14  Figure 1-2: C a - m e d i a t e d signaling events d u r i n g T cell activation. 2+  Upon TCR engagement by peptide-MHC, protein tyrosine kinases (designated as T K ) proximal to the T C R activate P L C - y l , which cleaves PIP from plasma membrane phospholipids to generate D A G and IP3. Elevated levels of IP3 in the cytosol lead to the release of C a from IP R C a channels located in the ER. TCR stimulation also leads to the generation of cADPR that binds and opens RyR C a channels also located in the ER, leading to further C a release. Through an undetermined mechanism, C a release from the ER causes sustained Ca" influx from the extracellular space through plasma membrane C R A C channels. Elevated intracellular C a activates calcineurin and several transcription factors, including N F A T , N F K B and MEF2. The level of cytoplasmic C a is regulated through the removal of C a by sarco-ER Ca -ATPase (SERCA) and plasma membrane Ca -ATPase (PMCA) pumps. Intracellular C a is redistributed by the mitochondria to prevent Ca -dependent inactivation of the C R A C channels. Plasma membrane potential is maintained by the efflux of K from Kv and Ca -activated K 2  2 +  2+  3  2+  2+  2+  +  2+  2 +  2+  2+  2+  2 +  2+  +  2+  channels. This Ca  2+  +  signaling diagram was adapted from Lewis et al. (10).  15  conserved calcineurin-binding sequence motif, PxIxIT, where P denotes a proline, I is isoleucine, T is threonine and x is any amino acid (38). Dephosphorylation of N F A T c leads to the uncovering of a nuclear localization signal in the regulatory domain, which permits the activated form of N F A T c to translocate from the cytoplasm to the nucleus (43) . Since there is no delay in NFATc dephosphorylation and nuclear translocation it has been suggested that N F A T c and calcineurin associate in resting T cells (35). Prolonged levels of intracellular C a  2 +  are required to maintain NFATc activation and nuclear  retention; however it is unclear how NFATc remains dephosphorylated in the nucleus. One possibility is that a small amount of calcineurin translocates with NFATc into the nucleus (38). Alternatively, it has also been suggested that a portion of calcineurin may reside in the nucleus of resting cells prior to TCR stimulation (38). Once in the nucleus, NFATc associates with the newly synthesized nuclear subunit of N F A T (NFATn) (44). The NFATc and NFATn complex regulates the expression  of  several  genes,  through binding to  response  elements  in gene  promoter/enhancer regions, usually in association with AP-1 (41). The established cytokine gene targets of N F A T in T cells are IL-2, IL-3, IL-4, IL-5, IL-8, IL-13, granulocyte-macrophage colony-stimulating factor, and interferon-y. Other gene targets of N F A T are the cell surface receptors; CD40 ligand, Fas ligand and IL-2 receptor (IL2R)oc (41). It should be noted that CsA and FK506 prevent NFATc nuclear translocation, and consequently block NFAT-mediated transcription of cytokine and cell surface receptor genes (41). CsA and FK506 do not block the synthesis of the NFATn component (44) .  16  In T cells, the existence of three calcineurin regulated NFATc proteins that are capable of recognizing the same D N A element was thought to reflect redundancy of function, but recent evidence supports the idea that the family members may have genespecific activities. The argument for gene-specific  activities of N F A T has been  strengthened by the phenotypic analysis of NFATcl" ", NFATc2 ~, and NFATc3~ mice. 7  7  /_  NFATcl" " mice have an impaired ability to produce IL-4 in T cells, whereas IL-2 7  production was only slightly affected (41). The T cells from NFATc2 " mice have v  increased IL-4 production and hyperproliferative responses to TCR engagement, which is likely due to a lack of Fas ligand expression (42). Lymphocytes from mice lacking both NFATc2 and NFATc3 have a greatly enhanced Th2-type cytokine phenotype and acute allergic responses (45). Therefore the phenotypic analysis suggests that NFATc 1 is a positive regulator of IL-4 transcription, but NFATc2 and NFATc3 may eventually repress IL-4 gene expression through a negative-regulatory feedback mechanism (41). Upon TCR disengagement and the subsequent absence of sustained intracellular Ca  2+  signaling, N F A T c proteins are rapidly exported from the nucleus and N F A T c -  dependent gene transcription is terminated. Prior to nuclear export, NFATc  is  phosphorylated in three conserved serine/proline rich motifs in the amino-terminus predominantly by the constitutively active glycogen synthase kinase-3p (GSK-3(3) (34, 46). Phosphorylation of N F A T c is speculated to expose a nuclear export signal, permitting NFATc to rapidly leave the nucleus through the nuclear export receptor, Crml (41). Although nuclear inhibitors of N F A T c activity have been identified, the regulation of these protein kinases is not clear. However, it has been established that the activity of  17  GSK-3p is rapidly downregulated to about 50% of basal levels upon TCR stimulation (47). Additional transcription factors regulated by Ca /calcineurin in T lymphocytes are N F K B and MEF2 (38). Previous studies have shown that the calcineurin inhibitors CsA and FK506 are capable of blocking upstream IKB kinase activation, as well as TCRinduced N F K B activity (48, 49). As expected, Ca -independent activation of N F K B is not 2+  inhibited by CsA and FK506 following tumor necrosis factor a (TNFa) stimulation (50). Although the mechanism of Ca /calcineurin-induced N F K B activation has not been 2+  clearly identified in T lymphocytes, it has been suggested that calcineurin-mediated IKB dephosphorylation induces the specific degradation of IKB (38). As a result of IKB degradation, active N F K B is released, allowing N F K B to translocate into the nucleus and bind to promoter/enhancer regions. Unlike N F A T and N F K B , MEF2 is a transcription factor in lymphocytes that is constitutively bound to DNA-response elements in the nucleus (51). MEF2, which is expressed in several tissues including brain and muscle, was recently identified as a Ca /calcineurin-dependent transcription factor in T cells 2+  (52).  Interestingly,  generation  of  MEF2-dependent transcription requires  direct  association with NFATc2 (52). In coordination with NFAT, MEF2 is capable of inducing transcriptional activation of IL-2 (51). In summary, T cell activation is a multi-step process involving numerous intricately woven signal transduction pathways, many of which are dependent upon the elevation of [Ca ]j. 2+  The mechanisms of how increased intracellular C a  2 +  levels are generated add to  the complexity of the activation process.  18  1.3 Intracellular Calcium Release and Regulation in T Cells  During T cell activation, there are several mechanisms that introduce bursts of C a into the intracellular space. Immediately following TCR engagement, C a released from ER C a  2 +  2 +  is transiently  stores. As a direct consequence of intracellular C a  depletion, a sustained influx of C a space. This mechanism of C a  2 +  2 +  2 +  store-  2 +  enters into the cytoplasm from the extracellular  entry across the plasma membrane and subsequent store  refilling is termed "capacitative C a  2 +  entry" or "store-operated C a  enter the cytoplasm from either the ER C a  2+  2 +  entry" (35). C a  2 +  ions  stores or through the plasma membrane by  9+  specific Ca  channels. In T lymphocytes, there are two important second-messenger  operated C a  2+  and RyR C a  2+  channels that mediate C a  2 +  mobilization from intracellular ER stores;  IP3R  channels.  1.3.1 IP3 Receptor Calcium Channels Immediately following TCR/CD3 complex stimulation, cytoplasmic IP3 levels rapidly rise, and remain elevated within the first 10 to 20 minutes after T cell activation (10). IP3 binds to the IP R C a 3  2 +  channel located in the ER membrane, releasing C a  its intracellular store (Figure 1-2). IP3RS are large tetrameric intracellular C a  2+  2 +  from  channels,  composed of four subunits of approximately 2700 amino acids (-300 kDa) (53).  The  amino-terminus of each subunit contains a single IP3 binding site that resides in the cytoplasm. It has been proposed that the negatively charged phosphate groups of IP3 interact with the IP R through the basic residues that are scattered throughout the IP3 3  binding site (53). Several IP3 molecules are required to bind to the IP3R to induce a  19  relatively large conformational change, causing channel opening and Ca  release (54).  Near the cytoplasmic-residing carboxyl-terrninus there are six transmembrane domains that anchor the channel in the reticular membrane. The last two transmembrane domains and the membrane-associated loop between these domains form the pore of the channel (54). In between the IP3 binding site and the channel pore is the modulatory domain. This domain is composed of 1700 amino acids, and contains phosphorylation and protein binding sites that directly mediate channel opening (54). Three isoforms of human IP Rs have been identified in lymphocytes (55). The 3  human Jurkat T cell leukemia line, the chicken DT40 B cell line, and mouse thymocytes express type 1 IP R (IP R1), IP3R2, and IP R3, whereas rat thymocytes and splenocytes 3  3  3  only express IP R2 and IP R3 (56-58). IP R2 and IP R3 share approximately 69% and 3  3  3  3  64% identity, respectively, with the amino acid sequence of IP3RI (59). Interestingly, the IP3R isoforms show differences in their sensitivities to IP3 and their regulation by [Ca ] i . I P 3 R I is rapidly activated by cytosolic IP3 and shows a biphasic response to [ C a ] i (10). 2+  IP3RI  is activated by -300  nM  [Ca ]i, 2+  but low and high  inactivation, which leads to the generation of regular C a responds more effectively to IP3 and C a  2 +  2 +  [Ca ]i 2+  cause channel  oscillations (54, 60). IP3R2  than IP3RI (10). Surprisingly, both IP3R2 and  IP3R3 are not inhibited by Ca (10). Since the IP3R isoforms have distinct sensitivities to 2+  IP3  Ca  and 2+  [Ca ]i, 2+  their differential expression may confer unique patterns of oscillatory  signaling through IP3-sensitive and -insensitive C a  2+  pools in the ER (35, 60). 2+  The Src family of non-receptor PTKs also seems to regulate IP3R-controlled Ca release from intracellular stores in T lymphocytes. Src and Fyn were shown to increase channel pore opening through phosphorylation of tyrosine residues on the IP3RI Ca  2_|_  20  channel in Jurkat T cells (61). The IP3RI contains two potential tyrosine phosphorylation sites, one located adjacent to the putative channel pore and the other near the IP3 binding region (61). In vivo analysis with thymocytes from fyn ~ mice showed decreased _/  phosphorylation of IP3RI correlated with reduced intracellular C a  2 +  release and defective  TCR signaling (61). Although it is well established that three IP3R isoforms are expressed in T lymphocytes, the exact contribution of each IP3R isoform to the initial Ca relatively unclear. In addition, both homotetrameric and heterotetrameric  release is still  IP3RS  have been  detected (62). Several studies have been conducted examining the role of IP3RI during T cell activation. Inhibiting expression of IP3RI by stable transfection of antisense IP R1 3  complementary deoxyribonucleic acid (cDNA) in Jurkat T cells prevented an increase in intracellular C a  2 +  and IL-2 secretion after TCR stimulation (63). The predominant role of  IP3RI was then questioned when a later study demonstrated that the IP3RI antisense cDNA also partially decreased the expression of IP3R2 and IP3R3 in Jurkat T cells (64). The role of IP3RI as a regulator of C a  2 +  mobilization was further disputed when a  previous in vivo study concluded that IP3RI is not essential for T cell activation and function (65). To establish the in vivo role of I P 3 R I , bone marrow of I P 3 R I " " mice was transplanted into irradiated wild-type mice, since ^ R l " " mice died shortly after birth 7  (65). Contradictory to the previous in vitro results, T cells from mice lacking IP3RI were able to mobilize C a  2 +  from intracellular stores after TCR stimulation, and did not show  defective TCR signaling (65). Taken together, the results from these in vitro and in vivo studies suggest that the IP3R isoforms may exhibit functional redundancy in T lymphocytes.  21  Not only are release, but  1P3R.S  IP3R.S  implicated in promoting T cell proliferation through Ca  have also shown to regulate programmed cell death or apoptosis. As  previously mentioned, prolonged high intracellular C a  2+  levels can cause cell death  through the activation of endonucleases responsible for D N A fragmentation and subsequent apoptosis (55). Previously, it has been shown that IP R1 deficient Jurkat T 3  cells are resistant to apoptosis induced by dexamethasone (a glucocorticoid that induces DNA  damage), T C R stimulation, ionizing radiation and Fas/CD95 activation (64).  Therefore, prolonged C a  2 +  mechanism of how C a  regulates apoptotic signals is still unclear; however, it has been  proposed that C a  2 +  2+  release through the IP R1 may induce apoptosis. The exact 3  may activate Ca -dependent proteases and nucleases involved in the 2+  apoptotic pathway (64). From these previous studies on IP3R function, it is clear that IP R-mediated C a  2 +  3  mobilization is not as simplistic as it was once viewed. Functional redundancy of the IP3R isoforms has been demonstrated in the DT40 B cells lacking these channel isoforms. In the DT40 cells, complete inhibition of C a  2+  release following B cell antigen receptor  stimulation was only accomplished by deletion of all three IP3R isoforms (66). Based on these results, IP3RI, IP3R2, and IP3R3 may function together in coordinating C a mobilization from intracellular C a  2+  2+  pools in T lymphocytes. In addition, it remains a  puzzle how cytosolic IP3 levels that rapidly dissipate after 10 min can maintain a long-  2__| lasting Ca  signal for 1 to 2 h. One possibility is that the local IP3 concentration is high  enough to keep IP3R C a  2+  channels open (35). An alternative explanation is that IP3RS  may be involved in short-term C a responsible for prolonged C a  2 +  2+  signaling, while RyR C a  2 +  channels may be  release from the ER (10). Based on the evidence that IP R 3  22  Ca  channels are inactivated by high, localized IP3 concentrations, and the increasing  data demonstrating a role for RyR C a  2+  channels, the latter hypothesis is more plausible.  1.3.2 Ryanodine Receptor Calcium Channels 2+  RyR are the second critical second-messenger operated Ca for C a Ca  2+  2 +  release from intracellular C a  2+  channel responsible  stores in T lymphocytes. Unlike  IP3R,  the RyR  channel does not appear to be located in the ER since thapsigargin, an inhibitor of  the ER Ca -ATPase pump has no effect on C a 2+  2 +  release by RyR (67). The precise  intracellular location of the RyR has not been clearly identified in T cells. Thapsigarginresistant ER-localized C a RyR C a  2+  2 +  pools have been identified; therefore, it is plausible that the  channels are located in these regions of the E R (68). RyR C a  2 +  channels are  large homotetrameric protein complexes, consisting of monomers of approximately 5000 amino acid residues (-500 kDa) (69). The amino-terminus of the RyR (4000 amino acids) resides in the cytoplasm and contains many regulatory binding sites for nucleotides, calmodulin, and C a , as well as phosphorylation sites (70). Interestingly, the binding 2+  partner for the immunosuppressive agent FK506, FK506-binding protein 12, directly associates with RyRs in the ammo-terminal region of the channel (70). The carboxylterminus of the RyR lies in the cytoplasm, while the adjacent region (1000 amino acids) consists of four transmembrane domains, and the putative channel pore between transmembrane domains 3 and 4 (70). The carboxyl-terminal cytoplasmic tail and adjacent two transmembrane domains of the RyR C a homology to similar regions of the I P 3 R C a  2 +  2 +  channel share extensive sequence  channel (71).  23  Three mammalian RyR C a  2+  channel isoforms have been identified in the SR of  skeletal muscle (type 1 RyR), cardiac muscle (type 2 RyR), and the brain (type 3 RyR), as well as in various other tissues through cDNA cloning (70). R y R l , RyR2 and RyR3 display approximately 66-70% amino acid sequence identity to one another (70). Jurkat T cells express the RyR3 isoform, which overall, has 95% amino acid sequence identity to the rabbit brain RyR3 (72). The existence of a functional RyR C a  2 +  channel in T  lymphocytes was demonstrated by the presence of a ryanodine-sensitive intracellular Ca  2 +  pool in Jurkat T cells (72). A physiological role for RyR3 was further investigated  by knocking-down RyR3 expression in Jurkat T cells through the stable integration of RyR3 antisense ribonucleic acid (RNA) (73). In RyR3 deficient Jurkat T cells, the sustained phase of C a  2+  signaling was impaired following T C R engagement by the  agonistic anti-CD3 monoclonal antibody (mAb), OKT3 (73). Although the in vitro studies with Jurkat T cells suggest an important role for RyR3 in regulating intracellular Ca  release, splenocytes from RyR3"" mice appeared to proliferate normally in response  to concanavalin A (Con A), IL-2 and lipopolysaccharide (74). The endogenous activator of RyR C a  2 +  channels is the nicotinamide adenine  dinucleotide (NAD ) metabolite, cyclic adenosine diphosphate ribose (cADPR) (75). The +  second messenger, cADPR was originally found in sea urchin eggs, where it mobilized intracellular C a  2+  in an IP -independent mechanism via RyR C a 3  precise mechanism of how cADPR regulates C a  2 +  2 +  channels (75). The  release from RyRs is not clear. cADPR  may directly bind to RyRs or may interact with RyRs through a separate cADPR-binding protein (75). Several studies have utilized Jurkat T cells to examine the role of cADPRinduced C a  2+  release. In permeabilized Jurkat T cells, cADPR application induced C a  2 +  24  release from intracellular stores in a dose-dependent manner (67). The cADPR-dependent Ca  2 +  release was blocked by the chemical analogues of cADPR, 8-amino-cADPR and 8-  bromo-cADPR (67). Furthermore, Jurkat T cells stimulated with the OKT3 mAb showed increased levels of cytosolic cADPR that lasted for up to 60 min (76). It was also demonstrated that Jurkat T cells stably expressing RyR3 antisense R N A had decreased Ca  2+  release in response to cADPR treatment (73). The molecular mechanisms for cADPR generation are still unclear in T  lymphocytes. In higher eukaryotes, one of the ADP-ribosyl cyclases responsible for converting N A D to cADPR is the cell-surface transmembrane protein, CD38 (77). CD38 +  may be a potential source of cADPR in T cells since surface expression of CD38 is upregulated following lymphocyte activation (78). The problem with this model is that the enzymatic activity of CD38 is associated with the extracellular domain so it is unknown how the membrane-impermeable cADPR enters T cells (78). It is possible that dimerization or oligomerization after CD38 ligation may trigger internalization of the receptor with attached cADPR (77). The second model for cADPR-controlled C a  2+  release is that the activation of the TCR/CD3 complex stimulates a soluble ADP-ribosyl cyclase to produce cADPR (67). Guse et al. confirmed this hypothesis by demonstrating that T C R stimulation of Jurkat T cells resulted in the activation of a tyrosine-kinase regulated, cytosolic ADP-ribosyl cyclase, in association with a prolonged increase in intracellular cADPR levels (76). Although a soluble ADP-ribosyl cyclase has been identified in T cells, the possible generation of cADPR from CD38 cannot be disproved. In conclusion, the in vitro studies with Jurkat T cells suggest that IP3 is necessary for the initial C a  2 +  release from IP3R C a  2 +  channels, but not sufficient for sustained C a  2 +  25  signaling, while cADPR association with RyR Ca second phase of C a  2 +  channels is essential for a prolonged  release (75). Since the majority of the experiments with cADPR  and RyR3 have been conducted in Jurkat T cells, these models need to be confirmed with primary human T lymphocytes.  1.4 Communication between Calcium Store Release and Calcium Influx Pathways  2~l"  2+  In T lymphocytes, the emptying of intracellular Ca IP R and RyR C a 3  2 +  stores by Ca  channels leads to the opening of store-operated C a  2+  release through channels (SOCs)  in the plasma membrane. Through electrophysiological means, the mechanism of storeoperated C a  2+  entry has been extensively studied in numerous non-excitable cells. To  date, the best characterized SOC is the C a  2 +  release-activated C a  2 +  (CRAC) channel that  is localized to the plasma membrane of T lymphocytes, mast cells and other hematopoietic cells (79). The C a highly selective C a  2 +  2+  current gated by the C R A C channel, termed / C R A C , is a  current with an extremely low C a  dependency for store-depletion to induce lymphocytes. For instance,  /CRAC  /CRAC  2+  conductance (79). The  activation has been well established in T  is activated by the ER Ca -ATPase pump inhibitor, 2+  thapsigargin, that causes a rise in [Ca ]i by preventing E R store-refilling (10). 2+  also activated  by the  membrane permeant Ca  /CRAC  is  chelator, TM,N,N',N'-tetrakis(2-  pyridylmethyl)ethylene diamine (TPEN), that directly reduces the intraluminal [Ca ] in 2+  the ER (10). Although it is clear that emptying of intracellular C a  2 +  stores leads to  /CRAC  activation in T lymphocytes, the mechanism by which the fall of [Ca ] within the ER 2+  activates  /CRAC  still remains a mystery. Presently, there are three models linking store 26  depletion to C R A C channel activation, however none have received extensive support or have been discounted completely (79).  1.4.1 Diffusible Messenger Model The first and oldest hypothesis linking store-depletion to  /CRAC  activation is the  diffusible messenger model. This model postulates that a soluble signaling factor is newly  synthesized  and/or released  from the ER following  store  emptying and  subsequently diffuses to the plasma membrane where it activates C R A C channels (Figure 1-3A) (35). An early study by Randriamampita et al. identified a novel soluble mediator, termed the Ca -influx factor (C1F) that stimulated C a 2+  Jurkat T cells following depletion of intracellular C a  2+  2 +  influx in mitogen-activated  stores (80). This model gained  further support when cytosolic extracts containing CIF from thapsigargin-treated Jurkat T cells induced C a  2 +  influx when injected into Xenopus laevis oocytes (81). CIF extracted  from the cytosol of Jurkat T cells was proposed to be a small (less than 500 Da), nonprotein-like, phosphorylated compound (80). However, the precise identity of CIF from Jurkat T cells is unknown since investigators have been unable to purify this compound. CIF has also been postulated to induce C a  2+  influx through SOCs in cells other  than T lymphocytes. For example, a novel SOC in vascular smooth muscle cells was shown to be activated by CIF, and not by a variety of other intracellular secondmessengers, including IP3 (82). In the study by Trepakova et al., CIF extracted from pmrl mutant yeast that lack the PMR1 Ca -ATPase pump or CIF isolated from human 2+  platelets was able to activate the novel store-operated channel in smooth muscle, as well as elicit C a  2 +  influx in Xenopus oocytes and Jurkat T cells (82). Recently, a study  27  Figure 1-3: Three proposed models linking E R C a channel activation.  store-depletion to C R A C  (A) The diffusible messenger model predicts that a soluble messenger, CIF is either newly synthesized or released from the ER following store emptying and then diffuses to the plasma membrane, activating C R A C channels. (B) The conformational coupling model suggests that either the IP3R Ca' channel or another ER resident protein physically associates with the C R A C channel to induce its opening. (C) The secretion model predicts that activated C R A C channels are inserted into plasma membrane through vesicle fusion following store-depletion. This diagram was adapted from Winslow et al. (19).  28  examining SOCs in human neutrophils identified that sphingosine 1-phosphate acts as a soluble messenger, linking store-depletion to store-operated C a  2 +  entry through the  plasma membrane (83). Sphingosine 1-phosphate is a bioactive, small (380 Da) molecule that is generated from the metabolism of sphingolipids by sphingosine kinase (83). In human neutrophils, thapsigargin-induced store emptying induced the synthesis of sphingosine 1-phosphate, which in turn stimulated C a  2 +  entry (83). Intriguingly,  sphingosine 1 -phosphate has many similar biochemical properties to the CIF originally identified in Jurkat T cells. Since sphingosine 1-phosphate is capable of increasing [Ca ]j 2+  in Jurkat T cells (84), it will be interesting to learn whether sphingosine 1-phosphate is newly synthesized in Jurkat T cells following store-depletion and if this molecule can activate  /CRAC  in these cells.  1.4.2 Conformational Coupling Model The conformational coupling model postulates that conformational changes in the IP3R  Ca  2+  channel elicited by store-depletion cause  /CRAC  activation through direct  physical interaction between IP R in the ER and C R A C channels in the plasma 3  membrane (Figure 1-3B) (35). In contrast to the other two models, there appears to be a similar number of studies either supporting or refuting the conformational coupling model. This model is based on experiments demonstrating the functional coupling of voltage-gated C a  2 +  channels to RyR C a  2+  channels in skeletal muscle (79). In support of  the coupling model it has been shown that when a physical barrier is formed between the plasma membrane and the ER through dense polymerization of F-actin in smooth muscle cells, C a  2 +  influx is blocked after thapsigargin-induced store-depletion (85). Further  29  acceptance of this model was gained by the demonstration that the IP3R inhibitor, 2aminoethoxyborane (2-APB) prevented thapsigargin-induced C a /CRAC  influx and inhibited  2 +  in Jurkat T cells (79). Transient receptor potential (TRP) C a  2 +  channels, which are  one of the molecular candidates for C R A C channels, have also been shown to directly interact with IP3RS through coimmunoprecipitation of the TRP family member, TRP3, and IP3R (86). Although the studies involving TRP3 suggest that TRP3 activation requires interaction with  IP3R,  TRP3 does not exhibit all of the biophysical properties of  a C R A C channel (10). The studies disputing the conformational coupling model involve the examination of store-operated C a  2 +  entry pathways in the DT40 B cell line lacking all three IP3R  isoforms. In the DT40 IP3R deficient cells, thapsigargin-induced C a  2+  and  /_  /CRAC  influx is normal  is still present (66). In addition, treatment of the DT40 IP R cells with 2-APB  also inhibits  3  /CRAC,  suggesting that 2-APB does not selectively block IP Rs (79). Based 3  on these studies, it has been postulated that ER resident proteins, other than the IP3R, may physically couple to the C R A C channel (79). One alternative candidate for the coupling model may be RyR C a  2+  channels since it has been shown that the pharmacological  inhibitors of RyRs block thapsigargin-induced C a cells (87). RyR C a  2 +  2+  influx in the IP3R deficient DT40  channels are an attractive candidate for this model, especially  because knocking-down RyR3 expression in Jurkat T cells prevents sustained C a  2+  influx  (73).  30  1.4.3 Secretion Model Finally, the secretion model postulates that store-depletion initiates vesicular transport of activated C R A C channels to the plasma membrane followed by insertion of the channels into the membrane via vesicle fusion (Figure 1-3C) (19). Several studies have provided evidence to support the secretion mechanism by demonstrating that inhibitors of the secretion/exocytosis pathway prevent C a  2 +  influx in a variety of non-  excitable cells. The secretion mechanism is based on the original studies showing that /CRAC  activation was blocked by the vesicular transport inhibitors, GTPyS and primaquine  (88, 89). In addition, Yao et al. recently established that botulinum neurotoxin A and dominant negative mutants of SNAP-25 (synaptosome-associated protein of MW25), both inhibitors of SNAP-25 function in vesicle fusion, prevented Xenopus  /CRAC  activation in  oocytes (90). The previously described study that showed cortical actin  polymerization blocked store-operated C a  2 +  influx in smooth muscle cells may also be in  favor of the secretion model (85). The physical barrier formed by actin polymerization may have prevented exocytosis of vesicles containing the activated SOCs (19). Although there are many studies supporting the secretion model, this mechanism remains controversial since  /CRAC  activation is not affected by other clostridial neurotoxins that  disrupt vesicle secretion through cleavage of SNAP receptor proteins (10). At the present time, it is difficult to ascribe a particular mechanism connecting store-depletion to C a  2 +  influx in T lymphocytes. The studies that support either the  conformational coupling or secretion models seem to argue against the diffusible messenger model (91). Unraveling the mystery behind C R A C channel activation will generate a better understanding of Ca  signaling mechanisms in T lymphocytes, and may  31  lead to the development of specific pharmacological agents that mediate this process (35). Ultimately, discovering the molecular identity of the C R A C channel expressed in T lymphocytes will vastly aid in determining the mechanisms controlling store-operated Ca  2 +  influx.  1.5 Calcium Influx Pathways in T Cells  Although the C a Ca  2 +  2 +  channels that regulate stored C a  2 +  release from the intracellular  stores within T lymphocytes are well characterized, the molecular identity of the  C R A C channel that modulates C a  2 +  influx across the plasma membrane still remains  elusive despite the fact that it contributes to the majority of elevated intracellular Ca during T lymphocyte activation. The search for the T lymphocyte C R A C channel, displaying identical biophysical properties to / C R A C , has been partially hampered since the commonly used heterologous expression systems, such as Xenopus oocytes, express endogenous SOCs (10). Overexpression of an exogenous channel cDNA may cause the association of transfected channels to endogenous SOCs with variable stoichiometry, leading to the generation of a nonphysiological current that does not resemble  /CRAC  (10).  A further challenge in identifying the C R A C channel has been the lack of high-affinity ligands or blockers of this channel (10). There is also the possibility that a single gene product may be unable to form  /CRAC  on its own, and that  heteromultimeric channel complex (19). Several C a  2 +  /CRAC  rnay be gated by a  channels that may gate C a  2 +  influx  across the plasma membrane have been identified in T lymphocytes, including IP R Ca 3  channels, mammalian homologues of the Drosophila  melanogaster TRP C a  2 +  channels,  32  and L-type VDCCs. Presently, it is still under debate whether one of these Ca is solely responsible for  channels  /CRAC-  1.5.1 IP3 Receptor Calcium Channels Initially, investigators suggested that plasma membrane IP R C a  2+  3  similar to the IP3RS found in the ER, were responsible for C a As previously mentioned, IP3 binding to the 1P R C a 3  2 +  2 +  channels,  influx in T lymphocytes.  channel in the ER induces a  conformational change, causing the IP3R channel to open. It was hypothesized that as Ca  2 +  in the ER is depleted,  IP R may change 3  its conformation and this new  conformational state would communicate to plasma membrane IP3R C a  2+  channels,  causing the plasma membrane channels to open (71). A study conducted by Khan et al. identified UVsensitive C a  2 +  channels in the plasma membrane of Jurkat T cells through  patch-clamp recordings, and detected cell surface iodinated IP3RS through immunoblot analysis using an anti-IP R Ab (92). Immunohistochemical staining of Jurkat T cells and 3  human T lymphocytes with the anti-IP R Ab also identified plasma membrane localized 3  IP3RS and capping of IP3RS following Con A stimulation (92). Recently, a study confirmed that Jurkat T cells express IP3R C a  2 +  channels as integral plasma membrane  proteins (93). By labeling Jurkat T cells with the membrane-impermeant biotinylating reagent, sulfo-N-hydroxysuccinimide biotin, significant amounts of I P 3 R I , IP3R2, and IP3R3 were detected in the plasma membrane with isoform-specific Abs (93). However, the IP3R isoforms exhibit functional redundancy and are also abundantly expressed in the ER; defining the respective contributions of these channels to C a  2 +  influx during T cell  activation has therefore been difficult (93).  33  1.5.2 Transient Receptor Potential Calcium Channels The most well studied molecular candidates for the T lymphocyte C R A C channel are the mammalian homologues of the Drosophila years ago, TRP C a  2+  TRP C a  2 +  channels. Approximately 15  channels were first identified in mutant flies with impaired vision  that responded to continuous light with a "transient receptor potential" (94). Shortly thereafter, the mutated gene was identified as a C a  2 +  channel and called trp because of the  electrical phenotype of the fly mutants (95). In the Drosophila  retina, TRP Ca  channels  are activated through a PLC-p-dependent process (36). Once open, TRP C a  channels  mediate C a  2+  2+  influx that modulates a light-induced depolarizing current essential for  visual transduction (36). Early studies on Drosophila  TRP C a  2+  channels suggested that  TRPs may behave as SOCs through the demonstration that TRP channels open in response to store-depletion (96). The finding that TRP activation may be store-dependent initiated a search for TRP homologues. This led to the discovery of a large superfamily of TRP C a  2+  channel homologues that are ubiquitously expressed in worms, mice and  humans (97). Interestingly, during this time, an increasing amount of evidence began to demonstrate that Drosophila  TRP C a  2+  channels are activated through store-independent  mechanisms rather than by store-depletion (98). Mammalian homologues of the Drosophila  TRP C a  2 +  channels have been  categorized into three subfamilies based on sequence and structural homologies (Figure 1-4). The TRP canonical (TRPC) subfamily members display the greatest degree of sequence homology (30-47% over the amino-terminus) to the Drosophila  TRP C a  2 +  channels (99). Members of the TRP vanilloid (TRPV) subfamily are related to the vanilloid receptor 1 (TRPV1), whereas members of the TRP melastatin (TRPM)  34  The TRP superfamily • TRPC2  • TRPC4 . TRPC5 • TRPC1 • TRPC3 . TRPC7 • TRPC6  canonical A  . TRPVS • . • • •  vanilloid  melastatin  I  I  TRPV1 TRPV2 TRPV4 TRPV5 TRPvB  TRPM2 TRPM8 TRPM4 i TRPM5 • TRPM1 TRPM3 • TRPM6 • TRPM7  Figure 1-4: Phylogenic tree of the three mammalian TRP subfamilies. The arrangement of the TRP members in the phylogenic tree is based on sequence and structural homologies. TRP canonical (TRPC) subfamily members have the highest degree of homology to Drosophila TRP. TRP vanilloid (TRPV) subfamily members are most homologous to the vanilloid receptor 1. TRP melastatin (TRPM) subfamily members are most closely related to the tumor suppressor melastatin. The figure was taken from Nilius et al. ( 9 7 ) .  35  subfamily, formerly known as long-TRPs (LTRPs), have the highest sequence similarity to the tumor suppressor melastatin, known as TRPM1 (97). The membrane topology of the TRP C a  2 +  channels from all three subfamilies closely resembles the structure of  VDCCs. Each TRP protein consists of six transmembrane segments with the TRP channel pore formed between transmembrane segments 5 and 6, and the amino- and carboxyl-termini located in the cytoplasm (Figure 1-5) (97). Four TRP proteins are required to form a functional channel unit (100). One notable difference between the basic structure of TRPs and VDCCs is that TRP C a  2+  channels lack the conserved  positively charged amino acids in the fourth transmembrane segment that contribute to the voltage sensing properties of VDCCs (36). TRPC and TRPV members, but not TRPM, contain ankyrin-like binding repeats in the amino-terminus that are believed to aid in the association with the cytoskeleton (36). Multiple TRP subtypes are expressed in most cells, and consequently, it has been shown that TRP heteromultimers are formed both in vitro and in vivo (101, 102). In addition, the TRP subtypes are known to respond to a variety of stimuli, including temperature, pain, oxidative stress, hormones and light (36). Currently, the TRPV6 channel, also known as CaTl and ECaC2, appears to be the primary TRP gene candidate for the C R A C channel in T lymphocytes. Yue et al. first demonstrated that CaTl exhibited many biophysical properties of the C R A C channel when overexpressed in Chinese hamster ovary-Kl cells (103). Similar to displayed a high C a  2+  /CRAC,  CaTl  selectivity, small 40-picoSiemens (pS) single-channel conductance  (a measure of how many ions per second pass through a single channel, where conductance is the inverse of resistance, 1 Amp/1 Volt = IS), and appeared to be  36  N TRPC channel  N TRPV channel  TRPM channel  Figure 1-5: Membrane topology and structure of the three mammalian T R P subfamilies. The TRPC, TRPV, and TRPM subfamily members each consist of six transmembrane segments with the pore loop region between transmembrane segments 5 and 6. The amino-terminus of TRPC and TRPV contain ankyrin-like repeats (designated as A) that may allow association of the channels with cytoskeletal elements. The carboxyl-terminus of TRPC and TRPM contain a small, highly conserved stretch of 25 amino acids called the TRP domain (designated as T) (97). The endogenous kinase domain (shown in red) in TRPM members regulates the activity of the channel (36). This figure was taken from Venkatachalam et al. (36).  37  activated by depletion of Ca  stores (103, 104). Subsequently, CaTl transcripts were  detected in Jurkat T cells and human spleen through reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis (105). Further support for CaTl was achieved when a dominant negative pore-region mutant of C a T l , overexpressed in Jurkat T cells, partially suppressed endogenous  /CRAC  following thapsigargin-induced store-  depletion (105). Although these studies suggested that CaTl formed part of or the entire C R A C channel, Voets et al. revealed that rat basophil leukemia and human embryonic kidney cells overexpressing CaTl displayed many biophysical properties distinct from /CRAC  (106). Central differences between CaTl and C R A C were that CaTl is five times  more permeant to monovalent cations compared to the C R A C channel, and that the CaTl pore is blocked by intracellular magnesium ions, whereas in the C R A C channel this gating mechanism is absent (106). Further work on  /CRAC  has shown that the C R A C  single-channel conductance is only 0.2-pS, and not 40-pS as previously described by Yue et al. (107). It is now accepted that the 40-pS single-channel conductance is attributed to endogenous magnesium-inhibited cation channels (79). These discrepancies between CaTl  and C R A C have lead investigators to reinterpret previous results on C a T l ,  proposing that CaTl is not solely responsible for / C R A C (97). Even though the CaTl gene product does not appear to exhibit all of the electrophysiological properties associated with  /CRAC,  it still remains possible that CaTl may participate with additional TRP  subtypes, such as the highly homologous TRPV5 that is also expressed in Jurkat T cells, to form a functional C R A C channel (79). Further work on CaTl is required to definitely establish whether CaTl can recapitulate  /CRAC  when overexpressed with other TRP  subtypes.  38  TRP subfamily members that are activated by store-independent mechanisms have also been identified in T lymphocytes. The TRPM2 subfamily member, LTRPC2, was recently detected in Jurkat T cells and peripheral blood through RT-PCR (108). In T lymphocytes, LTRPC2 is a non-selective Ca channel that mediates C a 2+  2+  influx in  response to the intracellular second messengers, ADPR and N A D , but is not activated by +  store-depletion (108). A recent study by Gamberucci et al. demonstrated that the second messenger, D A G , is capable of generating influx of C a independent of intracellular C a  2 +  2+  in Jurkat T cells that was  store-depletion. Since the TRPC subfamily members,  TRPC3 and TRPC6 are known to be activated by D A G the expression of TRPCs was examined in Jurkat T cells and human PBTs (109). Although TRPC1, 3, 4 and 6 transcripts were identified through RT-PCR, only the TRPC6 protein was detected in purified plasma membrane fractions, suggesting that TRPC6 is activated by D A G in T lymphocytes (110). Taken together, these recent studies on TRP channel function demonstrate that store-independent C a may also participate in sustained C a  2 +  2 +  influx through LTRPC2 and TRPC6 channels  signaling during T cell activation. It is still under  debate whether LTRPC2 and TRPC6 C a  2 +  influx pathways are activated through a TCR-  dependent mechanism.  1.5.3 Voltage-Dependent Calcium Channels There is also evidence to support the existence of voltage-dependent-like C a  2 +  channels in the plasma membrane of T lymphocytes. The basis of the V D C C model is that non-excitable cells, such as T lymphocytes, may express a C a  2 +  channel that shares  common structural features with a V D C C of electrically excitable cells but is not gated  39  by changes in membrane potential. VDCCs are abundantly expressed in electrically excitable cells, such as neurons and muscle cells, and activated in response to depolarization of the plasma membrane (111). Membrane depolarization is a change in the electrical potential across the plasma membrane that renders the membrane less polarized (less negative) than the resting potential. In excitable cell types, C a  2 +  influx  through VDCCs acts as a second messenger, controlling many intracellular events such as contraction, secretion, synaptic transmission, and gene expression (112). Through patch-clamp and pharmacological studies, VDCCs have been classified into different channel families based on their distinct C a voltage-activated C a  2 +  2 +  currents (113). The high  channels that are activated at more positive membrane potentials  include L - (long lasting current), P- (Purkinje cell), Q- (granular cell), N - (neuronal), and R- (toxin-resistant) type channels (Figure 1-6) (113). The T-type (transient current) channels are designated as the low voltage-activated C a  2 +  channels due to their activation  at negative membrane potentials (112). VDCCs are heteromultimeric protein complexes consisting of the channel-forming ai-subunit and at least three auxiliary subunits called P, 0^5, and y, which control the structure and activity of the oti-subunit (111) (Figure 17). The primary structure of the ai-subunit is composed of four repeated motifs (I to IV), each consisting of six a-helical transmembrane segments (SI to S6) and a loop between S5 and S6 transmembrane segments that is membrane-associated and forms the channel pore (112). The S4 transmembrane segments, containing conserved positively charged amino acids, are termed the voltage sensors and are believed to move outwards upon membrane depolarization, opening the C a  2+  channel (114). The amino- and carboxyl-  termini of VDCCs reside in the cytoplasm.  40  Protein  Gene  Chromosome  Primary tissues  Calcium current  Cayl.T (a ) Ca.1.2 (a, ).  CACNA1S CACNA1C  1q32 12p13.3  L-type L-type  Ca,1.3 (a )  CACNA1D  3p14.3 .  Ca 1.4(a, ) Ca„2:1 (a, ) Ca„2.2 (a,„) Ca„2.3 (ai ).  CACNA1F CACNA1A CACNA1B CACNA1E  Ca»3.1(a, ,) Cav3,2(a, ) Ca„3.3 (a„)  CACNA1G CACNA1H CACNA1I  skeletal muscle heart smooth muscle : brain, heart, pituitary, adrenal brain, pancreas, kidney, ovary, cochlea retina brain, cochlea, pituitary brain, nervous system brain, cochlea, retina, heart, pituitary brain, nervous system brain, heart, kidney, liver brain  1s  c  1D  v  F  4  E  -  1  ••  1  20.  :  40  60  •  •  80  r  H  .  .  Xp11.23 . 19p13 9q34 1q25-31 . 17q22 16p13.3 22q12.3-13-2  L-type L-type . P/Q-type. N-type R-type T-type T-type T-type  . 100  Matching percentage using C L U S T A L  Figure 1-6: Nomenclature, chromosome location and tissue expression of the different channel-forming ai-subunits. Based on amino acid sequence identity and physiological properties of the cti -subunits of VDCCs, a cohesive nomenclature for the ai-subunits was established (115). High voltage-activated C a channels include the Cayl family of oti-subunits that conduct L type C a currents and the Cay2 family conduct that either P/Q-, N- or R-type currents. The Cay3 channel family conducts the low voltage-activated C a currents, which are Ttype currents, aj-subunits in the Cayl family have 75% amino acid identity to the skeletal muscle ocis-subunit (Cavl.l). The Cayl channel family in its entirety has approximately 40-50% amino acid identity to the Cay2 family, whereas the Cay3 family has less than 25% identity to both Cayl and Cay2 families (112). The figure was taken from Jurkat-Rott et al. (114). 2+  2 +  2 +  41  Figure 1-7: Model of the subunit structure and composition for V D C C s . VDCCs are oligomeric protein complexes that consist of the 170-250 kDa channelforming ai-subunit, as well as the auxiliary subunits; 50-78 kDa p-subunit, -170 kDa a25-subunit, and -36 kDa y-subunit (113). The ai-subunit is composed of four motifs (I to IV), which each contain six a-helical transmembrane segments that are represented as cylinders. The S4 transmembrane segments (red cylinders) in the a- -subunit contain evenly spaced, positively charge amino acids that contribute to the voltage sensing properties of the channel. The CLj and 8 proteins are heavily glycosylated and disulfidelinked (116). The auxiliary subunits modulate the activation and inactivation properties of the ai-subunit, as well as aid in targeting the ai-subunit to the plasma membrane (113). The figure was adapted from Catterall et al. (112).  42  Even though the 0Ci-subunits are similar in structure, the Ca' currents gated by these channels control many different cellular functions in electrically excitable cells. The L-type otiD-subunit (also designated as Cayl.3) in endocrine cells initiates the release of hormones, whereas the L-type aiF-subunit (Cayl.4) in retina controls neurotransmitter release (112). Other L-type C a  2 +  currents, including the currents gated by (Xis-subunit  (Cayl.l) in skeletal muscle and ocic-subunit (Cayl.2) in cardiac muscle, promote excitation-contraction coupling (112). The ( X I A - ,  OCIB-,  and R-type currents, respectively, are the primary C a  2 +  and aie-subunits that gate P / Q - , N currents in neurons (112).  Initial support for the presence of voltage-dependent-like C a  2 +  channels in T  lymphocytes came when Densmore et al. identified an electrically responsive current in the plasma membrane of Jurkat T cells through patch-clamp recordings (117, 118). This "voltage-operable" current in Jurkat T cells was activated through the TCR/CD3 complex and C a  2 +  store-depletion (117, 118). RT-PCR analysis has also shown that transcripts of  the pore-forming otic- and otis-subunits of L-type VDCCs are expressed in Jurkat T cells (119). Savignac et al. demonstrated that murine T cell hybridomas express L-type C a channel  messenger  R N A (mRNA)  and  protein  (120).  In  addition,  2 +  several  pharmacological studies examining the effects of DHPs, a class of synthetic derivatives that specifically modulate L-type V D C C function, have provided further evidence to support the existence of VDCCs in T lymphocytes. Although T lymphocytes are considered to be non-excitable cells, the functional significance of L-type VDCCs and their potential contribution to  /CRAC  has not been thoroughly investigated. Furthermore,  the hypothesis that voltage-dependent-like C a  2+  channels may control a novel C a  2 +  influx  pathway in T lymphocytes is an attractive model that has not received a great deal of  43  attention in recent years compared to TRP Ca  channels. To address these questions,  over the next three chapters, the expression of one channel-forming ai-subunit of L-type VDCCs and its putative role during T cell activation will be evaluated through both molecular and pharmacological analyses. Despite the importance of C a and regulation of C a  2 +  2 +  influx in T lymphocyte activation, the mechanism  entry into T cells is still under debate. A combination of the  proposed channel models may be necessary to maintain intracellular C a  2 +  levels required  for T cell activation. It is clear that future investigations, such as elucidating the mechanism coupling store-depletion to C R A C channel activation and the molecular identification of the C R A C channel, are necessary to clarify the mysteries surrounding Ca  2 +  entry in T lymphocytes.  44  CHAPTER 2: MATERIALS AND METHODS  2.1 Cell Lines and Culture Conditions The human retinoblastoma WERI-Rb-1 cell line and the human T cell leukemia line Jurkat clone E6-1 were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and maintained in RPMI (Roswell Park Memorial Institute) 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 2 m M glutamine, 20 m M 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid (HEPES) and 1 m M sodium pyruvate. The human cervical carcinoma HeLa cell line (ATCC) was maintained D M E M (Dubelco's Modified Eagle Medium) (Invitrogen) supplemented with 10% FBS, 2 m M glutamine, 20 mM HEPES, 1 m M sodium pyruvate, 100 U/ml penicillin and 100 U/ml streptomycin (StemCell Technologies Inc., Vancouver, Canada).  2.2 Isolation and Culture of Human Peripheral Blood T Lymphocytes Whole blood (10-50 ml) was collected from healthy human male and female donors (n=15). Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation at 900 x g for 30 min at 18-20°C over a Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ) gradient. The resulting P B M C layer was washed and resuspended in RPMI supplemented with 10% FBS, 2 m M glutamine, 20 m M HEPES and 1 mM sodium pyruvate. PBMCs were then stimulated for 24 h with 10 ug/ml platebound anti-CD3 mAb, OKT3 (ATCC), and resuspended in RPMI containing 5 ng/ml  45  recombinant human IL-2 (rhIL-2) (Serologicals Corporation, Norcross, GA). After growing for 7 d in rhIL-2, the purity of the human PBTs was analyzed on a fluorescentactivated cell sorter (FACS)Calibur cytometer (BD Biosciences, San Jose, CA) with fluorescein  isothiocyanate  (FITC)-conjugated OKT3, FITC-conjugated mouse anti-  immunoglobulin (Ig)G2a isotype control (Caltag, Burlingame, CA), Cy-Chrome-5conjugated anti-CD4 mAb (Pharmingen, San Diego, CA), phycoerythrin (PE)-conjugated anti-CD8 mAb (Pharmingen), allophycocyanin (APC)-conjugated anti-CD14 mAb (Pharmingen), FITC-conjugated anti-CD15 mAb (Pharmingen), and PE-conjugated antiCD19 mAb (Pharmingen). Experiments with the activated human PBTs were conducted with cells from day 8-14 in culture. The cultured human PBTs were used in assays where large numbers of T cells were required, such as measurement of C a  2 +  kinetics,  immunoblotting, IL-2 assay and IL-2R expression.  2.3 Cell Separations Whole blood (~20 ml) was collected from healthy human male and female donors (w=3). PBMCs were separated by centrifugation at 900 x g for 30 min at 18-20°C over a Ficoll-Paque PLUS  (Amersham Biosciences),  washed  and incubated with FITC-  conjugated OKT3, PE-conjugated anti-CD19 mAb (Pharmingen), or APC-conjugated anti-CD14 mAb (Pharmingen) to selectively label naive T cells, B cells and monocytes, respectively. The FACSVantage Sort Enhanced (SE) flow cytometry system (BD Biosciences) was used to sort the labeled cells into individual populations and assess the purity of the sorted cells. Magnetic cell sorting (MACS) CD4 and CD8 microbeads (Miltenyi Biotec, Auburn, CA) were used to positively select and separate human C D 4  +  46  and CD8  T cells from PBTs that were isolated and cultured from whole blood as  previously described. The purity of the C D 4 and C D 8 T cell populations was analyzed +  +  on a FACSCalibur cytometer (BD Biosciences) with FITC-conjugated OKT3, FITCconjugated mouse anti-lgG2 isotype control (Caltag), Cy-Chrome-5-conjugated antia  CD4 mAb (Pharmingen), and PE-conjugated anti-CD8 mAb (Pharmingen). The sorted naive T cells, B cells, monocytes, and C D 4 and C D 8 T cells were used in the nested +  +  RT-PCR experiments. For the study involving renal disease patients, whole blood (~10 ml) was obtained from the renal patients (n-3) prior to hemodialysis by Drs. Adeera Levin and Kathryn Tinckman in the Nephrology Division at St. Paul's Hospital, Vancouver, Canada. Whole blood (~10 ml) was also collected from a healthy human female donor («=1). Human CD3 PBTs were separated from whole blood using the Human T cell RosetteSep™ Ab cocktail (StemCell Technologies).  In brief, RosetteSep™ Ab cocktail (50 u,l per ml  blood) containing bispecific Ab complexes to CD16, CD19, CD36 and CD56 and glycophorin A was incubated with whole blood for 20 min at room temperature (RT) to cross-link unwanted cells to red blood cells. Enriched human C D 3 PBTs were isolated +  by centrifugation at 900 x g for 30 min at 18-20°C over a Ficoll-Paque PLUS (Amersham Pharmacia) gradient, removed from the density medium/plasma interface, and washed. The purity of the human C D 3 PBTs was then analyzed on a FACSCalibur cytometer +  (BD Biosciences) with FITC-conjugated OKT3, FITC-conjugated mouse anti-lgG2  a  isotype control, Cy-Chrome-5-conjugated anti-CD4 mAb, and PE-conjugated anti-CD8 mAb.  47  2.4 Nested R T - P C R o f oCiF-Subunit and DNA Sequencing First strand cDNAs were synthesized with an oligo 2'-deoxy-thymidine (dT) primer using 1 pig of total R N A extracted from WERI-Rb-1 cells, Jurkat T cells, human PBTs, C D 4 T cells, and C D 8 T cells with the RNeasy Kit (Qiagen, Alameda, CA). +  +  Marathon-ready human retina and human spleen cDNA were purchased from Clontech (Palo Alto, CA) and FirstChoice PCR-Ready human liver cDNA was from Ambion (Austin, TX). RT-PCR fragments spanning exons 28 to 35 of the ociF-subunit cDNA sequence,  (GenBank accession  Biotechnology  Information  number AF067227 from the National Centre for  database),  were  GGACCATGGCCCCATCTATAATTACCG-3')  generated and  with  sense  antisense  primer primer  (5'(5'-  C C T G A A G A G C C A C C T T G C C G A A C - 3 ' ) . For nested amplification of the oc, -subunit F  cDNA sequence, PCR fragments of -180 base pairs (bp) spanning exons 29 to 30 were generated with sense primer ( 5 ' - G A A C C C G C A T C A G T A T C G T G - 3 ' ) primer  (5'-AATAGTGAAGAGGCCAGTGAAGACC-3').  and antisense  The housekeeping  gene,  rig/Si 5, which encodes a small ribosomal subunit protein that is constitutively expressed in all tissues, was amplified with sense primer ( 5 ' - T T C C G C A A G T T C A C C T A C C - 3 ' ) and  antisense primer ( 5 ' - C G G G C C G G C C A T G C T T T A C G - 3 ' )  (121). RT-PCR and  nested PCR reactions were performed with Platinum Tag polymerase (Invitrogen) and were conducted in a Whatman Biometra UnoII Thermocycler at 94°C for 1 min, then 30 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 1 min, followed by a 10 min extension at 72°C. PCR fragments were resolved on a 1% agarose gel and visualized by staining with ethidium bromide. The resulting 178 bp fragments were subcloned into  48  pCR2.1-T0P0 vector (Invitrogen) and the nucleotide sequence determined using standard ml3R primer at the D N A Sequencing C O R E Facility, University of Florida, FL. Nested RT-PCR was also used to investigate the mRNA expression of the two splice variants of the aiF-subunit in Jurkat T cells, human PBTs, C D 4 T cells, C D 8 T +  +  cells, and peripheral blood B cells and monocytes. Total R N A was extracted from the cells using the RNeasy Kit (Qiagen). 1 ug of total R N A was used to synthesize first strand cDNAs with an oligo(dT) primer. RT-PCR fragments of the aiF-subunit cDNA sequence were generated as previously described. For nested amplification of the voltage negative splice variant of the oc-F-subunit, PCR fragments (-240 bp) spanning exons 29 to 35 were generated with sense primer (5 ' - C C C A A G A A C C C G C A T C A G T A T C G T G - 3 ' ) and an antisense primer ( 5 ' - A G C C A C C T T G C C G A A C A T C T T G G G C T T - 3 ' ) , which was designed to specifically overlap the exon 30 to 35 splice junction. To amplify the voltage positive splice variant of the a-F-subunit, nested PCR was conducted using the sense primer ( 5 ' - C C C A A G A A C C C G C A T C A G T A T C G T G - 3 ' ) and the antisense primer (5'A C T G A G A A G C T T G A C C A G C C G C A T A A C - 3 ' ) to generate a -380 bp PCR fragment spanning exons 29 to 33. The rig/Si 5 house-keeping gene served as a loading control. RT-PCR and nested PCR reactions were performed with Advantage-GC 2 polymerase (Clontech) and were conducted in a Whatman Biometra Unoll Thermocycler at 94°C for 1 min, then 40-45 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min, followed by a 10 min extension at 72°C. An ethidium bromide stained 1 % agarose gel was used to resolve the PCR fragments. The PCR products were subcloned into pCR2.1TOPO vector (Invitrogen) and sequenced using the ml3R primer at the Florida D N A Sequencing CORE Facility.  49  2.5 Cloning of (XiF-Subunit cDNA from Human Retina and Spleen Libraries Marathon-ready human retina and human spleen cDNA libraries (Clontech, Palo Alto, CA) were used to clone the wild type and alternatively spliced oci F-subunit cDNA sequences, respectively. The 3'-terminal sequence of the aiF-subunit cDNA sequence, corresponding to nucleotides 3328-5813 (nucleotides numbered according to GenBank accession number AF067227), from both libraries was cloned using rapid amplification of cDNA ends (RACE) with gene specific sense primers, adaptor antisense primers, and the Advantage-GC 2 polymerase in the presence of 0.5 M G C Melt (Clontech). The gene specific  sense  primer  for  the  GGACCATGGCCCCATGTATAATTACCG-3'  first and for the  PCR  was  nested PCR was  5'5'-  C C C A A G A A C C C G C A T C A G T A T C G T G - 3 ' . The remainder of the a, -subunit cDNA F  sequence from human retina and spleen libraries was cloned by a series of nested PCR reactions with sense and antisense primers overlapping exon/exon boundaries using the Advantage-GC 2 polymerase. Three individual PCR products were generated from the nested PCR reactions, which correspond to nucleotides 1106-1898, 1750-2635 and 24423546 of the ctiF-subunit cDNA sequence (Figure 2-1). The 5'-terminal sequence of the (Xi F-subunit cDNA (1-1320 bp) was cloned using nested PCR with a 60-oligonucleotide sense  primer  beginning  with  the  5'-ATG  start  codon,  5'-  ATGTCGGAATCTGAAGGCGGGAAAGGTGAGAGAATCCTTCCATCCCTGCAGA C C C T T G G A - 3 ' , and an antisense primer overlapping an exon/exon boundary. A l l PCR products were subcloned into pCR2.1-TOPO vector (Invitrogen) and sequenced using standard ml3R and T7 primers at the Florida D N A Sequencing C O R E Facility. Subsequently, the full-length cDNAs of the retina ai F-subunit and voltage negative  OCIF-  50  cx1 F-Subunit cDNA Sequence:  5813 bp 1000  2000  3000  4000  5000  *Bbsl(3487 bp) *Sacll (2598 bp)  1750>-2635 bp *Sphl (1826 bp) 1106 M 898 bp  *Aatll (1151 bp)  Figure 2-1: PCR cloning strategy used to isolate otiF-subunit cDNA sequences from human retina and spleen cDNA libraries. The coding region of the human retina (XiF-subunit is 5813 bp. Five PCR fragments, represented as black boxes, correspond to different coding regions of the 0Ci F-subunit that were amplified from both the human retina and spleen cDNA libraries, using a i F-subunit specific primers. The PCR fragments specifically correspond to 1-1320 bp, 1106-1898 bp, 1750-2635 bp and 2442-3546 bp and 3328-5813 bp of the a, -subunit cDNA sequence (GenBank accession number AF067227). Unique, endogenous restriction enzyme sites, including Aatll, SphI, SacII and Bbsl, were used to ligate the five PCR fragments together to form full-length cDNA sequences for human retina and spleen (voltage negative splice variant) aiF-subunits. The location of each restriction enzyme site in the otiF-subunit cDNA sequence is indicated by the bp position. F  51  subunit splice isoform amplified from human spleen were constructed through ligation of the individual nested PCR products by a single, common restriction enzyme site in the overlapping regions (Figure 2-1). Once the individual PCR products were ligated together, the full-length cDNAs were subcloned into pCR2.1-TOPO vector.  2.6 Production of Carboxyl-Terminal FLAG-tagged otiF-Subunit The coding regions of retina 0CiF-subunit previously subcloned in pCR2.1-TOPO vectors (Invitrogen) was amplified by PCR using the NotI containing sense primer (5'GCAAGCGGCCGCCATGTCGGAATCTGAAGGCGGGAA-3')  and  the  Sail  containing antisense primer (5 ' - C G C G T C G A C T G A G G G C G T G G A C G C A G G C C A T - 3 ' ) . A PCR fragment of ~5.8 kilobases (kb) with the T A G stop codon removed was generated for the retina aiF-subunit, using the Advantage-GC 2 polymerase (Clontech). PCR reactions were conducted in a Whatman Biometra UnoII Thermocycler at 94°C for 3 min, then 25 cycles of 94°C for 1 min, 68°C for 1 min, and 72°C for 6 min, followed by a 10 min extension at 72°C. After digestion with NotI and Sail (New England BioLabs), the amplified retina aiF-subunit was subcloned in-frame with sequences encoding a F L A G tag into the pIRES-hrGFP-1 a mammalian expression vector (pGFP) (Stratagene, La Jolla, CA) (Figure 2-2). The resulting pGFP/Retina ociF-subunit vector dicistronically expressed the retina aiF-subunit with a FLAG-tag at the carboxyl-terminus and humanized recombinant green fluorescent protein (hrGFP), which served as a marker for transfected cells. After ligation into the pGFP vector, the nucleotide sequence of the retina a , F  subunit insert was determined by primer walking at the Florida D N A Sequencing C O R E Facility.  52  C M V promoter 1 6 0 2 multiple cloning site 651-715 3x FLAG tag 7 1 6 - 7 8 7 internal ribosome entry site 8 2 3 - 1 3 9 7 hrGFP ORF 1 4 0 7 - 2 1 2 3 SV40polyA 2188-2571 fl origin 2 7 0 9 - 3 0 1 5 LoxP sequence 3178-3211 ampicillin resistance (bla) ORF 3 2 5 6 4 1 1 3 p U C origin 4 2 6 0 - 4 9 2 7  pIRES-hrGFP-la Multiple Cloning Site Region (sequence shown 651-727) Sad" I  Sac II* I  NotI* I  I Smo !/Xmo I I I  BamH I I  EcoR I I  GA G C T C C A C C G C G G T G G C G G C C G C T C T A G C C C GGG C 6 G A T C C G A A T T C  . . .  STOP-  Sph I I ...GC  Sai I I  Xho I I  S  |  a r t a  I  f py^o tag * ;  A T G CGT CGA C T C GAG GAC T A C A A G GAT  Figure 2-2: The p I R E S - h r G F P - l a mammalian expression vector. The pIRES-hrGFP-1 a vector (Stratagene) contains a dicistronic expression cassette where the multiple cloning site is followed by an internal ribosomal entry site (IRES) linked to the hrGFP coding sequence. The aiF-subunit was ligated into the multiple cloning site through NotI and Sail restriction enzyme sites, and the expression of the ai -subunit was under the control of the cytomegalovirus (CMV) promoter. The carboxyl-terminus of the OtiF-subunit was also fused in-frame to three contiguous copies of the F L A G epitope (DYKDDDDK). The pIRES-hrGFP-la vector lacks drug-resistant markers for stable expression in mammalian cells. F  53  2.7 Immunofluorescence Staining 2.5x10 HeLa cells grown on coverslips were transiently transfected with either 2 4  p.g of pGFP or pGFP/Retina 0Ci F-subunit vectors by using FuGene 6 (Roche). 72 h after transfection, FLAG-tagged protein expression was determined following the methods of Moise et al. (122). In brief, cells were fixed with 2% paraformaldehyde for 20 min and permeabilized with 0.1% saponin in 2% bovine serum albumin (BSA) for 15 min, where indicated. Cells that were not permeabilized were incubated with 15% B S A for 15 min. 2% B S A was used to block cells for 1 h, followed by staining with the mouse M2 antiF L A G mAb (Sigma) in 2% BSA for 30 min. After mAb incubation, cells were washed thoroughly with 2% BSA, stained in the dark with Alexa Fluor 568 goat anti-mouse Ab (Molecular Probes, Eugene, OR) in 2% BSA for 30 min at RT, and washed with 2% BSA. Cells were then treated with SlowFade Antifade (Molecular Probes), and staining was analyzed by confocal microscopy with a Bio-Rad Radiance Plus on an inverted Zeiss Axiovert with DIC optics and Lasersharp software (Bio-Rad).  2.8 Immunoprecipitation Analysis of ocir-Subunit 20-30x10  6  WERI-Rb-1 cells, Jurkat T cells (untreated or OKT3 and 12-0-  tetradecanoylphorbol 13-acetate (TPA) stimulated) and human PBTs were washed and detergent-lysed in 500 ul of solubilization buffer containing 50 m M Tris-HCl (pH 7.5), 300 m M NaCl and 0.5% Triton X-100 in the presence of 10 jig/ml soybean trypsin inhibitor, pepstatin, and 40 |J.g/ml phenylmethanesulfonyl fluoride (PMSF) (Sigma, St. Louise, MO) for 45 min on ice. Protein concentration of the lysates was quantified by  54  Bicinchonic acid protein assay (Pierce, Rockford, IL).  Affinity-purified antibodies  against a human retina 0Ci -subunit peptide (85-90 amino acids of cytosolic domain I-II F  interlinker sequence) were raised in rabbit and used to detect cci F-subunit (provided by Dr. John Mcrory, University of Calgary, Calgary, Canada). Aliquots of the membraneenriched lysates (1.5 mg of protein) were incubated overnight at 4°C with either 2 u\g of ai F-subunit Ab or affinity-purified rabbit IgG Ab (Sigma) as a control. 30 ul of recombinant protein G sepharose (Amersham Pharmacia) was added for 1 h at 4 ° C and Ab/oti F-subunit complexes bound to protein G were then washed 2 x with 10 m M TrisHCl (pH 7.5), 0.15 M NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.2 % Nonidet P-40, 1 x with 10 m M Tris-HCl (pH 7.5). 0.5 M NaCl, 2 mM E D T A , 0.2 % Nonidet P-40, and 1 x with 10 m M Tris-HCl (pH 7.5) at 4°C. Samples were denatured by boiling in sodium dodecyl sulfate (SDS) sample buffer, run on 8% SDS-polyacrylamide gel electrophoresis (PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membrane. Western blot analysis was performed with affinity-purified cci F-subunit rabbit polyclonal Ab. Simultaneously, a Western blot for glyceraldehyde-3-phosphate  dehydrogenase  (GAPDH) expression was performed as a protein loading control. In brief, 10 pig of each protein lysate was denatured by boiling in SDS sample buffer, loaded on 12% SDSP A G E gel and transferred to nitrocellulose  membrane. Western blot analysis was  conducted with mouse anti-GAPDH mAb (Chemicon, Temecula, CA). ai -Subunit F  protein expression in Jurkat T cells stably expressing pSUPER, pSUPER-aiF-subunit constructs  or  pEYFP  alone  were  also  analyzed  through  aiF-subunt  protein  immunoprecipitation.  55  2.9 Flow Cytometry Analysis of (XiF-Subunit aiF-Subunit protein expression was determined through flow cytometry by staining 0.1 % saponin (Sigma) treated or untreated WERI-Rb-1 cells, Jurkat T cells or human PBTs with affinity-purified ctiF-subunit rabbit polyclonal Ab and FITC-conjugated goat anti-rabbit IgG Ab (Jackson ImmunoResearch, West Grove, PA). As a control for Ab specificity, ociF-subunit Ab was preincubated for 15 min at RT with the immungen (85-90 amino acid peptide of domain I—II interlinker region of human retina cciF-subunit conjugated to the Caulobactor  RSA protein) in a 1:1 ratio. Ab/immunogen complex was  directly added to saponin treated cells and FL1 fluorescence was detected by the addition of FITC-conjugated goat anti-rabbit IgG Ab. As an additional control, cells treated with saponin were also stained with affinity-purified rabbit IgG Ab (Sigma) followed by FITC-conjugated goat anti-rabbit IgG Ab. All data acquisition was performed on a FACSCalibur cytometer (BD Biosciences).  2.10 Real-Time PCR Quantitative detection of the voltage negative and positive splice variants of the cci F-subunit, and the LTRPC2 Ca  channel was carried out by real-time PCR with SYBR  Green Taq Readymix (Sigma) on a LightCycler Instrument (Roche, Indianapolis, IN). For the real-time PCR experiments, the Human T cell RosetteSep™ Ab cocktail (StemCell Technologies) was used to isolate naive human PBTs from whole blood (20 ml) of healthy human donors (n-3), as previously described. Jurkat T cells or naive human PBTs at l x l O cells/ml were either untreated or stimulated with 10 p.g/ml soluble 6  56  0KT3 and 10 nM TPA (Sigma) for 1 min, 5 min, 10 min, 1 h, and 4 h at 37°C. Following stimulation, total RNA was extracted from cells using the RNeasy Kit (Qiagen) and 1 (ig of total R N A was used to synthesize first strand cDNAs with an oligo(dT) primer. For amplification of the voltage negative and positive splice variants of the oti F-subunit, nested PCR was performed on RT-PCR reactions (diluted 1:20 for Jurkat T cells and 1:10 for PBTs) with the previously described splice variant specific primers. The LTRPC2 gene was amplified from cDNA (diluted 1:20 for Jurkat T cells and 1:10 for PBTs) with sense primer ( 5 ' - T C T C C G G C G C A G C A A C A G C A - 3 ' ) and antisense  primer (5'-  C C C T C G C G G C G G T G G A C A G T - 3 ' ) to generate a -660 bp PCR product (108). The rig/Si 5 gene amplified using 1:20 diluted Jurkat T cell and 1:10 diluted PBT cDNA, served as a control to normalize expression of the C a  2 +  channel mRNA. Real-time PCR  was conducted at 95°C for 300 sec, then 30 cycles of 95°C for 5 sec, 55-66°C for 5 sec (depending on the cDNA sequence amplified), and 72°C for 30 sec, followed by a 30 sec cooling to 40°C. PCR products were resolved on a 1% agarose gel and visualized by staining with ethidium bromide to confirm fragment size. Real-time PCR data analysis was conducted by first calculating the ACt value (ACt = Ct (cDNA of interest) - Ct (SI 5 cDNA)) of each sample, where Ct is defined as the cycle number at which fluorescence passes through the fixed threshold, and is inversely proportional to the template starting copy number. The AACt value is then calculated by subtracting the ACt of the untreated control from the ACt of the treated sample (AACt - ACt (treated sample) - ACt (untreated sample)). induction is derived from the equation 2"  AACt  Finally, the fold  , where in each experiment the untreated  57  sample has a fold induction of one. A l l formulations were derived from Applied Biosystems, Foster City, C A .  2.11 Construction of Plasmids for Stable siRNA Expression The mammalian expression vector, pSUPER (suppression of endogenous RNA), was used for stable expression of small interfering R N A (siRNA) in Jurkat T cells (provided by Dr. Reuven Agami, Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, Netherlands) (Figure 2-3). Four siRNA target sequences (19 nucleotides in length) against the oc-F-subunit mRNA sequence were chosen using Ambion and Oligoengine (Seattle, WA) Web-Based siRNA Target Finder and Design Tools. The 19 nucleotide target sequences began with two adenine nucleotides at the 5'terminus, contained 40-60% G C nucleotides, and were located in the coding region of the 0CiF-subunit mRNA at least 100 bp after the start of translation. As a control for successful mRNA knockdown, a siRNA target sequence directed against p53 mRNA was also selected (123). The Blastn Program confirmed that the chosen 19 nucleotide sequences did not cross-react with other mRNA sequences. According to the methods of Brummelkamp et al, the siRNA sequences were each synthesized as forward and reverse 64 bp synthetic D N A oligonucleotides (Sigma), containing the 19 nucleotide sense target sequence, which was separated by a 9 nucleotide non-complementary spacer (tctcttgaa) from the reverse complement of the same 19 nucleotide target sequence (Table 2-1) (123). The 64 bp forward and reverse oligonuleotides were annealed at 70°C, phosphorylated with T4 polynucleotide kinase (Invitrogen), and ligated into the pSUPER vector after digestion with Bglll and Hindlll (New England BioLabs Inc., Beverly, MA).  58  H1-RNA Promoter  Target Sequence (sense strand shown)  Figure 2-3: The mammalian expression pSUPER directs stable synthesis of siRNA transcripts. The polymerase 111 H I - R N A gene promoter in the pSUPER vector directs the synthesis of siRNA transcripts that form short hair-pin structures with a 19 bp double stranded region and a short 9 bp loop formed from the spacer region. The siRNA transcript lacks a polyadenosine tail, but contains a termination signal consisting of a row of five thymidines. The hairpin transcript also contains two uridines at the 5'-end of the 9 bp loop, as well as two uridines at the 3'-terminus of the transcript. When the pSUPER vector is expressed, the hairpin loop is cleaved by the endogenous Dicer enzyme and a final siRNA transcript is formed with 5'- and 3'-UU overhangs, which is an optimal siRNA structure for targeted gene silencing. The pSUPER vector also lacks drug-resistant markers for stable expression in mammalian cells, and therefore requires co-transfection with pEYFP.  59  Forward and Reverse 64 bp siRNA Oligonucleotides  pSUPER Constructs  Target mRNA Region  pSUPERaiF-subunit-1  288-306 bp  pSUPERaiF-subunit-2  489-507 bp  pSUPERaiF-subunit-3  600-618 bp  TCCCAGGAAGTAGCACGTCr7T7TGGA4^-3'  pSUPERaiF-subunit-4  750-768 bp  CAGCATGGCGAAGAAGAAGrrrrrGG^^-3'  pSUPERp53  838-856 bp  5'-GJ7rCCCTGGCTGGAACCTACTCGACttcaagaga GTCGAGTAGGTTCCAGCCA7y7T7;GGA44-3' 5-AGCTTTTCCAAAAATGGCTGGAACCTACTCGAC tctcttgaaGTCGAGTAGGTTCCAGCCAGGG-3' 5-G^rCCCCTTCCATCATGAAGGCTCTGttcaagaga CAGAGCCTTCATGATGGAA7TZ'7TGGA4-3' 5 -yiGCrrrrCC^^^TTCCATCATGAAGGCTCTG tctcttgaaCAGAGCCTTCATGATGGAAGGG-3' 5'-GJrCCCCGACGTGCTACTTCCTGGGAttcaagaga 5' -A GCTTTTCCAAAAA GACGTGCTACTTCCTGGGA tctcttgaaTCCCAGGAAGTAGCACGTCGGG-3' 5-GytrCCCCCTTCTTCTTCGCCATGCTGttcaagaga 5 -JGCrrrrCOt^^CTTCTTCTTCGCCATGCTG tctcttgaaCAGCATGGCGAAGAAGAAGGGG-3' 5'-GA r C C C C G A C T C C A G T G G T A A T C T A C t t c a a g a g a GTAGATTACCACTGGAGTCT7T7TGGX4.4-3' 5' -A GCTTTTCCAAAAA GACTCCAGTGGTAATCTA tctcttgaaGTAGATTACCACTGGAGTCGGG-3'  Table 2-1: pSUPER constructs used for stable expression of siRNA in Jurkat T cells. siRNA target sequences were generated against four regions of the human retina O C I F subunit mRNA sequence (GenBank accession number AF067227), and one region of the human p53 mRNA sequence (GenBank accession number AB082923). The target mRNA region corresponds to the number of nucleotides downstream of the translation start site of either the aiF-subunit or p53 mRNA, where appropriate. For the 64 bp oligonucleotides, the bold, capitalized text refers to the sense and antisense 19 nucleotide siRNA target sequences, whereas the lower cased text corresponds to the 9 nucleotide spacer. The italicized text refers to nucleotides at the 5'- and 3'-termini of each 64 bp oligonucleotide.  60  If the insertion of siRNA sequences into the pSUPER vector was successful, vectors digested with EcoRl and Hindlll (New England BioLabs) contained inserts of 300 bp. For further verification, the inserted siRNAs were sequenced with standard T3 and T7 primers at the Florida D N A Sequencing C O R E Facility.  2.12 Generation of Stable Cell Lines Expressing siRNA l x l O Jurkat T cells were washed and resuspended in Opti-MEM (Invitrogen). 7  Cells were co-transfected with a 10:1 ratio of Sca-I (New England BioLabs) linearized pSUPER constructs (2 pg) and non-linearized pEYFP-Nl vector (0.2 u\g) (Clontech) by electroporation using a Bio-Rad Gene Pulser Electroporator (Hercules, CA) set at 250 V, 975 p.F. As controls, Jurkat T cells were also transfected with either 2 u.g pSUPER without a siRNA insert, or 0.2 p:g pEYFP-Nl vector alone. The pEYFP vector was used since expression of the amino-terminus of the enhanced yellow fluorescent protein (EYFP) served as a transfection marker and the neomycin resistance cassette served as a selectable marker. Bulk cultures of transfected Jurkat T cells were sorted twice by FACSVantage SE flow cytometer (BD Biosciences) based on EYFP expression, and stable cultures were selected with 2 p:g/ml Geneticin (Invitrogen). Clonal cell lines were generated by a third sort of transfected cells into 96-well plates by FACSVantage SE flow cytometer. After the sort, cell lines continued to grow in 2 p.g/ml Geneticin. To check for stable integration of pSUPER constructs, genomic D N A was purified from the clonal cell lines using the Genomic D N A Isolation Kit (Sigma). For amplification of the pSUPER construct from genomic DNA, PCR fragments of -550 bp spanning either side of  the  64  bp  inserts  were  generated  with  sense  primer  (5'-  61  CACGACGTTGTA AAACGACGG-3')  and  antisense  primer  (5'-  A C T T T A T G C T T C C G G C T C G T A T G - 3 ' ) . PCR reactions were performed with Platinum Taq polymerase (Invitrogen) and were conducted in a Whatman Biometra UnoII Thermocycler at 94°C for 1 min, then 30 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 1 min, followed by a 10 min extension at 72°C. PCR fragments were resolved on a 1% agarose gel and visualized by staining with ethidium bromide. To assess the gene silencing effects of siRNA, p53 and 0CiF-subunit protein expression were analyzed through immunoprecipitation experiments.  2.13 Immunoprecipitation Analysis of p53 20-30x10 Jurkat T cells stably expressing pSUPER, pSUPER-p53 or pEYFP alone 6  were washed and lysed in 500 ul of solubilization buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% NP-40, 5 mM E D T A in the presence of 10 LXg/ml soybean trypsin inhibitor, pepstatin, and 40 p.g/ml PMSF. Protein concentration of the lysates was quantified by Bicinchonic acid protein assay (Pierce). 6.0 mg aliquots of lysate were incubated overnight at 4°C with 10 ug of mouse anti-human p53 mAb, clone BP53-12 (Upstate Biotechnology, Lake Placid, NY). 30 ul of recombinant protein G sepharose (Amersham Pharmacia) was added for 1 h at 4 ° C and mAb/p53 complexes bound to protein G were then washed 2 x with 10 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 2 mM E D T A , 0.2 % Nonidet P-40, 1 x with 10 mM Tris-HCl (pH 7.5). 0.5 M NaCl, 2 mM E D T A , 0.2 % Nonidet P-40, and 1 x with 10 mM Tris-HCl (pH 7.5) at 4°C. Samples were denatured by boiling in SDS sample buffer, run on 12% SDS-PAGE gel and  62  transferred to nitrocellulose membrane. Western blot analysis was performed with affinity-purified p53 rabbit polyclonal Ab (Cell Signaling Technology, Beverly, MA). Western blot for G A P D H expression was also performed as a protein loading control, as previously described.  2.14 Measurement of Intracellular Calcium Levels Intracellular C a 1  acetoxymethyl  2+  levels were measured using the ratiometric C a  ester  dye  (Molecular  Probes)  according  to  2+  indicator indomanufacturer's  recommendations. In brief, Jurkat T cells or human PBTs (donors, n=3) at l x l 0 cells/ml 7  were loaded with 1 u M indo-1 for 1 h at 37°C in M E M (Minimum Essential Medium) (Invitrogen). For analysis, 100 ui of cell suspension (lxlO cells) was added to either 1.9 6  ml of M E M or Ca -free S - M E M (Invitrogen). Indo-1 loaded T cells were then examined 2+  for 10 min time periods following induction at the 2 min mark with either 10-100 LtM (+/-) Bay K 8644 (Calbiochem, San Diego, CA), 2 | l M ionomycin (Calbiochem) or dimethyl sulfoxide (DMSO) solvent using a FACSVantage SE flow cytometer system (BD Biosciences). Jurkat T cells and human PBTs loaded with indo-1 were also preincubated with 1200 U.M nifedipine (Calbiochem) or DMSO with or without extracellular C a  2 +  in the  medium for 10 min. At the 2 min mark, Jurkat T cells were stimulated with 10 Lig/ml soluble OKT3, whereas human PBTs required a combination of 2 Ltg/ml soluble antiCD28 mAb (Sigma), 10 Ltg/ml soluble OKT3 and 40 Ltg/ml soluble rabbit anti-mouse IgG polyclonal Ab (Sigma), which served as a cross-linking Ab, to activate C a  2 +  influx.  63  Anti-CD3 stimulation using the OKT3 mAb alone did not activate Ca The change in  [Ca ]i 2+  influx in PBTs.  was determined by calculating the average ratio of emission signals  of indo-1 at 405 nm and 485 nm, representing the ratio of Ca -bound to Ca -free indo-1, 2+  2+  respectively, using FlowJo software. In all experiments, the amount of DMSO solvent was equal to or less than 0.5% of the total treatment volume. (+/-) Bay K 8644 and nifedipine were both prepared in the dark as a 200 m M stock solution dissolved in DMSO.  2.15 Immunoblot Analysis of Phospho-p44/p42 M A P Kinase Jurkat T cells or human PBTs were washed, resuspended at l x l O cells/ml in RPMI 7  and incubated for 4 h at 37°C. Cells were then preincubated with or without 2 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) for 15 min to chelate C a , followed by 10 min stimulation with either (+/-) Bay K 8644 or 2 U.M 2+  ionomycin at 37°C. Jurkat T cells were also preincubated with either DMSO, 100 (iM or 200 u M nifedipine for 1 h, followed by 10 min stimulation with 100 | l M (+/-) Bay K 8644 at 37°C. Additionally, as a positive control for phospho-p44/42 mitogen-activated protein (MAP) kinase activation, Jurkat T cells were stimulated with 10 |lg/ml soluble OKT3 for 10 min at 37°C. Following stimulation, cells were lysed in 200 u.1 of lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% NP-40, 5 mM E D T A , 1 m M sodium vandadate, 5 m M sodium fluoride, 1 m M sodium molybdate, and 5 mM f3 glycerol phosphate, in the presence of 10 |lg/ml soybean trypsin inhibitor, pepstatin, and 40 |lg/ml PMSF. Cell lysates were denatured by boiling in SDS sample  64  buffer, run on 12% SDS-PAGE gel and transferred to nitrocellulose membrane. Western blot analysis was performed with phospho-p44/42 M A P kinase rabbit polyclonal Ab (Cell Signaling Technology). After development, the blots were stripped in 62.5 m M Tris-HCl (pH 7.5), 0.2% SDS and 100 mM 2-mercaptoethanol for 30 min at 50°C and then reprobed with Erkl/2 (K-23) polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA) as a protein loading control.  2.16 NFAT-Luciferase Assay l x l O Jurkat T cells were washed and resuspended in Opti-MEM (Invitrogen). 7  Cells were incubated with either 20 (ig of pNFAT-TA-Luc or pTA-Luc (Clontech) (Figure 2-4) for 5 min at 4°C and transfected by electroporation using a Bio-Rad Gene Pulser Electroporator set at 280 V , 975 up. 40-48 h after transfection, cells at l x l 0  6  cells/ml were incubated with nifedipine (1-200 U.M) or DMSO for 1 h at 37°C, followed by stimulation with 10 |ig/ml soluble OKT3 for 6 h at 37°C. NFAT-dependent luciferase activity was assayed on 1x10 cells/100 ul using the procedures outlined in the Brights  Glo Luciferase Assay System (Promega, Madison, WI). Luciferase activity was measured in a microplate luminometer.  2.17 IL-2 Assay l x l O Jurkat T cells or human PBTs in 1.0 ml RPMI were incubated with DMSO 6  or nifedipine (1-200 U.M) for 1 h at 37°C. Cells were then transferred to a 24-well plate immobilized with 10 pig/ml OKT3, 10 nM TPA was added, and cells were incubated at  65  Nhei (22)  A. = NFAT c/s-acting enhancer element TB = Transcription Blocker .  Figure 2-4: The pNFAT-TA-Luc vector monitors NFAT-mediated signaling transduction pathways in mammalian cells. The pNFAT-TA-Luc vector (Clontech) contains three copies of the N F A T consensus sequence located upstream of the T A promoter, which consists of the T A T A box from the herpes simplex virus thymidine kinase promoter. Following the promoter sequence is the firefly luciferase reporter gene. Transcription of the luciferase reporter gene is activated once endogenous N F A T transcription factors bind to the N F A T cw-acting enhancer element. To ensure efficient processing of the luciferase transcript in mammalian cells, the SV40 late polyadenylation signal is downstream of the luciferase gene. The pTA-Luc vector (Clontech) is similar to pNFAT-TA-Luc, except for it lacks the NFAT consensus sequences. pTA-Luc is used as a control for monitoring constitutive levels of luciferase activity in transfected cells.  66  37°C. After 24 h, supernatants were quantified for IL-2 concentration by a standard sandwich Enzyme-Linked Immunosorbent Assay (ELISA) technique (R&D Systems, 2_|_  Minneapolis, MN). To determine whether the Ca  ionophore, ionomycin, could reverse  the inhibitory effect of nifedipine, Jurkat T cells or human PBTs at l x l O cells/ml were 6  incubated with either DMSO or 1-50 u M nifedipine for 1 h. Cells were then stimulated for 24 h with 10 ug/ml plate-bound OKT3, 10 n M TPA and, where appropriate, 2 u M ionomycin with nifedipine remaining in the medium. The concentration of IL-2 in the supernatants was quantified by sandwich ELISA. To assess T cell function in renal patients administered DHPs, normal PBTs from a healthy donor and PBTs isolated from renal disease patients at l x l O  6  cells/ml were  stimulated for 24 h at 37°C with 10 ug/ml plate-bound OKT3 and 10 nM TPA. Following the 24 h incubation, the sandwich ELISA was used to quantify IL-2 concentration in the supernatants. To determine the effects of uremic serum on T cell function, normal and uremic serum were separated from whole, coagulated blood of a healthy donor and renal disease patients, respectively, by centrifugation at 900 x g for 30 min. Normal PBTs (from a healthy donor grown in culture for 1 week with 5 ng/ml rhlL-2) at l x l 0 cells/ml 6  were incubated with medium containing either 10% FBS alone, or medium with FBS supplemented with 10% normal serum or 10% uremic serum for 1 h at 37°C. With the normal human and uremic serum remaining in the medium, cells were then transferred to a 24-well plate immobilized with 10 pig/ml OKT3, 10 nM TPA was added, and cells were further incubated at 37°C for 24 h. Supernatants were quantified for IL-2 concentration by the sandwich ELISA.  67  2.18 Flow Cytometry Analysis of IL-2R and CD69 IL-2R and CD69 expression were determined through flow cytometry on a FACSCalibur cytometer (BD Biosciences) by separately staining T cells (from the IL-2 assay) with either human IL-2Ra mAb, clone 7G7/B6 (Upstate Biotechnology) or human CD69 mAb, clone FN50 (Pharmingen), respectively, followed by incubation with FITCconjugated goat anti-mouse IgG Ab (Jackson ImmunoResearch). Cell viability was assessed by staining dead cells with 2 ug/ml propidium iodide (PI) (Sigma).  2.19 Mice C57B1/6 female mice bearing a transgenic (Tg) TCRcu3 receptor specific for the male antigen H-Y were provided by Dr. Philippe Poussier at Sunnybrook and Women's College, Health Sciences Centre, Toronto, Canada. Balb/c and C57B1/6 mice (Charles River Laboratories, Wilmington, MA) were housed in the animal facilities at University of British Columbia and were used between 8 to 12 wk of age. All mice studies were approved by the Committee on Animal Care at the University of British Columbia using the guidelines set out by the Canadian Council on Animal Care.  2.20 Mixed Lymphocyte Reaction Splenocytes from C57B1/6 (H-2 ) mice at 2xl0 b  6  cells were incubated with  nifedipine for 1 h at 37°C. C57B1/6 splenocytes were then stimulated with 2000 Radirradiated stimulator splenocytes  at 4x10  6  cells from allogeneic Balb/c (H-2 ) or d  syngeneic C57B1/6 (H-2 ) mice for 5 to 6 d at 37°C with nifedipine remaining in the b  68  culture medium. Splenocytes were grown in RPMI supplemented with 10% FBS, 2 m M glutamine, 50 nM 2-mercaptoethanol, and 100 U/ml each of penicillin and streptomycin. Proliferation was evaluated by using a flow cytometer-based bead assay (124). In brief, after 5 to 6 days splenocyte cultures were combined with an equal volume of phosphate buffered saline (PBS) containing 2 ug/ml PI and 2x10 cells/ml latex beads with a 2 u M 6  diameter (Interfacial Dynamics Corp., Portland, OR). PI positive or dead cells were removed from the analysis on the basis of F L 3  hlgh  staining and forward light scatter  gating on a FACSCalibur cytometer (BD Biosciences). Forward light scatter and side light scatter were then used to visualize the latex beads, viable small splenocytes and proliferating blasts. Following data acquisition, gated analysis with the CellQuest software (BD Biosciences) was used to identify the ratios of latex beads to blasts. Using this ratio, the number of proliferating blast cells was then quantified in triplicate for each treatment with Microsoft Excel software.  2.21 In Vivo Proliferation Assay Thymocytes from C57B1/6 female mice bearing a Tg TCRocp receptor that is M H C Class I restricted and specific for the male H-Y antigen were loaded with 5 u M 5(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes) for 7 min at RT. 20-30x10 CFSE loaded Tg thymocytes were intravenous (i.v.) injected into 6  the tail vein of female or male C57B1/6 recipients, followed by intraperitoneal (i.p.) injection of either vehicle control or 15 mg/kg nifedipine into the C57B1/6 males (Figure 2-5). Nifedipine was prepared as a 1 mg/ml stock solution, dissolved in PBS containing 5% ethanol and 1% Tween-80 (125). 40 h after the initial i.v. injection, spleens were  69  Female C57BL/6 HY-specific T C R - T g  C F S E - l a b e l e d thymocytes i.v. injected into normal male C57BL76  thymocytes l a b e l e d with C F S E  Viable T Cell Blasts  Male mice i.p. injected with 1 5 m g / k g nifedipine  C D 8 + T C R « + C F S E + Proliferating Transplanted Female T Cells  CD8+TCRa+ T Cells  3  200  400  600  FSC  800 1000  10«  101  102  103  TCRct-Tg  4 0 - 4 8 h later sacrifice m i c e and analyze spleens for tranplanted C F S E T cells  s  100  10  1  10  103  Z  10<  CFSE  Figure 2-5: Experimental design for the in vivo proliferation assay. (A) Female H-Y-specific TCR-Tg thymocytes were loaded with C F S E and i.v. injected into male C57B1/6 recipient mice. Following the cell injection, male mice were i.p. injected with 15 mg/kg nifedipine or the vehicle control, and approximately 40 h later spleens were analyzed for transplanted H-Y-specific TCR-Tg C D 8 C F S E T cells. (B) Flow cytometry was used to quantify the % total of gated viable, C F S E , C D 8 and Tg TCR cells in the male mice. In the histogram, M l represents the number of nondividing cells, M2 (1 cell division), M3 (2 cell divisions), and M4 (3 cell divisions). From this analysis, the proliferation of female H-Y-specific TCRap receptor CD8 T cells was determined in each successive cell division. +  +  +  +  h l g h  +  7 0  harvested and single cell suspensions were prepared. For cell staining, splenocytes were suspended in D M E M and labeled with PE-conjugated anti-CD8a mAb (BD Biosciences) and biotin-conjugated anti-TCRoc mAb, clone T3.70 (provided by Dr. Hung-sia Teh, University of British Columbia, Vancouver, Canada), which is specific for Tg TCRoc. Cells were then stained with Cy-Chrome conjugated-streptavidin (BD Biosciences), washed and analyzed on a FACSCalibur cytometer (BD Biosciences). Proliferation of female H-Y-specific TCRocp receptor C D 8 T cells in vivo was quantified by determining +  the % total of gated viable, C F S E , C D 8 +  +  and Tg T C R  h i g h  cells in successive cell  divisions using CellQuest software (Figure 2-5). As a result of CFSE labeling being distributed equally between daughter cells, a halving of cellular fluorescence intensity marked each successive cell division among proliferating cells. The total number of female H-Y-specific TCR-Tg C D 8 C F S E T cells that recovered from the spleens was +  +  determined by using the flow cytometry-based bead assay as previously described.  2.22 Statistical Analysis Statistical significance was determined by the Analysis of Variance (ANOVA) test, using two-factorial design without replication with Microsoft Excel software. For all tests, P<0.01 was considered to indicate statistical significance. A l l error bars shown represent the standard deviation (SD).  71  CHAPTER 3: M O L E C U L A R IDENTIFICATION  OF L-TYPE  VOLTAGE-  DEPENDENT CALCIUM CHANNELS IN T LYMPHOCYTES  3.1 Introduction  TCR  engagement by peptide-MHC complexes on the surface of APCs initiates  the depletion of C a  2+  ions from intracellular C a  2 +  stores in T lymphocytes. The rapid rise  in [Ca ]j activates the opening of SOCs in the plasma membrane sustaining C a 2+  for 1 to 2 h (126). The C a activated C a  2+  2 +  2 +  influx  current gated by SOCs, which is termed the Ca -release 2+  current (/CRAC) in T lymphocytes, has been extensively characterized  through patch-clamping Jurkat T cells (127-129) and human PBTs (130). As a result of the electrophysiological studies a biophysical "fingerprint" for /CRAC has been generated 2_|_  which has following characteristics; activation by intracellular Ca small single-channel conductance, high selectivity for C a  2 +  [Ca ]i-dependent modulation of channel activity (10, 2+  store-depletion, very  over monovalent cations, and 19, 131). Even though the  properties of /CRAC have been explicitly defined through electrophysiological means, a candidate protein for the C R A C channel has not been clearly identified. Finding a protein for /CRAC has proven to be very difficult, as many of the potential C a  2+  channel candidates lack one or more of the characteristics of the /CRAC  "fingerprint". To further complicate matters several different C a  2+  channels together may  conduct /CRAC during T cell activation lending to the possibility that a single candidate protein may not be solely responsible for /CRAC (132). One of the current viewpoints for the /CRAC model is that the C R A C channel consists of one or multiple members of the 72  TRP family of Ca TRP candidate for  channels originally found in Drosophila /CRAC  (99). The most plausible  is C a T l , a TRPV subfamily member, that when overexpressed  in Jurkat T cells exhibits many biophysical properties of endogenous  /CRAC,  but is only  partially regulated by store-depletion (105). Although Cui et al. provided evidence supporting that CaTl comprises all or part of the C R A C channel (105), an earlier study disputed CaTl forms  /CRAC  since there are additional biophysical properties of CaTl that  are distinct from / C R A C (106). The reported differences between membrane C a generation of  2+  /CRAC  and CaTl suggest that other plasma  channels expressed in T lymphocytes may also be involved in the  /CRAC-  Interestingly, many of the characteristics of the  /CRAC  "fingerprint"  are shared with L-type VDCCs, even though the gating mechanisms for channel activation  differ (store-depletion  versus  membrane depolarization)  channels, like L-type VDCCs have a high selectivity for C a which is dependent on C a  2 +  2+  (128).  CRAC  over monovalent cations,  binding to high affinity sites on the channels (133).  Furthermore, lowering the extracellular [Ca ] to micromolar levels reduces the C a 2+  selectivity of C R A C channels and VDCCs (127,  2 +  133). In a study comparing the  biophysical properties of C R A C channels to VDCCs, Kerschbaum et al. demonstrated through probing the channels with various organic cations of differing sizes that the predicted diameter of the channel pore is ~0.6 nm for both channels (128). Other similarities shared between C R A C channels and VDCCs are comparable sensitivities to external and internal pH (128). Although VDCCs are typically expressed and function in electrically excitable cell types, several studies have reported the expression of a variety of channel-forming  73  (Xi -subunits  and auxiliary p-subunits  of L-type VDCCs  in human and mouse  lymphocytes. For instance, Brereton et al. demonstrated through RT-PCR analysis that the (Xic- and ocis-subunits of L-type VDCCs are expressed in Jurkat T cells, but whether these channels contribute to TCR-induced C a  2+  influx was not addressed (119). Savignac  et al. reported that an L-type V D C C transcript is expressed in the 2G12.1 murine T cell hybridoma line where the channel is thought to mediate  Ca -dependent gene 2+  transcription of IL-4 (120). Through an oligonucleotide array analysis comparing gene expression profiles in human Thl and Th2 cells, it was shown that the auxiliary P-subunit of VDCCs had 5.4-fold increased expression level in Thl compared to Th2 cells (134). In addition, a previous study by Grafton et al. showed that the aic-subunit mRNA and protein and p-subunit protein are expressed in various human B and T cell lines (135). Grafton et al. further demonstrated that an Ab raised against an extracellular region of the aic-subunit stimulated sustained C a  2 +  influx when added to the human L3055 B cell line  (135). Taken together, the previous studies demonstrate that several (Xi -subunits are expressed in T lymphocytes; however, they did not address the functional contribution of each L-type a- -subunit to C a  2 +  influx during T cell activation.  The investigation on the role of an L-type ai-subunit in T lymphocytes was continued here by examining the functional expression of the oc-p-subunit of L-type VDCCs in T lymphocytes. The otiF-subunit gene, CACNA1F, was originally cloned from 2"!"  human retina (136). In the retina, the oci -subunit appears to mediate Ca F  entry into the  photoreceptors, promoting tonic neurotransmitter release (137). Through Northern blot analysis it was found that the aiF-subunit mRNA is not only highly expressed in human retina, but also present in human skeletal muscle, kidney and pancreas at lower levels 74  (137). Interestingly, mutations in the C A C N A 1 F gene are responsible for the retinal disorder, incomplete X-linked congenital stationary night blindness (CSNB) (138, 139). Even though the original Northern blot analysis revealed that the otiF-subunit is not exclusively expressed in the retina, there are no detailed reports examining the function of the ai F-subunit in other human tissues. Therefore, the investigation presented here began by determining whether ai F-subunit mRNA and protein are present in human T cells. The examination on ocip-subunit expression revealed that two unique splice isoforms of the retinal channel-forming 0Ci F-subunit are expressed in various human leukocytes, including T lymphocytes, but are not found in normal human retina or in retina of humans with CSNB reported thus far. To determine whether the oci F-subunit splice isoforms regulated C a  2 +  influx during T cell activation, several experiments were  conducted, aimed at examining the expression of the splice isoforms following TCRinduced C a  2+  influx in Jurkat T cells and normal human PBTs. The results in this study  collectively established that the L-type 0Ci F-subunit is functionally expressed in T lymphocytes, and the expression is partially regulated through the TCR, lending to the possibility that a V D C C may play a role in generating  /CRAC-  75  3.2 Results  3.2.1 L-Type (XiF-Subunit mRNA Transcript is Expressed in T Cells Through a nested RT-PCR based assay with primers designed to specifically amplify the pore-forming oti F-subunit of an L-type V D C C , a PCR product spanning exons 29 to 30 o f the (Xi F-subunit was detected in human spleen, the human Jurkat T cell leukemia line, and human PBTs (Figure 3-1 A) (140). Human retina and WERI-Rbl retinoblastoma cDNAs were used as positive controls for the PCR assay since the complete ai -subunit gene, C A C N A 1 F , was previously isolated from human, rat and F  mouse retina, where it is expressed at high levels (136, 137, 141, 142). In human retina, ctiF-subunit mRNA is approximately 5.8 kb in length, consists o f 48 exons, and overall the oci F-subunit protein has 55-62% amino acid sequence identity to other L-type C a  2 +  channel (Xi-subunits (137). In the initial studies examining a i F-subunit expression, the a -subunit mRNA ]F  was not detected in lymphoid tissue (137). As demonstrated here, the aiF-subunit is expressed  at low  levels in T lymphocytes  therefore,  0Ci F-subunit expression in  lymphoblastoid tissue may have been overlooked by the lack o f a nested RT-PCR-based assay. Using nucleotide sequencing, it was confirmed that the -180 bp amplified PCR product from Jurkat T cells, human spleen, human PBTs, C D 4 and C D 8 T lymphocytes +  +  shares 100% nucleotide identity to the L-type V D C C a i F-subunit gene expressed in human retina and WERI-Rbl retinoblastoma (Figure 3-1B). The ai -subunit was not F  expressed ubiquitously in human cells since 0Ci F-subunit expression was not detected in  76  6? L-type a 1 F - S u b u n i t Calcium Channel  mmm mmm  mm*  mmm  cr  o  S 1 5 Ribosomal Subunit  B Retina  GAACCCGCATCAGTATCGTGTGTGGGCCACTGTGAACTCTGCTGCCTTTGAGTACCTGAT  t.O  Wer i  GAACCCGCATCAGTATCGTGTGTGGGCCACTGTGAACTC'TGCTGCCTTTGAGTACCTGAT  60  Spleen  GAACCCGCATCAGTATCGTGTGTGGGCCACTGTGAACTCTGCTGCCTTTGAGTACCTGAT  60  Jurkat  GAACCCGCATCAGTATCGTGTGTGGGCCACTGTGAACTCTGCTGCCTTTGAGTACCTGAT  60 6Q  PET  GAACCCGCATCAGTATCGTGTGTGGGCCACTGTGAACTCTGCTGCCTTTGAGTAC'CTGAT  CD4  GAACCCGCATCAGTATCGTGTGTGGGCCACTGTGAACTCTGCTGCCTTTGAGTACCTGAT  60  CD8  GAACCCGCATCAGTATCGTGTGTGGGCCACTGTGAACTCTGCTGCCTTTGAGTACCTGAT  60  ************************************ Ret i n a  GTTCCTGCTCATCCTGCTCAACACAGTTGCCCTAGCCATGCAGCACTATGAGCAGACTGC  120  We r i  GTTCCTGCTCATCCTGCTCAAC ACAGTTGCCCTAGCCATGC AGC ACTATGAGCAGACTGC  120  Spl een  GTTCCTGCTCATCCTGCTCAACACAGTTGCCCTAGCCATGCAGCACTATGAGCAGACTGC  120  Jurkat  GTTC'CTGCTCATCCTGCTCAACACAGTTGCCCTAGCCATGCAGCACTATGAGCAGACTGC  120  PBT  GTTCCTGCTCATCCTGCTCAACACAGTTGCCCTAGCCATGCAGCACTATGAGCAGACTGC  120  CD4  GTTCCTGCTCATCCTGCTCAACACAGTTGCCCTAGCCATGCAGCACTATGAGCAGACTGC  120  CDS  GTTCCTGCTCATCCTGCTCAACACAGTTGCCCTAGCCATGCAGCACTATGAGCAGACTGC  120  ************************************************************ Ret ina  TCCCTTCAACTATGCCATGGACATCCTCAACATGGTCTTCACTGGCCTCTTCACTATT  We r i  TCCCTTCAACT ATGCC ATGGAC ATCCTCAACATGGTCTTCACTGGCCTCTTC ACTATT  178 178  Spleen  TCCCTTCAACT ATGCCATGGACATCCTCAACATGGTCTTCACTGGCCTCTTC ACTATT  178  Jurkat  TCCCTTCAACT ATGCCATGGACATCCTCAACATGGTCTTCACTGGCCTCTTCACTATT  17 8  PBT  TCCCTTCAACT ATGCCATGGACATCCTCAACATGGTCTTCACTGGCCTCTTCACTATT  178  CD4  TCCCTTCAACT ATGCCATGGACATCCTCAACATGGTCTTCACTGGCCTCTTC ACTATT  178  CD8  TCCCTTCAACTATGCCATGGACATCCTCAACATGGTCTTCACTGGCCTCTTCACTATT **********************************************************  178  Figure 3-1: The channel-forming an-subunit of L-type V D C C s is expressed in T lymphocytes. (A) Using a nested RT-PCR reaction, a -180 bp PCR product corresponding to exons 29 to 30 of the channel-forming ai F-subunit of an L-type V D C C was isolated from human cell lines and tissues, including retina, WERI-Rbl retinoblastoma, spleen, Jurkat T cells, PBTs, C D 4 and C D 8 T cells, but was not expressed in normal human liver (top panel). The SI5 ribosomal subunit PCR served as a loading control (bottom panel). PCR products were resolved on a 1% agarose gel and visualized by staining with ethidium bromide. (B) Nucleotide sequence alignment of the ~180 bp PCR product amplified from retina, WERI-Rbl retinoblastoma and T lymphocytes using the European Bioinformatics Institute ClustalW alignment program. The PCR product from human T lymphocytes shares 100% nucleotide identity to the L-type 0 C | F-subunit V D C C isolated from human retina. +  +  77  normal human liver. The expression of the otiF-subunit in lymphoid tissue was confirmed by a recent study by McRory et al, demonstrating that aiF-subunit mRNA expression is not confined to the retina, but is also present in human spleen, thymus, and bone marrow (143).  3.2.2 Identification of Novel Alternative Splice Variants of the oc<F-Subunit in T Cells Since very little is understood about the role of VDCCs during lymphocyte activation, the complete cDNA sequence of the T lymphocyte aiF-subunit was sought after for functional analysis of the channel in human T cells. Using the 3'-RACE reaction, a PCR product corresponding to the 3'-terminal sequence of the OtiF-subunit was isolated from a human spleen cDNA library, which served as an enriched source of human T lymphocytes. In parallel, the complete ai -subunit cDNA sequence from a F  human retina library was also isolated. It was found that the 3'-terminal cDNA sequence of the aiF-subunit from spleen was approximately 430 bp smaller than the cDNA isolated from retina even though identical PCR primers were used in the 3'-RACE reaction (Figure 3-2A). D N A sequence analysis revealed that a novel splice variant of the (XIFsubunit was expressed in human spleen, which lacked exons 31, 32, 33, 34, and 37 due to alternative splicing (Figure 3-2B). The excision of these exons caused the deletion of transmembrane segments S3, S4, S5 and half of S6 in motif IV of the spleen ai p-subunit. As a result, a new membrane topology of the channel protein at the carboxyl-terminus was formed (Figure 3-2C). Removal of transmembrane segment IVS4 (exon 33) through  78  Notl/Spel Digest  EcoRI Digest  Precursor Pre-mRNA (Motif IV):  Voltage  I  DHP site & EF Hand  DHP site 36  S6  \  S6  EF Hand 38  I  Splice site  Mature mRNA (Motif IV):  Alternative Splicing  s  P  l i c e  site  Forward Primer \  I  2S1 9  I  30  S2  I k  3S Reverse Primer  1  3fi  S6  T~33~  Protein: Motif I  alF-Subunit alS-Subunit  Motif II  1284 PSGIWEAVPTPSGLQETCGNBHAPGLRWDGDIQRHTLCPGPDIPEDQNRR 1333 1281 PLGPGKFCPHRVACTAAGGHEHAPEQRRHSHLQCHTLCPGPHGTQDQDGR 1330 * * * * **** * * ******* ** *  79  Figure 3-2: Schematic representation of mRNA splice sites and putative protein topology of the voltage negative splice variant of 0CiF-subunit isolated from human spleen. (A) Restriction enzyme digests of pCR2.1-TOPO cloning vectors containing either the human retina or spleen 3'-RACE products, which correspond to the 3'terminal cDNA sequence of the voltage negative aiF-subunit splice variant. Digestion of the pCR2.1TOPO vectors with NotI and Spel enzymes reveals that the 3'-terminus of aiF-subunit cloned from spleen is significantly smaller than the retina aiF-subunit 3'-terminus. The EcoRl enzyme digest further reveals that the 3'-terminus of otiF-subunit from spleen lacks a single EcoRI restriction site compared to the retina cDNA sequence. (B) Alternative splicing of exons 31, 32, 33, 34 and 37 leads to the deletion of exons encoding transmembrane segments IVS3, S4, S5 and half of S6 of the pore-forming aiF-subunit. Exons encoding transmembrane segments are highlighted in bold boxes with the segment number written below the respective box. Introns are represented as lines in between exon boxes. (C) Diagram of putative channel topology of the voltage negative splice variant of the aiF-subunit. Motifs I-Ill are comprised of six transmembrane segments, whereas due to alternative splicing motif IV only contains two transmembrane segments. Removal of exon 37 through splicing leads to a reading frameshift resulting in early termination of the carboxyl-terminus of the channel protein. The reading frameshift causes a portion of the carboxyl-terminus to no longer be homologous to the a-F-subunit, but instead to have 40% amino acid homology to the human ocis-subunit.  80  splicing led to the deletion of a voltage sensor domain, whereas splicing of segment IVS6 (exon 37) resulted in the deletion of a DHP binding site and an EF-hand Ca -binding 2+  motif. The spleen a-F-subunit was termed the voltage negative splice variant since it no longer contained the S4 voltage sensor domain in motif IV (see Appendix A for the nucleotide sequence and Appendix B for the amino acid sequence of the voltage negative variant). Further sequence analysis of the 3'-terminal sequence demonstrated that the deletion of exon 37 led to the joining of four successive guanines at the exon 36 and 38 junction in the voltage negative splice variant instead of the three guanines that are normally found at the beginning of exon 38 in the retina aiF-subunit cDNA sequence. The four consecutive guanines caused a frameshift in the nucleotide and amino acid sequence in the spleen aiF-subunit, leading to an early in-frame T A G stop codon (at -4.1 kb downstream from the 5'-ATG start codon) and premature termination of channel protein translation at the carboxyl-terminus. In addition, the four guanines in a row changed the amino acid sequence downstream of the frameshift. Instead of having 95% amino acid identity to the aiF-subunit, extraordinarily, the carboxyl-terminus has 40% amino acid identity to the human ais-subunit L-type V D C C found in skeletal muscle (GenBank accession number XP001910) (Figure 3-2C). To my understanding, this is the first reported example of a naturally-occurring chimeric L-type V D C C . In general, this discovery has exciting implications for ion channel biology. A previous study by Brereton et al. also detected the a-s-subunit transcript in Jurkat T cells through RT-PCR (119). The a-s-subunit transcript identified by Brereton et al. was not identical to the  OCIF-  subunit splice variants discovered here since the oc-s-subunit transcript had nucleotide  81  identity to the IVS2 to IVS6 regions of the skeletal a-s-subunit and no identity to the CCIFsubunit (119). Therefore, it appeared that alternative splicing lead to the formation of a novel aiF-subunit carboxyl-terminus, possibly residing in the cytoplasm. The deletion of exon 37 and the resulting reading frameshift was confirmed by repeatedly isolating and sequencing this unique OtiF-subunit cDNA sequence from many different T cell mRNA sources, including human spleen, Jurkat T cells, mouse splenocytes and thymocytes, and naive human PBTs. The unique 3'-terminal cDNA sequence was not found in the same region of CL\ p-subunit isolated from human retina. In addition to the voltage negative splice variant of the ajF-subunit, a second novel splice variant of the aiF-subunit channel was detected in the human spleen cDNA library using the 3'-RACE reaction. Although the second splice variant lacked exons 32 and  37  through alternative  splicing, it retained the  voltage sensor domain in  transmembrane segment IVS4. Therefore, this variant was termed the voltage positive splice variant (Figure 3-3A) (see Appendix A for the nucleotide sequence and Appendix B for the amino acid sequence of the voltage positive variant). Splicing out of the short exon 32, encoding 7 amino acids caused a deletion of part of the extracellular loop between segments IVS3-S4. The deletion of exon 37 in the voltage positive variant also generated a repeat of four guanines in the nucleotide sequence at the exon 36 and 38 junction, resulting in an identical nucleotide and reading frameshift as in the voltage negative splice variant. The voltage positive variant also contained a carboxyl-terminus with 40% amino acid identity to the ais-subunit, as well as an early stop codon causing premature truncation of the channel protein (Figure 3-3B). Furthermore, one striking difference between the two variants, other than the spliced exons, is that the carboxyl-  82  A  Precursor Pre-mRNA (Motif IV): I  Voltage sensor  29  . . •'  35 I  S1  . DHP site DHP site & EF Hand . EF.Hand .  FIE  S6  H ~ 3 7 T - I 38 " ^ - ^ S 6  Splice site  Splice site Alternative Spiicing Mature mRNA (Motif IV):  I  B  29  I  30 1 31 I  33  1 34 II  S1  '.  S2  S4  S5 .  S3  35  I  36  ~r^8~  S6  .  Protei Motif I  OClF-Subunit-  1284 'PSGIWEAVPTPSGLQETCGNEHAPGLRWDGDIQRHTLCPGPDIPEDQNRR. 1333-  alS-Subunif  1281  PLGFGKFCPHRVACTAAGGHEHAPEQRRHSHLQCHTLCPGPHGTQDQDGR.1330  Figure 3-3: Schematic representation of mRNA splice sites and putative protein topology of the voltage positive splice variant of c c i F-subunit isolated from human spleen. (A) Alternative splicing of exons 32 and 37 occurs in motif IV of the voltage positive splice variant of the pore-forming oci -subunit. The voltage sensor domain encoded by exon 33 is not deleted by splicing, and therefore this variant is termed voltage positive. Exons encoding transmembrane segments are highlighted in bold boxes with the segment number written below the respective box. nitrons are represented as lines in between exon boxes. (B) Diagram of putative channel topology of the voltage positive splice variant of the c<i F-subunit. Alternative splicing of exon 32 leads to the deletion of a portion of the extracellular loop linking segments IVS3 and IVS4, and deletion of exon 37 removes half of the transmembrane segment IVS6 of the pore-forming ai F-subunit. Removal of exon 37 through splicing also leads to a reading frameshift resulting in early termination of the carboxyl-terminus of the channel protein in this splice variant. The reading frameshift causes a portion of the carboxyl-terminus to no longer be homologous to the ctiF-subunit, but instead to have 40% amino acid homology to the human ctissubunit. F  83  terminus of the voltage positive splice variant may reside extracellularly instead of in the cytoplasm as a result of fewer exons being excised in this isoform. The remaining 5'-cDNA sequences of the voltage negative and positive splice variants amplified from the human spleen library were identical to the retinal ai -subunit F  cDNA sequence. Both splice isoforms had 99% nucleotide and 95% amino acid sequence identity to the a-F-subunit from human retina. There were no additional differential splice sites found upstream of exon 31 in the voltage negative and positive splice variants of the aiF-subunit. Therefore, the 0Ci -subunit splice variants expressed in human spleen contain F  novel structural features at the carboxyl-terminus which may have a unique impact on the Ca  2 +  kinetics gated by these channels in T lymphocytes. The full-length cDNA sequences  of the retinal aiF-subunit and the voltage negative spliced aiF-subunit were generated from ligation of the individual nested PCR products.  3.2.3 Carboxyl-Terminus of the Retina (XiF-Subunit Resides in the Cytoplasm Although the  carboxyl-termini of channel-forming  L-type a- -subunits  are  normally located in the cytoplasm of different cell types, it has not been previously established whether this was also the case for the human retina oti -subunit. In addition, F  determining the membrane topology of the retina a-F-subunit would aid in distinguishing the localization of the alternatively spliced carboxyl-termini of the T lymphocyte splice variants. To begin this analysis, the complete cDNA sequence of the retina oc-F-subunit was subcloned into the pGFP mammalian expression vector. The pGFP vector not only allowed for the identification of transfected cells due to the dicistronically expressed  84  hrGFP, but also FLAG-tagged the carboxyl-terminus of the retina ai F-subunit. The carboxyl-terminal FLAG-tagged 0Ci F-subunit was used to directly study the topology of the retina oc-F-subunit in transfected cells. To address whether the retina oci -subunit could be overexpressed in mammalian F  cells, HeLa cells were transiently transfected with either pGFP vector alone or pGFP/Retina a- F-subunit. After 72 h, the cells were stained for the FLAG-tagged  OCIF-  subunit. First, it was demonstrated that the anti-FLAG mAb did not non-specifically stain transfected HeLa cells since there was only weak, background staining with the mAb in cells transfected with pGFP vector alone (Figure 3-4A). The staining of pGFP/Retina otiF-subunit transfected cells treated with or without saponin was then examined. When HeLa cells were saponin treated, the anti-FLAG mAb labeled intracellular membranes as well as the intracellular side of the plasma membrane (Figure 3-4B). Intracellular membrane compartments were stained with the anti-FLAG mAb since it has been previously established that co-expression of the a- -subunit with the appropriate auxiliary (3-subunit is required to localize all of the ectopically expressed oti-subunit to the plasma membrane (142, 144). In the presence of saponin, it was also observed here that the staining of the FLAG-tagged a-F-subunit was not cytoplasmic since mAb labeling did not co-localize  with  hrGFP  expression.  As expected,  HeLa cells  transfected  with  pGFP/Retina ai F-subunit without saponin treatment showed no staining with the antiF L A G mAb (Figure 3-4C). These experiments demonstrated for the first time that the retina 0Ci F-subunit was expressed in mammalian cells with the carboxyl-terminus residing in the cytoplasm. It was also observed that HeLa cells transfected with the retina  OCIF-  subunit rapidly deteriorated in health compared to cells transfected with the pGFP vector  85  GFP  Anti-FLAG  Merge  pGFP + Saponin  B  pGFP/Retina oc-| p-Subunit + Saponin  pGFP/Retina a-| p-Subunit Saponin  Figure 3-4: FLAG-tagged retina 0*1 p-subunit is expressed in HeLa cells. (A) HeLa cells were transfected with pGFP vector alone, saponin treated and stained with M2 anti-FLAG mAb, followed by Alexa 568-conjugated anti-mouse Ab (magenta). The pGFP vector emits green fluorescence in transfected cells. HeLa cells were transfected with the pGFP/Retina a- F-subunit construct and treated (B) or not treated (C) with saponin. Cells were then stained with M2 anti-FLAG mAb and Alexa 568-conjugated anti-mouse Ab (magenta). Carboxyl-terminal FLAG-tagged retina oti F-subunit was only detected intracellularly in cells permeabilized with saponin and not detected extracellularly in nonpermeabilized cells. The "+" designates saponin treated, permeabilized cells, whereas the "-" designates cells not treated with saponin.  86  alone. Therefore overexpression of this Ca  channel may alter Ca  homeostasis in  transfected mammalian cells.  3.2.4 Alternative Splice Variants of (XiF-Subunit are Differentially Expressed in Human Leukocytes Through cDNA library cloning, it was established that two unique splice variants of the aiF-subunit are expressed in human T lymphocytes. The next step of the investigation was to examine whether the alternative splice variants are differentially expressed in different leukocyte populations found in spleen. Differential expression of the splice variants may provide new insights into the reported C a  2+  kinetic differences in  physiologically distinct leukocyte populations. For instance, it has been demonstrated that the C a  2 +  response in human C D 4 T cells stimulated by APCs begins after a short delay +  (145), whereas the interaction of cytotoxic C D 8 T cells with target cells results in a rapid +  increase in intracellular C a  2 +  that is maximal within 30 to 60 s (146). Fanger et al.  observed that the rise in intracellular C a  2 +  following store-depletion with thapsigargin  treatment is significantly lower in mouse Th2 cells compared to Thl cells (147). It is also well documented that there are differences in C a  2 +  influx between tolerant versus  activated lymphocytes (148). Using a nested RT-PCR assay with splice variant specific primers (Figures 3-2B and 3-3A depict primer binding sites), the expression of the splice variants in Jurkat T cells and in freshly isolated human PBTs, C D 4 and C D 8 T cells, B cells and monocytes +  +  (also isolated from human peripheral blood) was examined. Both the voltage negative and positive splice variants were expressed in Jurkat T cells, and the heterogeneous  87  population of PBTs, as well as in purified CD4 However,  and CD8  T cells (Figure 3-5A).  voltage negative splice variant expression appeared to be limited to  lymphocytes, as this variant was present in B cells, but not found in monocytes (Figure 35B). Intriguingly, the voltage positive splice variant was expressed in both peripheral blood B cells and monocytes.  3.2.5 CtiF-Subunit Protein is Expressed in Human T Cells To demonstrate that the otiF-subunit protein is expressed in T lymphocytes, immunoprecipitations from Triton X-100 membrane solubilized lysates of Jurkat T cells and human PBTs with an Ab specific to the human retina oti F-subunit were carried out. The immunoprecipitated protein was then analyzed by Western blotting with the  CCIF-  subunit Ab. As illustrated in Figure 3-6A, the aiF-subunit Ab detected a single band at -200 kDa, corresponding to the ai F-subunit protein in both Jurkat T cells and in PBTs isolated from two different human donors. The human retinoblastoma WERl-Rb-1 cell line was used as a positive control for ai F-subunit expression since the ai F-subunit protein is robustly expressed in the retina (137). In a control experiment, it was demonstrated that the c<i F-subunit Ab specifically immunoprecipitated the ai F-subunit protein since a protein of -200 kDa was not immunoprecipitated using affinity-purified rabbit IgG Abs (Figure 3-6A). The cCiF-subunit Ab did not appear to detect two protein bands, representing the alternatively spliced otiF-subunit isoforms in either Jurkat T cells or human PBTs. Posttranslational carbohydrate modifications, such as N-linked and O-linked glycosylation,  88  V  A  Voltage Negative a1 F-Subunit Splice Variant Voltage Positive a1 F-Subunit Splice Variant S15 Ribosomal Subunit  B  Voltage Negative a1 F-Subunit Splice Variant  o  mwm mm- mm- mm  • H H H H H H H H H U H H H H H H mwm mm-  «**"•"*  IP  ^ <o°>  Voltage Positive a1 F-Subunit Splice Variant S15 Ribosomal Subunit  Figure 3-5: Expression of alternatively spliced isoforms of 0Ci F-subunit mRNA in human leukocytes. (A) Nested RT-PCR detected the voltage negative and positive splice variants of the ctiFsubunit in Jurkat T cells, human PBTs, and CD4 and C D 8 T cells. (B) Peripheral blood B cells expressed both the voltage negative and positive splice variants, whereas peripheral blood monocytes only expressed the voltage positive isoform as determined through nested PCR. The S15 ribosomal subunit PCR served as a control for loading and integrity of cDNA samples. PCR products were analyzed on a 1% ethidium bromidestained agarose gel. Prior to nested PCR, the purity of the cell populations was assessed by flow cytometry. The human PBTs contained 11.5% C D 3 C D 4 C D 8 \ 86.5% C D 3 C D 4 C D 8 , 1.0% CD3 CD4"CD8 , and 1.0% CD3CD4CD8". The C D 4 and C D 8 T cells were >99% pure, whereas B cell and monocyte cell populations were 96% pure. The results are representative of three independent experiments. +  +  +  +  +  +  _  +  +  +  89  90  Figure 3-6: Detection of (XiF-subunit protein in Jurkat T cells and human PBTs. (A) aiF-Subunit protein (~200 kDa) was immunoprecipitated using affinity-purified Abs against human a- -subunit from 1.5 mg of membrane protein solubilized lysates of WERI-Rb-1 cells, Jurkat T cells and human PBTs isolated from two different donors (top panel). a-F-subunit protein was not immunoprecipitated with affinity-purified rabbit IgG Ab (Control IP, top panel). The immunoprecipitated protein was analyzed by Western blotting with the a- F-subunit Ab. The position of the prestained molecular weight marker is indicated on the right-hand side of the 0Ci F-subunit panel. As a loading control, Western blot analysis was performed on the samples with an Ab directed against G A P D H (bottom panel). The human PBTs from Donor A contained 36.5% CD3 CD4 CD8", 57.5% C D 3 C D 4 C D 8 , 5.0% CD3 CD4"CD8", and 1.0% CD3CD4CD8", and from Donor B contained 24.8% CD3 CD4 CD8", 72.2% CD3 CD4"CD8 , 1.5% CD3 CD4"CD8", and 1.5% CD3"CD4"CD8". (B) Flow cytometry analysis detected ai F-subunit protein expression in WERI-Rb-1 cells, Jurkat T cells and human PBTs. Saponin-treated cells were either stained with a- F-subunit Ab (bold solid line) or rabbit IgG Ab (thin solid line), or the cells were not saponin-treated and stained with ai F-subunit Ab (dotted line) (top panel). In the bottom panel the bold solid and thin solid lines are the same as before, but the dotted line is saponin-treated cells stained with a- -subunit Ab, which had been preincubated with the antigenic peptide. The human PBTs contained 28.1% CD3 CD4 CD8", 54.2% C D 3 C D 4 C D 8 , 9.7% CD3+CD4CD8", and 8.0% CD3CD4" CD8". Results depicted are representative of three independent experiments. F  +  +  +  +  +  +  +  +  +  +  F  +  +  +  +  91  may obscure the size differences between the (X] F-subunit splice variant proteins. Using the Expert Protein Analysis System (ExPASy) proteomics program, a single putative relinked glycosylation site at asparagine 230, (NQTE) was identified in both of the  OCIF-  subunit splice isoforms, whereas only one O-linked glycosylation site at threonine 1207 was predicted in the voltage positive splice isoform. Due to the additional splice sites in the voltage negative splice variant, there were no predicted O-linked sites in this isoform. In contrast, the ExPASy program identified two putative N-linked glycosylation sites at asparagines 230 (NQTE) and 1459 (NATL), and two O-linked glycosylation sites at threonines 1207 and 1637 in the retinal a-F-subunit. These potential differences in glycosylation sites may cause the a- -subunit splice isoforms to have similar molecular F  weights to one another and the retinal ajp-subunit, and therefore may not be resolved as two smaller, individual protein bands on the SDS-PAGE mini-gel. To further confirm the presence of the ai F-subunit protein in T lymphocytes and to demonstrate ai F-subunit Ab specificity, a series of flow cytometry experiments with the a- F-subunit Ab were conducted. First, it was demonstrated that the a- F-subunit Ab bound to its intracellular epitope by labeling either untreated or saponin-treated WERIRb-1 cells, Jurkat T cells and PBTs with the a-F-subunit Ab. Only cells pretreated with saponin showed 0Ci -subunit Ab labeling as indicated by an increase in mean FL1 F  fluorescence (Figure 3-6B, top panel). Next, it was determined that the ai -subunit Ab F  was binding specifically to the ai F-subunit protein by preincubating the Ab with the antigenic peptide prior to cell labeling. Preincubation with the antigenic peptide completely abolished 0Ci F-subunit Ab binding in all cells tested (Figure 3-6B, bottom panel). In the flow cytometry experiments, the WERI-Rb-1 cells showed a larger increase  92  in mean FL1 fluorescence compared to Jurkat T cells or PBTs. This correlated with the immunoprecipitation data, which also showed a significantly greater amount of  OCIF-  subunit protein expression in WERI-Rb-1 cells compared to T cells.  3.2.6 Comparison of cciF-Subunit Alternative Splice Variant mRNA Expression in Resting and Activated T Cells After establishing that the mRNA and protein of the 0Ci F-subunit are expressed in T lymphocytes, it was addressed whether the endogenous expression of the a-F-subunit splice isoforms in T cells is regulated by TCR-induced activation. To investigate the effect of activation on a-F-subunit splice variant mRNA expression, real-time PCR using splice variant specific primers was performed on Jurkat T cells and naive human PBTs either untreated or treated with an anti-CD3 mAb, OKT3 and the phorbol ester TPA to induce C a  2+  influx (Figure 3-7). T cells were activated for a minimum time period of 1  min to a maximum of 4 h since C a  2 +  influx is an immediate event following T C R  ligation, which is maintained for 1 to 2 h to drive downstream signaling events such as IL-2 expression (126,  149). Therefore, time periods during T cell activation that  encompass critical changes in C a  2 +  influx mediated through plasma membrane C a  2 +  channels were analyzed. First, the mRNA expression of the voltage negative splice variant of the a i F subunit in resting and activated Jurkat T cells was investigated. It was observed that treatment with OKT3 and TPA for 1 min and 2 min time periods induced a large 380and 620-fold increase, respectively, in the mRNA expression of the voltage negative variant (Figure 3-7A). However, slightly longer activation times of 5 and 10 min caused  93  B  2000  10 0)  ft  •B > cc s  ^  c W <  •—z  6  O <D ^ > CC O = p  1000 c  •-P  E  CU  8  • . o  ° w < 1500  4-  0. |.  500  11  CO >*  2 I! 1  ^ ^ ^ N  1«< .  V  A ^  # D  1-5-)  a  29 o  ra  ^  :!  1  1:5  S3 — a. 2  O  1.0  CC  H  ^  1.&  ^  a>  f  > 0.5-  ^  < ^  2.5 2.0  ^  f 1.1 '*  <•>•«• • > n R  <  r  2.0, °co <  rr  o  ^  N  cs ^ 1.0 £ CD C — Z co  °  #  V C>  2.0  CD  .2  <# ^  A-S" ^  isS>  2.5 o <  5 z  20  •o r r 'fi •(=• 1-5  •S  ° 1n CL 1-0  cc  0.5 0  ^ # ## ^ ^^  1  0.5 0  £# ^  ^ ^^  Figure 3-7: Anti-CD3 stimulation alters mRNA expression levels of otiF-subunit splice variants and LTRPC2 channel in Jurkat T cells and human PBTs. Jurkat T cells (A, B, E) and human PBTs (donors, «=3) (C, D, F) were either not treated (NT) or stimulated with 10 ug/ml soluble OKT3 and 10 nM TPA for 1 min, 5 min, 10 min, 1 h and 4 h. Real-time PCR was used to quantify the mRNA abundance of voltage negative and positive splice variants of the ai F-subunit, as well as the LTRPC2 channel in resting and activated T cells. The SI 5 ribosomal subunit PCR served as a control for integrity of cDNA samples and to normalize the C a channel mRNA expression. The graphical analysis represents the fold induction or reduction of mRNA expression in the treated samples compared to the untreated sample. The fold induction of the untreated sample equals one in all experiments. The human PBTs contained 55.8% CD3 CD4 CD8", 34.9% C D 3 C D 4 C D 8 , 8.3% CD3+CD4CD8", and 1.0% CD3CD4" CD8". Each bar represents the mean and SD of assays from duplicate samples. The results shown here are representative of three independent experiments. 2+  +  +  +  +  94  the mRNA expression of this splice variant to return to levels similar to the no treatment (NT) control. When Jurkat T cells were stimulated for 1 h, the voltage negative splice variant underwent a robust 1347-fold increase in mRNA expression, which again returned to the baseline level after 4 h of OKT3 and TPA stimulation. Inducible mRNA expression has been previously observed in T lymphocytes. A regulatory mechanism termed activation-induced splicing is responsible for the 100-fold increase in T N F a mRNA following naive T lymphocyte activation (150). Activation-induced splicing of T N F a mRNA involves the accumulation of T N F a pre-mRNA  in resting T cells,  followed by the rapid processing of pre-mRNA into mature T N F a mRNA following TCR-induced activation (150). Since the PCR primers amplified a region in the a i F subunit mRNA overlapping a -810 bp intronic sequence, real-time PCR products were analyzed on a 1% agarose gel to determine whether activation-induced splicing may regulate the expression of the voltage negative splice variant. It was found that the expected 240 bp PCR product of the mature mRNA of voltage negative isoform was weakly expressed in untreated Jurkat T cells and present at higher levels following 1 and 2 min of activation (Figure 3-8A). The expected PCR product of the unprocessed premRNA of the voltage negative isoform is -1050 bp. In the absence of T C R stimulation, there was an additional PCR product present at -450 bp, and after 5 min of stimulation a -1050 bp PCR product, possibly corresponding to the pre-mRNA, was observed. The 450 and 1050 bp PCR products were not a result of genomic D N A amplification since prior to cDNA synthesis, total R N A preparations were treated with RNase-free DNase to remove contaminating genomic D N A . Therefore, activation-induced splicing may  95  A  Voltage Negative a, -Subunit Splice Variant f  —1050  bp (Voltage negative pre-mRNA)  bp — 240 bp (Voltage negative m R N A ) —  450  SI5 Ribosomal Subunit  pj  Voltage Positive a, -Subunit Splice Variant F  — 380 bp (Voltage positive m R N A )  S 1 5 Ribosomal Subunit  Figure 3-8: Activation-induced splicing may control the mRNA expression of the voltage negative splice variant of the cc-F-subunit in Jurkat T cells. Jurkat T cells were either untreated or stimulated with 10 Ltg/ml soluble OKT3 and 10 nM TPA for 1 min, 2 min, 5 min, 10 min, 1 h and 4 h in duplicate samples. (A) Real-time PCR was used to quantify the mRNA abundance of voltage negative splice variant of the (XiF-subunit. PCR fragments of -240 bp (voltage negative mature mRNA), 450 bp, and 1050 bp (voltage negative pre-mRNA) were amplified using splice variant specific primers that spanned exon 29 to the exon 30 and 35 splice junction. (B) Real-time PCR was used to quantify the mRNA abundance of voltage positive splice variant of the ( X I F subunit. A single PCR fragment of -380 bp, corresponding to the voltage positive mature mRNA, was detected using splice variant specific primers that spanned exons 29 to 33. The SI 5 ribosomal subunit PCR served as a loading control. PCR products were analyzed on a 1% ethidium bromide-stained agarose gel. The results are representative of three independent experiments.  96  partially regulate the mRNA expression of the voltage negative splice variant of the a - F subunit. The expression of the voltage positive splice variant of the ai F-subunit in Jurkat T cells was then examined, and it was found that the mRNA expression was also altered following activation, but not to the same extent as the voltage negative variant. A 4-fold increase in the voltage positive variant expression was induced after 1 min of OKT3 and TPA treatment, which quickly returned to baseline levels at 2 and 5 min (Figure 3-7B). At later activation time periods of 10 min, 1 h and 4 h the expression of the voltage positive variant increased 2.4-, 2.8- and 7.6-fold, respectively. Pre-mRNA corresponding to the voltage positive splice variant was not observed in resting or activated Jurkat T cells (Figure 3-8B). Interestingly, the two a- p-subunit splice isoforms appeared to have different expression profiles in Jurkat T cells following TCR-induced activation. Next, the effect of activation on ai F-subunit splice variant expression in Jurkat T cells to the untransformed naive human PBTs was directly compared. It was found that the mRNA expression of the voltage negative and positive splice variants of the a - F subunit was markedly different when comparing activated Jurkat T cells to human PBTs. When PBTs were stimulated with OKT3 and TPA, the mRNA expression of the voltage negative and positive splice variants was not upregulated at the activation time periods tested (Figure 3-7, C and D, respectively). Instead the expression of both of the a i F subunit splice variants remained relatively constitutive after anti-CD3 stimulation with a consistent yet small decline in expression after 4 h of activation. PCR products representing voltage negative and positive pre-mRNAs were not present in either untreated or stimulated human PBTs.  97  Finally, the mRNA expression profiles of the oci p-subunit splice variants were compared to one of the recently identified TRP C a  2 +  channels, LTRPC2, that is expressed  in Jurkat T cells, human PBTs and weakly in human spleen (Figure 3-9A). It was previously demonstrated that the LTRPC2 C a  channel is a novel mediator of C a  2+  2 +  influx in immunocytes, including Jurkat T cells and peripheral blood lymphocytes (108). Therefore, it was addressed whether LTRPC2 C a  2 +  channel expression was differentially  regulated in comparison to the cti p-subunit splice variants following TCR-induced activation. As illustrated in Figure 3-7, LTRPC2 mRNA expression in resting and activated Jurkat T cells (Figure 3-7E) and human PBTs (Figure 3-7F) was substantially different compared to the expression of the two oci F-subunit splice variants. It was also found that there were slight differences in LTRPC2 expression between Jurkat T cells and naive PBTs. In Jurkat T cells, the expression ofthe LTRPC2 C a  z  channel gradually  decreased over increasing activation time, whereas in PBTs there was an immediate 2fold increase in LTRPC2 expression after 1 min of activation followed by a slow decline  2__| in expression of this Ca channel. In addition to the LTRPC2 C a CaTl  2 +  channel, other TRP C a  and TRPC6, were reported to contribute to C a  2 +  2+  channels, including  influx pathways during T  lymphocyte activation (105, 110). Because of their significant roles in mediating Ca entrance, it was worthwhile examining the mRNA expression of both CaTl and TRPC6 Ca  2 +  channels in resting and TCR-stimulated Jurkat T cells and naive human PBTs  through real-time PCR. Surprisingly, after repeated PCR attempts with several primer pairs, including published primer sequences, and different PCR reaction parameters, PCR products of the correct size were not amplified for either CaTl (Figure 3-9B) or TRPC6  98  $f  <f>  660 b p -  A  S  B  v  <fr  C$  ^  650 bp — 300 bp. 270 bp'  ^ <p  *<v  600 bp350 bp100 b p Figure 3-9: mRNA expression of different T R P subfamily members in Jurkat T cells, human PBTs and human spleen. (A) Using a RT-PCR reaction with human LTRPC2 specific primers, a -660 bp PCR product corresponding to the LTRPC2 C a channel was isolated from Jurkat T cells, human PBTs and human spleen. (B) Human CaTl specific primers designed by Cui et al. did not detect the expected PCR fragment of 271 bp from the human T cell mRNAs tested, instead two non-specific PCR fragments of 650 and 300 bp were amplified (105). (C) The predicted 116 bp PCR fragment, corresponding to human TRPC6, was not amplified using TRPC6 specific primers designed by Gamberucci et al. (110). Instead non-specific PCR fragments of -600 bp and 350 bp that did not correspond to TRPC6 were detected. A l l PCR products were resolved on an ethidium bromide stained 1% agarose gel. The results shown here are representative of two independent experiments with the abovementioned primer sets. 2+  99  (Figure 3-9C) from Jurkat T cells, human PBTs and human spleen.  Since the  amplification of multiple PCR products hinder real-time PCR analysis, the expression of CaTl and TRPC6 could not be evaluated using this technique.  3.2.7 Comparison of ctiF-Subunit Protein Expression in Resting and Activated Jurkat T Cells After thoroughly examining the mRNA expression of the aiF-subunit splice variants in Jurkat T cells, it was then important to determine whether the observed increases in mRNA expression triggered by T C R stimulation lead to augmented  OCIF-  subunit protein expression. To determine the effects of activation on a, F-subunit protein levels, Jurkat T cells were either untreated or stimulated with OKT3 and TPA for time periods of 24 h, 48 h and 72 h. In this experiment, longer activation times were used compared to the real-time PCR experiments, to ensure complete protein translation of the OCiF-subunit. Following OKT3/TPA treatment, immunoprecipitations from Triton X-100 membrane solubilized lysates of Jurkat T cells with an Ab specific to the human retina cti F-subunit were performed. The immunoprecipitated protein was then analyzed by Western blotting with the 0Ci F-subunit Ab. As  demonstrated in Figure 3-10, the ai F-subunit protein was expressed at  relatively low levels in untreated Jurkat T cells. In the presence of OKT3 and TPA,  OCIF-  subunit protein expression increased with increasing activation time, and appeared to reach maximal expression levels after 48 h of stimulation. The stability of the aiF-subunit protein was also examined. Jurkat T cells were stimulated for 24 h, 48 h and 72 h with  100  L-type a , -Subunit Calcium Channel C  fcw—"»  —200 kDa  Figure 3-10: Activated Jurkat T cells express increased levels of a i F-subunit protein. Jurkat T cells were either NT or stimulated with 10 plg/ml OKT3 and 10 nM TPA for 24 h, 48 h or 72 h. c<i F-Subunit protein (-200 kDa) was then immunoprecipitated using affinity-purified Abs against human otiF-subunit from 1.5 mg of membrane protein solubilized lysates of untreated and activated Jurkat T cells. The immunoprecipitated protein was analyzed by Western blotting with the ai F-subunit Ab. Equal protein loading was ensured by quantifying the protein concentration in each sample by the Bicinchonic acid protein assay. Results depicted are representative of two independent experiments.  101  0KT3  and TPA in the presence  of the protein translation inhibitor, 10  Lig/ml  cycloheximide. It was found that the ai F-subunit protein was weakly expressed at 48 h after activation and cycloheximide treatment, however, at 72 h the ai F-subunit protein could not be detected (data not shown). Overall, in activated Jurkat T cells, increases in oti p-subunit mRNA correlated with enhanced ai F-subunit protein expression. In addition, the expressed otiF-subunit protein was more stable than ai F-subunit mRNA expression in Jurkat T cells. Finally, it was examined whether TCR-dependent regulation of ai F-subunit mRNA and protein expression was due to a direct association of the 0 C i F-subunit and the TCR/CD3 complex. The possible association between the 0 C i F-subunit and the T C R was investigated by a coimmunoprecipitation assay with the <X\F-subunit Ab and the CD3 mAb, OKT3 using Jurkat T cells solubilized with 0.5% Triton X-100. The ai p-subunit protein could not be detected in CD3 immunoprecipitates, and CD3 was not present in aiF-subunit immunoprecipitates under the detergent conditions used (data not shown). Therefore, it appears that the TCR-dependent regulation was not due to physical association of the TCR/CD3 complex with the oti F-subunit channel variants.  3.3 Discussion  Since there are only 10 reported genes encoding 0C] -subunits, serving many diverse functions, including muscle contraction to initiation of gene transcription, alternative pre-mRNA splicing is one mechanism used to generate tissue specificity and  102  functional diversity of the channel-forming a,-subunits (114). In this study, it was established that two novel splice isoforms of the L-type ai F-subunit are expressed in human T lymphocytes. The truncated ai F-subunit variants are unlike other alternatively spliced ai-subunits that have been previously described, and structurally distinct from the ai F-subunit originally isolated from human retina. Although the functional expression of novel ai F-subunit isoforms in T lymphocytes has been characterized here, alternative usage of exons has been reported for other ai-subunits expressed in non-excitable cells. The murine erythroleukemia cell (MELC) line expresses a truncated form of the cardiac aic-subunit, which lacks the first four transmembrane segments S1-S4 in motif I (151). When overexpressed as a chimeric channel in Xenopus oocytes, the M E L C aic-subunit forms a functional C a  2 +  channel protein (151). In addition, Grafton et al. also provided  evidence that a similar truncated aic-subunit is functionally expressed in both T and B lymphocytes (135, 152). Therefore, it appears that alternative splicing of the ai-subunits leads to the expression of structurally unique ai-subunit proteins in non-excitable cell types that have adopted cell-specific functions, while maintaining sequence identity to ai-subunits present in electrically excitable cells. Excision of several functional domains in the T lymphocyte ai F-subunit channel variants by alternative splicing may alter characteristic properties of a V D C C , such as voltage sensing, drug binding and inactivation kinetics. First, it was found that the a i F  subunit splice isoforms undergo splicing which may render these channels insensitive to membrane depolarization. In the voltage negative splice variant, the splicing out of exon 33 leads to the deletion of the S4 voltage sensor domain in motif IV. Previous studies  103  using site-directed mutagenesis have revealed that the basic residues in the S4 domains significantly contribute to the voltage sensing properties of voltage-gated ion channels (153,  154).  When these basic residues are neutralized the oti-subunit becomes  unresponsive to membrane depolarization (153). Therefore, deletion of the entire 1VS4 domain in the voltage negative splice variant and consequential removal of several basic residues would presumably abolish gating charge dependence of this splice isoform even though 3 voltage sensor domains still remain intact. In the voltage positive splice variant, a voltage sensor domain is not deleted, however, splicing of a small exon that encodes part of the IVS3-S4 extracellular loop may alter the voltage sensing function of this channel isoform. Since the S4 voltage sensor domain moves upon membrane depolarization, it has been proposed that splicing of the IVS3-S4 domain interlinker may prevent S4 movement due to their close proximity (114, 155). The deletion of the short exon encoding part of the IVS3-S4 extracellular loop has also been reported in the otic-, otio-, and ais-subunits of L-type VDCCs (114). For instance, rat osteosarcoma cells express an 0CiD-subunit lacking the 1VS3-S4 linker that is capable of gating C a  2+  influx in response to the parathyroid  hormone, but not to membrane depolarization (156). In conjunction with the previous studies on voltage sensor function, the results presented here indicate that the deletion of either the IVS4 voltage sensor domain or the IVS3-S4 interlinker may prevent the  OCIF-  subunit isoforms from being gated by membrane depolarization. An alternative gating mechanism may be present in T lymphocytes, such as ER store-depletion or a direct signal from the TCR.  104  In addition to changes in voltage sensing, alternative splicing may also alter the putative DHP binding properties of the aj F-subunit isoforms. It was observed that excision of exon 37 removes 3 amino acids in a portion of the IVS6 transmembrane domain that have been proposed to confer DHP sensitivity and binding to the a i F-subunit (137). Previous analysis on the DHP binding site has revealed that DHPs bind to three separate sites on IIIS5, IIIS6 and IVS6 transmembrane domains of ai-subunits that are allosterically linked (157). Site-directed mutagenesis of amino acids that bind DHPs in the IVS6 domain leads to a 100-fold decrease in DHP binding sensitivity (158). It has also been shown that naturally-occurring splice variants of the human aic-subunit have different sensitivities to the inhibitory action of the DHP derivative, (+)-[ H]isradipine 3  (159). Therefore, the lack of at least 3 amino acids required for DHP sensitivity in the aiF-subunits splice isoforms may provide one explanation why it is necessary to apply 2_|_  large doses of DHPs, such as nifedipine, to completely block Ca -influx through L-type VDCCs in T lymphocytes (160). Along with domain interlinkers and the S6 transmembrane domains, the carboxylterminus is also not well conserved within the ai-subunit gene family, implying this region may generate further functional diversity of ai-subunit proteins (114). In this study, it was found that the deletion of exon 37 and the resulting reading frameshift significantly alter the carboxyl-tennini of both ai F-subunit splice isoforms. First, it was 2_|_  observed that the splicing out of exon 37 leads to the removal of an EF-hand Ca binding motif. Next, it was noted that the subsequent reading frameshift prevents the translation of a 4 amino acid cluster ( W A L in aiF-subunit, V V T L in aic-subunit) within the F helix of the EF-hand motif that is essential for Ca -dependent inactivation in high2+  105  voltage activated channels (161). Interestingly, it was recently reported that the retinal (Xi p-subunit,  unlike  other  L-type ai -subunits,  does not  display  Ca -dependent 2+  inactivation (143). The calmodulin-binding isoleucine-glutamine (IQ) motif located in the carboxyl-terminus of retinal (Xi F-subunit has also been suggested to confer C a 2+  dependent inactivation; however,  it has not been determined whether calmodulin  mediates Ca -dependent inactivation through this motif in the (XiF-subunit (143, 162). It should be noted that the IQ motif is absent in both ai F-subunit splice isoforms. Due to the reported lack of Ca -dependent inactivation, the removal of the EF-hand and IQ motifs 2+  through alternative splicing may not effect the biophysical properties of the ai p-subunit splice isoforms. It was also observed that the reading frameshift causes a portion of the carboxyltermini of the (Xi F-subunit splice isoforms to have 40% amino acid identity to the human skeletal muscle (Xis-subunit L-type V D C C . To my knowledge, this is the first report of this form of "splice conversion". Although the functional significance of the sequence identity switching to the otis-subunit is not clear, it may impart important consequences to the biological functioning of the channel proteins. Finally, it was observed that the reading frameshift also results in early truncation of the carboxyl-termini of both OCIFsubunit splice isoforms. There are several studies reporting the expression of truncated L type ai -subunits. Two-domain splice variants consisting of motifs I and II of the ocicsubunit and motifs I and IV of the ais-subunit exist in cardiac and skeletal muscle, respectively (163, 164). However, at the present time, none of these two-domain otisubunits have been functionally expressed (114). Additionally, a premature stop codon mutation located at amino acid 1459 in the carboxyl-termini of the retinal ocip-subunit  106  protein found in CSNB patients does not appear to alter the activation, inactivation, and conductance properties of the ai F-subunit (143). Based on the experiments with the mutated c<i F-subunit, it is plausible the oci F-subunit splice isoforms that are truncated at amino acid 1452 may have similar biophysical properties to the retinal aiF-subunit. In summary, deletion of exon 37 dramatically alters the carboxyl-termini of both  OCIF-  subunit isoforms. Determining the current gated by the oti F-subunit isoforms will ultimately provide insight into how alternative splicing effects the biophysical properties of the cciF-subunit channel. When the mRNA expression of the a, F-subunit splice isoforms in different human leukocyte populations was investigated, it was determined that the voltage negative and positive splice variants are expressed in both T and B lymphocytes, whereas monocytes only express the voltage positive variant. It is not surprising that T and B lymphocytes express the two 0 C i F-subunit splice isoforms since C a  2 +  influx is induced through antigen  receptor ligation and is dependent upon store-depletion in both cell types. However, it remains unclear whether this data supports the hypothesis that the reported C a  2 +  kinetic  differences in distinct lymphocyte populations are due to differential mRNA expression of the 0CiF-subunit splice isoforms. Quantitative real-time PCR analysis may reveal more subtle differences in the mRNA expression levels of both splice variants in peripheral blood C D 4 and C D 8 T cells, as well as B lymphocytes. The demonstration that human +  +  monocytes express the voltage positive splice variant is the first direct evidence that an Ltype V D C C lacking voltage sensing properties is expressed in monocytes. Interestingly, Willmott et al. reported that store-operated C a  2 +  influx in the U937 immature human  monocytic cell line was mediated through a DHP sensitive L-type V D C C , which was  107  insensitive to membrane depolarization (165). Therefore, the properties of the voltage positive splice isoform described here suggest that this 0Ci -subunit variant may also F  participate in C a  2 +  influx in human monocytes.  Subsequently, oci F-subunit protein expression was examined, and it was found that the ai p-subunit protein is definitively expressed in Jurkat T cells and human PBTs. The identification of the 0 C i F-subunit protein in T lymphocytes directly contrasts with previous findings  on  oti F-subunit  protein  expression  in  lymphoid  tissues.  Through  immunohistochemical analysis of human lymph node, thymus, and spleen, McRory et al. showed that T and B lymphocytes did not interact with an oti p-subunit Ab produced against amino acids 1658-1723 of the human retinal aiF-subunit protein (GenBank accession number N M 005183) (143). Consistent with our observations, the ai F-subunit Ab utilized by McRory et al. (directed against a portion of the carboxyl-tail of the retinal ai F-subunit that is absent in the voltage negative and positive splice isoforms due to a premature stop of protein translation), did not stain T and B lymphocytes positive for the ai F-subunit protein in the tissues analyzed (143). Although the voltage negative and positive splice variant proteins differ in size by -100 amino acids or 11 kDa, visual resolution of the splice isoforms as individual protein bands on a Western blot was not observed. The individual otiF-subunit splice isoforms may not have been visualized since one of the splice isoforms may be expressed at too low a level to be detected by immunoblotting. Alternatively, the predicted N-linked and O-linked glycosylation may cause the splice variants to have similar molecular weights, preventing the resolution of the ociF-subunit splice isoforms as two individual protein bands.  108  When the endogenous mRNA expression of the oti F-subunit splice isoforms following anti-CD3 stimulation was examined through real-time PCR, it was found that expression of both isoforms is regulated by TCR-induced activation in Jurkat T cells and to a lesser extent in human PBTs. In T lymphocytes, the mechanisms used to regulate immediate and early activation genes include alterations in the rate of transcription and mRNA stabilization (166). The rapid and large induction of mRNA expression of the voltage negative splice variant in Jurkat T cells activated for 1 and 2 min may be explained by a combinatory effect of increased transcription rate and mRNA stability, as well as "activation-induced splicing" of this splice isoform. Activation-induced splicing has been previously described as the regulatory mechanism responsible for large increases in T N F a mRNA following naive T lymphocyte activation (150). Yang et al. reported that T N F a pre-mRNA accumulates in naive mouse CD4 T cells and T C R engagement induces splicing of the T N F a pre-mRNA, resulting in a rapid rise in T N F a mRNA within 15 min of activation (150). When examining the mRNA expression of the voltage negative splice variant in Jurkat T cells, it was found the expected 240 bp PCR product was amplified, as well as two larger PCR fragments, one of which that may correspond to voltage negative variant pre-mRNA. This suggests that activation-induced splicing may play a role in the rapid mRNA expression of this ai F-subunit splice variant. In addition to activation-induced splicing, other genes have shown to undergo immediate alternative splicing due to increases in C a  2 +  influx. For instance, alternative  splicing of the plasma membrane Ca -ATPase pump occurs after only 1 min in response 2+  to increased [Ca ]i and can be suppressed by pretreatment with the membrane permanent 2+  Ca  2+  chelator, B A P T A - A M , in 1MR32 human neuroblastoma cells (167). In this study,  109  robust increases in the mRNA expression of voltage negative splice variant at 1 and 2 min, and 1 h of TCR stimulation, correlates with increases in [Ca ]i. Since it has been 2+  previously suggested that all surface TCR/CD3 complexes are recycled within 1 h, the 2+  observed increased in mRNA expression after 1 h may be due to increased [Ca ]; as a result of TCR recycling (168). Slight decreases in [Ca ]imay also be responsible for the 2+  decline in mRNA expression of the voltage negative variant at 5 and 10 min, as well as at the latest time point of 4 h. In comparison to the voltage negative variant, the voltage positive splice variant undergoes relatively small increases in mRNA expression levels at 1 min, 10 min, 1 h and 4h of activation in Jurkat T cells. The rapid increases and decreases in mRNA expression of both a i F-subunit splice isoforms suggest tightly controlled regulatory mechanisms of gene expression (169). In addition, the differences in mRNA expression of the (Xi F-subunit splice isoforms indicate that the voltage negative variant may contribute to immediate and sustained C a positive variant may regulate only sustained C a  2 +  2 +  entry, whereas the voltage  influx in activated Jurkat T cells. The  observed increase in a , F-subunit protein expression after 1 to 2 days of activation also supports the hypothesis for a role for both splice variants in contributing to sustained C a  2 +  influx. In contrast to Jurkat T cells, the mRNA expression of the voltage negative and positive splice variants in human PBTs did not appear to be tightly regulated by TCRinduced activation since the expression of both splice isoforms was not significantly altered following anti-CD3 treatment. The only consistent change was a small decrease in mRNA expression of both splice isoforms following 4 h of activation. The mRNA expression profiles suggest that the a.]F-subunit splice isoforms may contribute to a small,  110  sustained Ca  influx in PBTs, which dissipates slowly after 4 h. In addition to the data  shown here, there are other reported differences in C a  2 +  influx pathways when comparing  Jurkat T cells to untransformed PBTs. It has been previously demonstrated through electrophysiological means that in Jurkat T cells the number of C R A C channels is between 100 to 400 (129), whereas resting PBTs only express 15 C R A C channels (130). One further difference that was observed between Ca  channel expression m Jurkat T  cells and PBTs was an immediate increase in LTRPC2 C a  2+  channel mRNA expression  only in PBTs following activation. This suggests that LTRPC2 may significantly contribute to the initial C a  2+  response in PBTs but not to the same magnitude in Jurkat T  cells. Taken together, the unique mRNA expression profiles of the ai F-subunit splice isoforms and the LTRPC2 C a  2+  channel illustrate the complexity of the C a  2+  response  during crucial hours of T cell activation. Furthermore, the identification of novel voltagedependent-like C a  2 +  channels suggests that different C a  different phases of the C a current that resembles  2+  2 +  channels may be involved in  influx pathway, and together these channels may generate a  /CRAC-  In summary, the study on the expression of ai F-subunit provided extensive molecular and biochemical evidence for the presence of an L-type Ca  channel in T  lymphocytes. The data presented here provides a foundation for further exploration of the role  of the  alternatively  spliced  ociF-subunits  during  T lymphocyte  activation.  Overexpression of the voltage negative splice variant of the 0 C i F-subunit in Jurkat T cells will be conducted to establish a definitive role for this ai -subunit splice isoform during F  T cell activation. Further elucidation of the role of the L-type C a  2+  channel 0CiF-subunit  111  will provide the basis for a better understanding of the mechanisms controlling Ca' influx in T lymphocytes.  112  CHAPTER 4: DEFINING CHANNELS  TO  ACTIVATION IN  THE  CALCIUM  CONTRIBUTION INFLUX  OF  L-TYPE  DURING  T  CALCIUM  LYMPHOCYTE  VITRO  4.1 Introduction  Pharmacological  studies  pioneered  by  the  German  physiologist  Albrecht  Fleckenstein in 1964 lead to the discovery of two chemically distinct compounds, verapamil and prenylamine that were found to inhibit cardiac muscle excitationcontraction coupling in a Ca -dependent manner (170). Fleckenstein and his colleagues 2+  showed that the effect of these drugs on cardiac muscle mimicked C a  2+  withdrawal,  2_|_ which could be overcome by the addition of high concentrations of extracellular Ca (170). Shortly thereafter,  several compounds that were also potent inhibitors of  excitation-contraction coupling were synthesized, propelling the emergence of a new  2+ class of pharmacodynamic drugs termed "Ca  2"b  antagonists" (171). Ca  antagonists drew  immediate attention as a potential therapeutic treatment of cardiovascular diseases because of their cardiodepressant effects. In the mid 1970's, C a  2 +  antagonists, such as  verapamil, were first introduced to patients for the treatment of high blood pressure (172), cardiac arrhythmias (173,  174)  and angina pectoris (175).  researchers elucidated that the mode of action of the C a  2 +  Subsequently,  antagonists was to selectively  bind to L-type VDCCs in the plasma membrane, blocking extracellular C a  2 +  influx  through these channels, which results in the relaxation of cardiac and vascular smooth 113  muscle (171, 176). Following these studies Ca by some researchers as C a  2 +  antagonists were appropriately renamed  channel blockers (CCBs) (177). Currently, CCBs are still  widely prescribed by clinicians for the treatment of several cardiovascular diseases including hypertension, angina and some arrhythmias (157). From the time of the initial discovery of C a  2 +  antagonism by Fleckenstein, a  continually increasing number of selective second, third and fourth generation CCBs have evolved (178). CCBs are presently classified into three chemically distinct drug groups, including benzothiazepines (i.e. diltiazem), phenylalkylamines (i.e. verapamil), and DHPs (i.e. nifedipine) (Figure 4-1) (157). Although all three classes of CCBs bind and modulate L-type V D C C function, they show marked differences  in chemical  structure, binding sites to L-type VDCCs, tissue selectivity and clinical activity (157). The physical state of L-type VDCCs are either resting (closed), open (activated) or inactivated (135), and therefore C C B binding to L-type VDCCs is both voltage and channel state dependent (178). Unlike benzothiazepines and phenylalkylamines that require open channels for binding, DHP antagonists bind to the inactivated channel state and stabilize it. DHP agonists, such as (+/-) Bay K 8644 also favor binding to the open state ofthe channel (Figure 4-1) (135, 157). Diltiazem, verapamil, and nifedipine are all used to treat hypertension since they preferentially bind and inhibit the function of L-type VDCCs in vascular smooth muscle (157). In contrast to DHPs, verapamil and diltiazem also bind to L-type C a  2+  channels in cardiac muscle, therefore these drugs are an effective  treatment of cardiac arrhythmias (157). Since the synthesis of the first DHP in 1882 by Hantzsch, the DHP family has grown to contain the largest number of derivatives compared to benzothiazepines and  114  A  Verapamil  Dilitiazem  CH C.H2N(CH ) 2  3 2  Nifedipine  (+/-) Bay K 8644  Figure 4-1: Chemical Structures of L-Type Ca  Channel Modulators  (A) First generation CCBs with clinical importance in the treatment of cardiovascular disease are diltiazem (benzothiazepines group) verapamil (phenylalkylamines group) and nifedipine (DHP group). The C a agonist, (+/-) Bay K 8644 is a member of the DHP group. The structure of DHPs is based upon a core pyridine structure. (B) The orientation of the 4-phenyl ring determines whether the DHP derivative is a C a channel antagonist or agonist. The "up" position of the 4-phenyl ring confers antagonistic properties, whereas the "down" position confers agonistic properties to the DHP. This figure was taken from Striessnig et al. (179). 2+  2 +  115  phenylalkylamines (180). As a result, DHPs have become the most broadly studied class of CCBs. The specificity of action of DHPs has been confirmed by establishing that the DHP binding sites are located within the channel-forming ai-subunit of L-type VDCCs. Initially, several groups used photoreactive DHP reagents to covalently label and localize the DHP receptor site within the ai-subunit (179, 181). The DHP photolabeled a,subunits were proteolytically digested, and the resulting labeled peptides were identified by  immunoprecipitation with antipeptide antibodies (157). Studies performed by  Nakayama et al. and Striessnig et al. revealed that the photoreactive DHPs bound to transmembrane domain II1S6 and the domain interlinker connecting I11S5 and IIIS6 in the skeletal muscle ocis-subunit (179, 181). Chimeric C a  2 +  channels were then used to further  pinpoint the regions of DHP binding. Tang et al. constructed chimeric ai-subunits by replacing regions of domains 111 and IV in the cardiac muscle ocic-subunit that confer DHP sensitivity with identical portions of the aiA-subunit that are DHP insensitive (182). This study did not report any DHP binding to domain III, but did demonstrate highaffinity DHP binding to the transmembrane domain IVS6 and to the domain interlinker connecting 1VS5 and IVS6 (182). Following these studies, site-directed mutagenesis was used to identify individual amino acids that are the critical determinants for DHP binding. Mutagenesis studies also resolved discrepancies in the different DHP binding sites identified by photoreactive DHPs and channel chimeras (183, 184). Intensive analysis of the DHP binding site has revealed that amino acids in the I1IS5, IIIS6 and IVS6 transmembrane domains of 0 C i -subunits are allosterically linked and essential for DHP binding and action (177, 185).  116  Synthetic DHP derivatives are not only utilized as a therapeutic, but they are also 2"i_  powerful research tools used extensively to identify the presence of putative L-type Ca channels in the plasma membranes of numerous cell types, including T lymphocytes (157). It has been reported that the DHP L-type C a  2 +  channel antagonist, nifedipine, is a  potent suppressor of T lymphocyte proliferation. Based upon an in vitro [ H]-thymidine 3  uptake assay, Birx et al. demonstrated that 0.001 U.M to 100 uM nifedipine prevented the proliferation of human T lymphocytes in response to the mitogens, phytohemagglutinin (PHA) and Con A (186). In a similar study, human PBMCs stimulated with P H A were unable to proliferate in the presence of 10 uM to 200 u M nifedipine; the addition of IL-2 restored the proliferative response in the nifedipine-treated cells (187). Furthermore, it has been demonstrated through in vitro proliferation assays that nifedipine has a dosedependent inhibitory effect on T lymphocyte proliferation when added in combination with the immunosuppressive agent CsA (188, 189). Ricci et al. provided additional evidence that L-type VDCCs are present in lymphocytes by showing that the radiolabeled DHP antagonist (+)-[ H]isradipine bound to human peripheral blood lymphocytes with 3  high affinity and that (+)-[ H]isradipine binding was only weakly displaced by verapamil 3  or diltiazem (190). The aim of the work presented here was to further investigate the presence of L type C a  2+  channels in T lymphocytes and to understand the contribution of C a  through L-type VDCCs  2+  influx  during T lymphocyte activation and proliferation. After  establishing that the channel-forming ai F-subunit of L-type C a  2 +  channels is expressed in  T lymphocytes, the next objective was to determine whether L-type Ca  channels play a  critical role in TCR-induced activation. To define the contribution of the ai F-subunit  117  splice isoforms to TCR-induced Ca  influx, experiments were conducted to knock-down  ai F-subunit splice variant expression in Jurkat T cells through R N A interference (RNAi). Since the ai F-subunit and other DHP sensitive  ai-subunits are expressed in T  lymphocytes, it was hypothesized that DHP derivatives would directly bind to these aisubunits and effect C a  2 +  influx and Ca -dependent signaling in T cells. Therefore, the 2+  effects of (+/-) Bay K 8644 (a DHP agonist that induces L-type C a  2 +  channel opening)  2+  and nifedipine (a DHP antagonist that blocks L-type Ca lymphocyte  activation pathways  were  then  2+  channels) on Ca  examined.  influx and T  Although earlier  studies  demonstrated that nifedipine could block T cell proliferation, the investigators did not thoroughly explore the effects of nifedipine on the T lymphocyte activation process. Furthermore, while some of the previous reports on nifedipine have examined the nonspecific stimulation of T lymphocytes using the mitogenic lectins PHA and Con A , the focus of this study was on the specific activation of T lymphocytes through the TCR/CD3 complex using the anti-CD3 mAb, OKT3. In all experiments where possible the effects of (+/-) Bay K 8644 and nifedipine on the Jurkat T cell leukemia line were compared to T lymphocytes  isolated  from  human peripheral blood to establish  if there  were  discrepancies between T cell lines and untransformed T cells. The results in this study collectively suggest the presence of a DHP sensitive L-type V D C C in the plasma membrane of T lymphocytes that contributes to C a  2 +  entry during T cell activation.  118  4.2 Results  4.2.1 Examination of the Effects of siRNA on ctip-Subunit Expression in Jurkat T Cells RNAi is a powerful gene silencing technique that uses siRNA duplexes, 19 to 22 nucleotides in length and complementary to the gene of interest, to mediate specific mRNA degradation. To determine the contribution of the ai F-subunit to C a  2 +  influx  following TCR engagement, the pSUPER vector system was used to stably express 19 bp siRNA duplexes specifically targeted to the a, F-subunit in Jurkat T cells (Figure 2-3). The pSUPER constructs used in this study were designed to target the degradation of both a, F-subunit splice isoforms expressed in Jurkat T cells since the siRNA target sequences were located upstream of the spliced regions (Table 2-1). In addition, as a control for siRNA efficacy, Jurkat T cells were transfected with the pSUPER-p53 construct, which has been shown to specifically knock-down endogenous p53 mRNA and protein expression through RNAi in different cell types (123). After approximately two months growing under selection conditions, bulk cultures of Jurkat T cells co-transfected with the pEYFP vector and either pSUPER (lacking a siRNA insert), pSUPER-0CiF-subunit constructs, or pSUPER-p53 were analyzed for stable integration of the pSUPER constructs into the genomic D N A . As illustrated in Figure 4-2A, all of the pSUPER constructs were stably expressed by Jurkat T cells except for pSUPER-ociF-subunit-l. Subsequent transfections of pSUPER-aiFsubunit-1 into Jurkat T cells did not result in stable expression therefore only three  119  ### #  Kj  5  ffl^  & &> .e  6  g>4  X$» ! D  0  ^4 <J?  £ S  <r  —570 bp — 510bp  pSUPER  jo*"  <S-  a* F  ,  <r  GAPDH  Clone: 1  <r  p53  Genomic DNA  j  6  Clone: 1 2 3 **** ^1  • 200 kDa  0 *  1 2 3 1 2 3  « -Subunit«*«i***-*. **•» 1F  GAPDH « M »  GAPDH  — 200 kDa  —.-^^r  F i g u r e 4-2: S t a b l e e x p r e s s i o n o f s i R N A s t a r g e t e d to the ( X i r - s u b u n i t i n J u r k a t T cells did not k n o c k - d o w n a n -subunit protein expression.  (A) Bulk cultures of Jurkat T cells were examined for stable expression of pSUPER constructs. Using a PCR reaction, -570 bp PCR products, corresponding to pSUPER constructs containing siRNA inserts, were amplified from genomic D N A . The 510 bp PCR product corresponds to the pSUPER construct without a siRNA insert. A 570 bp PCR product was not amplified from Jurkat T cells expressing pSUPER-aiF-subunit-1, indicating a lack of stable integration of this vector. Untransfected refers to Jurkat T cells not transfected with any constructs. Genomic DNA and PCR products were analyzed on a 1% agarose gel stained with ethidium bromide. (B) Knock-down of p53 protein expression was detected in cloned Jurkat T cells expressing the pSUPER-p53 construct by the lack of p53 protein immunoprecipitated with a mAb against human p53. (C and D) Using affinity-purified Abs against human ai F-subunit, the ai p-subunit protein was immunoprecipitated from three different clones of Jurkat T cell lines expressing different pSUPER-a,F-subunit constructs, demonstrating expressed siRNAs did not knock-down aiF-subunit protein levels. As a loading control for all immunoprecipitation experiments, Western blot analysis was performed on the samples with an Ab directed against GAPDH. Results depicted are representative of at least three independent experiments.  120  pSUPER-aiF-subunit constructs were used in the remaining experiments. Next, the pSUPER expressing cells were cloned and, where appropriate, analyzed for either p53 or aiF-subunit protein levels. As shown in Figure 4-2B, the pSUPER-53 construct effectively knocked-down endogenous p53 protein expression in Jurkat T cells, whereas transfection of cells with pSUPER, or pEYFP alone did not effect the protein levels of p53. Several clonal Jurkat T cell lines, expressing siRNA to different regions of the 0 C i F  subunit, were then analyzed and it was found that none of the constructs reduced the levels of the a, F-subunit protein compared to 0 C i F-subunit expression in cells transfected with pSUPER or pEYFP alone (Figure 4-2, C and D). Additionally, it was observed that the protein levels of the 0Ci -subunit in cells transfected with pEYFP alone were reduced F  in one clone (pEYFP alone, Clone 1 in Figure 4-2D). Therefore, it was reasoned that although siRNA is an effective technique at knocking-down protein expression in Jurkat T cells, the siRNA target sequences chosen in this study did not successfully knock-down 0CiF-subunit protein expression.  4.2.2 Induction of Calcium Influx in Jurkat T Cells and Human PBTs by (+/-) Bay K8644 Since the pSUPER vector system did not effectively knock-down ai -subunit F  expression in Jurkat T cells, a different strategy was required to assess the functional contribution of L-type Ca channels to C a 2+  2+  influx during T cell activation. As an  alternative approach, the effect of the DHP derivative, (+/-) Bay K 8644, on C a  2 +  influx  in human T lymphocytes was tested. It has previously been reported that the treatment of  121  Jurkat T cells with Bay K 8644 in the range of 0.01-100 u M induces a small rise in intracellular C a , indicating the presence of a DHP sensitive C a 2+  2 +  influx pathway in  these cells (191). However, the report did not specify which enantiomer of Bay K 8644 was used. The (-)-enantiomer of Bay K 8644 has strong agonistic properties, whereas the (+)-enantiomer acts as a weak C a  2+  channel antagonist. Since the optical isomers of Bay  K 8644 induce opposing L-type V D C C activity, the experiment was repeated in this study with (+/-) Bay K 8644, a racemic mixture that has the net effect of enhancing C a influx through L-type C a effects of (+/-)  2 +  2+  channels (192). Additionally, a direct comparison of the  Bay K 8644 treatment on C a  2 +  influx in Jurkat T cells to the  untransformed PBTs was sought after since this has not been previously examined. When indo-1 loaded Jurkat T cells and human PBTs were treated with (+/-) Bay K 8644, a dose-dependent increase in the mean ratio of indo-1 bound to C a  2 +  (405  nm)/free indo-1 (485nm) was observed indicating an increase in intracellular C a  2 +  (Figure 4-3). In both Jurkat T cells and PBTs, 10 uM (+/-) Bay K 8644 generated a small, sustained rise in intracellular C a . However, treatment of Jurkat T cells and PBTs with 2+  higher concentrations of (+/-) Bay K 8644 caused different responses in Ca  influx, in  Jurkat T cells, 50 u M and 100 u M (+/-) Bay K 8644 induced a sustained increase in cytosolic C a  2+  (Figure 4-3A), whereas treatment of human PBTs with either 50 U.M or  100 u M (+/-) Bay K 8644 caused a transient C a  2+  influx that rapidly declined to below  baseline after the 10 min time period (Figure 4-3B). In the absence of extracellular C a in the medium, 100 u M (+/-) Bay K 8644 did not cause a rise in intracellular C a  2 +  2 +  in  either Jurkat T cells or human PBTs, indicating that (+/-) Bay K 8644 treatment with extracellular C a  2 +  specifically allowed C a  2 +  entry into these cells. Treatment with DMSO  122  A  B  • 0  ' 100  •  *  200  300  * 400  i •' * ' i  ' 500  0  100  1  *' i '  1 1  200  i  1  300  Time (sec)  ' r ' ' i*' ' 1  1  400  500  Time (sec)  Figure 4-3: (+/-) B a y K 8644 induces C a influx in a dose-dependent manner in the human J u r k a t T cell leukemia line and human P B T s . 2 +  Jurkat T cells (A) and human PBTs (donors, n=3) (B) were loaded with indo-1. The basal concentration of free intracellular C a was initially measured, after which 10-100 u M (+/-) Bay K 8644 was added to the sample and the analysis was resumed. Red line, DMSO solvent (Control); Yellow, 10 uM (+/-) Bay K 8644; Green, 50 uM (+/-) Bay K 8644; Grey, 100 u M (+/-) Bay K 8644; Purple, 100 u M (+/-) Bay K 8644 with no extracellular C a in medium. Indo-1 loading was assessed by stimulating C a influx with 2 uM ionomycin in Jurkat T cells (C) and human PBTs (D). The human PBTs contained 14% C D 3 C D 4 C D 8 , 79% CD3 CD4 CD8 , 6.5% CD3 CD4"CD8- and 0.5% CD3CD4CD8" cells. The results are representative of three independent experiments. 2+  2+  2+  +  +  +  +  +  123  solvent alone did not induce significant Ca well-characterized lipophilic C a  2 +  entry into T lymphocytes. The effects of the  ionophore, ionomycin, were also examined to ensure  efficient loading of the indo-1 dye into T lymphocytes. The addition 2 U.M ionomycin induced a rapid and sustained C a  2 +  influx in both Jurkat T cells and human PBTs (Figure  4-3, C and D).  4.2.3 Nifedipine Inhibits Anti-CD3 Induced Calcium Influx in Jurkat T Cells and Human PBTs Nifedipine is a blocker of L-type C a investigate the role of a DHP sensitive C a  2 +  channels and was used to further  2 +  channel in T lymphocytes. Pretreatment of  indo-1 loaded Jurkat T cells and human PBTs with nifedipine in the presence of extracellular C a bound to C a  2+  2 +  resulted in a dose-dependent decrease in the mean ratio of indo-1  (405 nm) versus free indo-1 (485nm) following anti-CD3 stimulation  (Figure 4-4, A and B). A decrease in the mean indo-1 ratio demonstrates that nifedipine consistently inhibited anti-CD3 induced C a  2+  influx in both Jurkat T cells and PBTs  whereas the DMSO solvent control had no effect. It should be noted that an anti-CD3 mAb was used to stimulate C a  2 +  influx in Jurkat T cells, whereas experiments conducted  with human PBTs required an anti-CD3 mAb as well as an anti-CD28 mAb and a crosslinking Ab to induce C a  2+  influx in these cells.  Although nifedipine clearly inhibited C a  2 +  influx in the cells tested, it was 2_|_  important to determine whether this inhibition was a partial or complete blockage of Ca influx. This would help distinguish whether L-type C a that mediate C a  2 +  2 +  channels are the only channels  influx or if other channels contribute to the C a  2 +  response during T 124  A  B  Figure 4-4: Nifedipine blocks C a and human PBTs.  2 +  influx in the human Jurkat T cell leukemia line  Jurkat T cells (A) and human PBTs (donors, n=3) (B) loaded with indo-1 were preincubated with 1-200 uM nifedipine in the presence of extracellular C a . For each sample, after the 10 min treatment with nifedipine baseline C a measurements were taken, cells were then stimulated at the 2 min mark, and the analysis was immediately resumed. C a influx was induced with an anti-CD3 mAb in Jurkat T cells and a combination of anti-CD3 mAb, anti-CD28 mAb and a cross-linking Ab in human PBTs. Indo-1 loaded Jurkat T cells (C) and human PBTs (D) were treated with 1-200 u M nifedipine and stimulated in the absence of extracellular Ca . Red line, DMSO solvent (Control); Light Blue, 1 uM Nifedipine; Yellow, 10 uM Nifedipine; Green, 50 uM Nifedipine; Grey, 100 uM Nifedipine; Dark Blue, 200 uM Nifedipine. The human PBTs contained 18% C D 3 C D 4 C D 8 \ 45% CD3 CD4"CD8 , 32% C D 3 C D 4 C D 8 and 5% CD3CD4CD8" cells. The results depicted are representative of three independent experiments. 2+  2+  2+  +  +  +  +  +  +  125  lymphocyte activation. To address this question, C a extracellular C a Ca  2 +  influx induced in the absence of  2 +  was compared to nifedipine-inhibited C a  2 +  2 +  influx with extracellular  present in the medium. In both anti-CD3 stimulated Jurkat T cells and PBTs, a  rapid, transient increase in intracellular C a , arising from intracellular C a 2+  observed when extracellular C a  2 +  stores, was  2+  was absent from the medium (Figure 4-4, C and D,  Control, red line). This transient C a  2+  spike was not observed in Jurkat T cells treated  with nifedipine in the presence of extracellular C a , indicating that nifedipine only 2+  partially blocked C a  2+  influx in these cells.  However, in human PBTs, higher  concentrations of nifedipine completely abolished C a no extracellular C a extracellular C a Finally,  2+  2+  2 +  influx since the C a  2+  trace with  was very similar to PBTs treated with 50-200 (iM nifedipine when  was present. since nifedipine  is  a lipophilic compound that can  intracellularly, nifedipine blockage of C a  2+  efflux from intracellular C a  2+  accumulate stores was  investigated (193). To examine this, Jurkat T cells and human PBTs were treated with nifedipine and anti-CD3 stimulated in the absence of extracellular C a  2 +  (Figure 4-4, C  and D). Nifedipine treatment did not significantly effect the transient rise in cytosolic Ca  2 +  from intracellular C a  2+  stores in Jurkat T cells, whereas in human PBTs 50-200 u M  nifedipine reduced the efflux of C a  2+  from intracellular stores in these cells. It should be  noted that (+/-) Bay K 8644 and nifedipine are typically used at 1-300 u M on both electrically excitable and non-excitable cell types. The concentration of DHPs used in these experiments  therefore  replicated the concentration  range of C a  2+  channel  modulators applied in other studies (165, 192).  126  4.2.4 (+/-) Bay K 8644 and Nifedipine Modulate Phospho-p44/p42 M A P Kinase Activation in Jurkat T Cells and Human PBTs The next step in the investigation was to determine whether C a L-type C a  2+  2+  influx through  channels could activate downstream, Ca -dependent signaling pathways 2+  involved in T lymphocyte activation. The expression of the p44/42 M A P kinase, also known as Erkl/2, was examined since a rise in intracellular C a  2 +  through C a  2 +  ionophores, such as ionomycin and A23187, can induce the activation of Erkl/2 in T lymphocytes (194). The addition of the specific C a  2 +  chelator, E G T A , prior to stimulation  of Jurkat T cells and human PBTs with ionomycin blocks activation of Erkl/2 demonstrating that one mode of Erkl/2 activation in T lymphocytes is through an 2_j_  increase in intracellular Ca  (194). Additionally, DHP derivatives are reported to  modulate the M A P kinase pathway in neurons, but this phenomenon has not been examined in T lymphocytes (195). In Jurkat T cells, 50 | l M and 100 | l M (+/-) Bay K 8644 stimulation resulted in a rapid and transient phosphorylation of both Erkl and 2, which was similar to the level of Erkl/2 activated with 2 U.M ionomycin (Figure 4-5A). The activation of Erkl/2 with (+/-) Bay K 8644 was blocked by pretreatment with E G T A . In human PBTs, (+/-) Bay K 8644 did not activate Erkl and only weakly activated Erk2 compared to the ionomycin control (Figure 4-5B). The activation of Erk2 with (+/-) Bay K 8644 was not blocked by pretreatment with E G T A . The treatment of Jurkat T cells and human PBTs with D M S O alone (Control) did not activate Erkl/2. It should be noted that the effects of 100 (iM (+/-) Bay K 8644 on naive human PBTs that were immediately isolated from PBMCs and not  127  A DMSO  2 uM Ionomycin  1 uM (+/-)Bay K 8 6 4 4  10 u M 50 u M 100UM <-/-)Bay K 8 6 4 4 ( + / - ) B a y K 8 6 4 4 ( + / - ) B a y K 8 6 4 4  2 mM EGTA: lERKI "ERK2 •ERK1 "ERK2  B 2uM 1 uM 10 u M I o n o m y c i n (+/-)Bay K 8 6 4 4 (+/-)Bay K 8 6 4 4  DMSO  50 u M 100 u M (+/-)Bay K 8 6 4 4 (+/-)Bay K 8 6 4 4  2 mM EGTA: • ERK1 "ERK2 .ERK1 •ERK2  AJ sP f^^gjM,:.  '^iPlf PBTs  —  EE  RK1  ""ERK2  Jurkat  Figure 4-5: (+/-) Bay K 8644 modulates phospho-p44/42 M A P kinase activation in Jurkat T cells and human PBTs. Jurkat T cells (A) and human PBTs (donors, «=3) (B) were preincubated with or without 2 mM E G T A followed by stimulation with either 2 (J.M ionomycin or (+/-) Bay K 8644 (10-100 |iM). Cells incubated with DMSO alone served as control. The human PBTs contained 18% CD3 CD4 CD8", 69% CD3 CD4"CD8 , 11.5% C D 3 C D 4 C D 8 - and 1.5% CD3CD4CD8" cells. (C) Naive human PBTs and Jurkat T cells were treated with either D M S O or stimulated with 100 U.M (+/-) Bay K 8644. Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were probed with a phosphospecific Ab to detect activated Erkl/2 (top panel). The membrane was stripped and reprobed with an Ab directed against Erk 112 to detect the total amount of kinase loaded in each lane (bottom panel). The results are representative of three independent experiments. +  +  +  +  +  128  previously cultured with 10 jig/ml plate-bound OKT3 and rhIL-2 were also examined. Unstimulated naive human PBTs had the same level of Erk2 activation following (+/-) Bay K 8644 treatment as human PBTs grown for 8 days in culture with OKT3 and rhIL-2 (Figure 4-5C). Since there is very little information on what aspects of the T lymphocyte activation process are effected by inhibiting C a  2 +  influx with nifedipine treatment, the  investigation of whether Erkl/2 activation induced by (+/-) Bay K 8644 could be specifically blocked by nifedipine was carried out. Pretreatment of Jurkat T cells with either 100 uM or 200 uM nifedipine, but not the DMSO solvent, significantly inhibited Erkl/2 activation induced by 100 u M (+/-) Bay K 8644 (Figure 4-6). As a positive control for Erkl/2 phosphorylation, Jurkat T cells were stimulated with soluble OKT3. Stimulation of Jurkat T cells through the TCR/CD3 complex induced robust activation of Erkl/2, showing that the C a  2+  influx induced by (+/-) Bay K 8644 supports only partial  Erkl/2 activation.  4.2.5 Nifedipine Blocks NFAT-Transcriptional Activity in Jurkat T Cells The activation of the transcription factor N F A T is dependent upon increased intracellular Ca (196). The investigation of whether the block in C a 2+  type C a  2 +  2 +  influx through L -  channels by nifedipine alters the transcriptional activity of N F A T was  conducted by transiently transfecting an NFAT-luciferase reporter plasmid into Jurkat T cells. Activation of transfected Jurkat T cells induces endogenous N F A T transcription factors to bind to the N F A T cw-acting enhancer element within the construct, and transcribe the reporter gene. First, it was determined that the maximum induction of  129  100 uM (+/-)Bay K 8644  **** ^  .</ •ERK1 *ERK2  ^Mfe^ugK .....  •.yj* u^; mm  .  .  .  ^  m  -  g  m  a  b  •ERK1 "ERK2  Figure 4-6: Nifedipine modulates phospho-p44/42 M A P kinase activation in Jurkat T cells. Cells were preincubated with either DMSO, 100 \iM or 200 u M nifedipine, or NT, preincubated with D M S O , 100 u M or 200 uM nifedipine followed by stimulation with 100 u M (+/-) Bay K 8644. Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were probed with a phospho-specific Ab to detect activated Erkl/2 (top panel). The membrane was stripped and reprobed with an Ab directed against Erkl/2 to detect the total amount of kinase loaded in each lane (bottom panel). The results are representative of three independent experiments.  130  N F A T when transfected Jurkat T cells were exposed to soluble OKT3 occurred between 5 to 8 h after stimulation (data not shown). Blocking C a  2+  channel activity by  pretreatment of Jurkat T cells with nifedipine resulted in inhibition of OKT3-induced NFAT activation in a dose-dependent manner (Figure 4-7). Low concentrations of nifedipine that weakly blocked C a  2 +  influx, such as 50 u M nifedipine, significantly  reduced the transcriptional activity of NFAT. Higher doses of nifedipine, such as 200 u M almost completely abolished N F A T activity. The DMSO solvent alone did not inhibit N F A T activation in Jurkat T cells. Additionally, OKT3 stimulated Jurkat T cells transiently transfected with a reporter construct that does not contain the N F A T cw-acting enhancer element (Control), did not activate NFAT.  4.2.6 IL-2 Production and IL-2R Expression is Inhibited by Nifedipine in Jurkat T Cells and Human PBTs Since IL-2 secretion is a definitive indicator of T cell activation, it was assessed whether blocking L-type C a  2+  channels with nifedipine can inhibit IL-2 production in  both anti-CD3 stimulated Jurkat T cells (Figure 4-8A) and human PBTs (Figure 4-8B). Nifedipine treatment significantly inhibited IL-2 secretion in both cell types and abolished IL-2 secretion completely with 200 u M nifedipine. This is in agreement with the results showing the inhibitory effects of nifedipine on C a  2 +  influx (Figure 4-4) and  N F A T activation (Figure 4-7). Treatment with the DMSO solvent did not significantly block IL-2 secretion from either Jurkat T cells or human PBTs. To ensure the block in IL-2 secretion by nifedipine was not due to cell death induced by drug cytotoxicity, both Jurkat T cells and human PBTs were stained with PI  131  350 •£•'  3001  c ~ 2501  it.  <u. > f l •hi  2  200i rfi  0)  rfi  g 150H  £ ^ 100-1 50 NT  DMSO'  1  10  50  100  200  (jiM Nifedipine)  Figure 4-7: Inhibition of NFAT transcription by nifedipine in the human Jurkat T cell leukemia line. Jurkat T cells were either transiently transfected with a N F A T reporter construct (open bars) or with a reporter construct lacking the NFAT cw-acting enhancer element (hatched bars), which monitors for constitutive levels of luciferase activity. After being cultured for 40-48 h, the cells were either untreated (NT), incubated with DMSO (Control) or 1200 (iM nifedipine and were then stimulated with soluble OKT3 (10 ug/ml). Cells were harvested and the N F A T activity was measured by luciferase activity assay. The results are presented as relative NFAT-dependent luciferase activity. Each bar represents the mean and SD of assays from triplicate wells. The results are representative of three independent experiments. P<0.01, relative to DMSO treated Jurkat T cells transfected with the N F A T reporter construct.  132  B 4000  .4000-  I  3500  3500  . 3000:  .3000  rfi  '. I 2500  2500-  CO  2000  <f 2000 ~  &  1500  -  1000-  1000  '.' 500 0  1500  . . : " 5oo NT. DMSO  10  50  100  "o  .200.  NT  DMSO . .1  (uM Nifedipine)  10  50  .100  200  ((iM Nifedipine)  D 100  100,  90  90  80  80  . 70  ; 70  ff  60  60  .50  50  40  40  .30  30  20  20 10  10 0-  NT  DMSO  1  10  50  . 100  0  200  NT  4000,  2000-  j  1.500-  100  200  .3000 1 2500cf) " 20001500  1000-  1000  500 • 0--  50  3500-  3000 2500-  10  4000-  3500  1  1  {^iM Nifedipine)  (uM Nifedipine)  f  DMSO  500 NT DMSO'DMSd  .1  10  10  (jiM Nifedipine)  50.  50  ;  0- NT  DMSODMSOI  10  10  50  50  ((jM Nifedipine)  133  Figure 4-8: Nifedipine prevents IL-2 secretion from Jurkat T cells and human PBTs. Jurkat T cells (A) or human PBTs (donors, n-3) (B) were incubated with 1-200 u M nifedipine and then stimulated with immobilized OKT3 (10 p.g/ml) and 10 nM TPA. After cells were stimulated for 24 h, the amount of IL-2 secreted in the supernatant was measured by standard sandwich ELISA. The control is Jurkat T cells or human PBTs treated with the DMSO solvent alone. The human PBTs contained 19% CD3 CD4 CD8~, 72% CD3 CD4"CD8 , 8.0% CD3 CD4"CD8" and 1.0% CD3"CD4"CD8" cells. PO.01, relative to the DMSO control. Viability of Jurkat T cells (C) and human PBTs (D) was assessed by staining with PI. P>0.01, relative to the DMSO control. To demonstrate whether additional C a can overcome the inhibitory effect of nifedipine, Jurkat T cells (E) and human PBTs (donors, « = 3 ) (F) were incubated with 1-50 u M nifedipine. Cells were stimulated with immobilized OKT3 (10 (ig/ml), 10 nM TPA and without (open bars) or with 2 uM ionomycin (hatched bars). The human PBTs in this experiment contained 11.5% CD3 CD4 CD8", 86.5% C D 3 C D 4 C D 8 , 1.0% CD3 CD4"CD8" and 1.0% CD3CD4CD8" cells. *, PO.01, as comparing samples with or without ionomycin added. Results depicted are representative of three independent experiments. Each bar represents the mean and SD of assays from triplicate wells. +  +  +  +  +  2 +  +  +  +  +  +  134  after culture supernatants were removed for assaying IL-2. The PI negative or viable cell population was then analyzed by flow cytometry. In Jurkat T cells and human PBTs, 1 200 u M nifedipine did not have a statistically significant impact on cell viability compared to the viability of cells stimulated with OKT3/TPA and treated with D M S O (Figure 4-8, C and D). It was then examined whether the inhibitory effect of nifedipine could be reversed by the addition of C a . Since ionomycin rapidly increases intracellular C a 2+  2+  in T cells,  2~f-  treatment with this ionophore was used to provide additional Ca  to the cells. Jurkat T  cells (Figure 4-8E) and human PBTs (Figure 4-8F) were treated with nifedipine and ionomycin where indicated and IL-2 secretion was again assayed as an indicator of T cell activation. In both cell types, inhibition of IL-2 secretion by 1 U.M and 10 uM nifedipine could be completely overcome by the addition of ionomycin. However, the treatment of T cells with 50 U.M nifedipine could only be partially reversed by ionomycin treatment. In all cases, reversing the inhibitory effect of nifedipine with ionomycin was more successful in human PBTs compared to Jurkat T cells. IL-2R expression was also examined on viable T cells and it was found that only 200 uM nifedipine significantly inhibited receptor expression. In Jurkat T cells (Figure 49A), 200 U.M nifedipine caused a 55% decrease in the log mean fluorescence intensity of IL-2R expression, whereas in human PBTs pretreated with 200 uM nifedipine (Figure 49B) a 70% decrease was observed compared to IL-2R expression of T cells stimulated with OKT3/TPA and treated with DMSO.  135  A  io°  i6  1  10 CD25 2  10'  10'  lit)  2  To ' 3  IC  CD25  B  10' CD25  CD25 :  10^ CD25  10°  i0 . 3  10  4  CD25  CD25  136  Figure 4-9: Decreased IL-2R expression in Jurkat T cells and human PBTs after treatment with nifedipine. Jurkat T cells (A) or human PBTs (donors, «=3) (B) were preincubated with nifedipine (1-200 uM) for 1 h, and then stimulated with immobilized OKT3 (10 (ig/ml) and 10 n M TPA for 24 h with nifedipine remaining in the culture medium. Cells were stained with PI to remove dead cells from analysis and human IL-2Rcc mAb followed by FITC conjugated goat anti-mouse Ab. Cells were treated with either DMSO (dotted line), with DMSO and OKT3/TPA (solid line), or with nifedipine and OKT3/TPA (bold solid line). The human PBTs contained 19% C D 3 C D 4 C D 8 , 72% C D 3 C D 4 C D 8 , 8.0% CD3 CD4"CD8" and 1.0% CD3CD4CD8" cells. The results shown here are representative of three independent experiments. +  +  +  +  +  137  4.2.7 Nifedipine Suppresses Splenocyte Proliferation To investigate whether nifedipine could block the proliferation of T lymphocytes, the effects of nifedipine on the proliferative response induced by a mixed lymphocyte reaction (MLR) was assayed. Nifedipine significantly suppressed the proliferation of splenocytes in a dose-dependent fashion (Figure 4-10). Low doses of nifedipine, including 1 u M and 10 uM, weakly inhibited splenocyte proliferation, whereas 100 u M and 200 u M nifedipine completely abrogated proliferation. There was no significant inhibition of splenocyte proliferation by treatment with DMSO solvent alone.  4.3 Discussion  Initially, in an attempt to clearly define the functional contribution of the OCIFsubunit variants to C a  2 +  entry, the gene-silencing technique, RNAi, was employed to  downregulate the expression of both splice isoforms in Jurkat T cells. Although there were concerns that the expression of the 200 kDa ai F-subunit membrane-localized protein could not be knocked-down by RNAi, a previous study reported that stable 2__ |  expression of antisense RNA knocked-down protein expression of the type 3 RyR Ca channel in Jurkat T cells (73). Nevertheless, the siRNA target sequences chosen in this study were not successful at knocking-down ocip-subunit protein expression in Jurkat T cells. Although RNAi is a potent technique used to suppress gene expression in mammalian cells, it requires the analysis of many different siRNA duplexes in order to successfully knock-down expression of the gene of interest. Future experiments 138  16  14-1 ^ X  12H  E  la-  CD  s'  O  I to  61  o_  NT  DMSO  10  50  100  (pM Nifedipine)  200 Autologous ~ MLR  Figure 4-10: Nifedipine suppresses splenocyte proliferation. Splenocytes from C57B1/6 mice were incubated with 1-200 LiM nifedipine in triplicate and then stimulated with irradiated Balb/c splenocytes. Five to six days later, each sample was assayed for proliferation as reflected as lymphocyte number determined by flow cytometry. The control is splenocytes treated with D M S O solvent alone. Each bar represents the mean and SD of assays from triplicate wells. P<0.01, relative to the Control.  139  examining the effects of siRNA should encompass a larger number of siRNA constructs targeted to different regions of the oti F-subunit. As an alternative strategy, the DHP derivatives, (+/-) Bay K 8644 and nifedipine, were used to further investigate the presence of L-type C a and to assess the contribution of C a  2 +  2 +  channels in T lymphocytes  influx by L-type C a  2 +  channels during the T  lymphocyte activation process. Previous studies investigating the role of L-type C a  2 +  channels have used synthetic DHP derivatives to study the function of these channels in the plasma membranes of numerous cell types (157, 165, 192). Through an intracellular Ca  assay, it was demonstrated here that (+/-) Bay K 8644 exerts an agonistic action on 9+  the Ca  channels of Jurkat T cells and human PBTs. However, treatment with (+/-) Bay  K 8644 did not induce maximal C a  2 +  influx in either cell type since anti-CD3 stimulation  of PBTs and Jurkat T cells resulted in a 2- to 4-fold larger increase in intracellular C a , 2+  respectively. An explanation for this discrepancy may be that the (-)- and (+)-enantiomers of Bay K 8644 may competitively bind to the cti-subunits expressed in T cells, dampening the overall agonistic action of the DHP derivative. It was also observed that (+/-) Bay K 8644 treatment resulted in a sustained C a transient C a  2+  flux was  induced in PBTs.  2 +  influx in Jurkat T cells and only a  Interestingly, previous studies  have  demonstrated through electrophysiological means that in Jurkat T cells the number of plasma membrane C R A C channels is between 100 to 400 (129), whereas resting PBTs only express 15 C R A C channels (130). If L-type C a discrepancy in (+/-) Bay K 8644 induced Ca  2 +  channels contribute to  /CRAC,  the  influx could be explained by the difference  in the number of channels expressed by both cell types.  140  The experiments examining the effects of nifedipine on C a nifedipine only partially blocks C a significantly affect C a  2 +  TCR/CD3  nifedipine do seem to affect C a  2 +  influx showed that  influx in Jurkat T cells and PBTs, and does not  2 +  release from intracellular C a  stimulation through the  2 +  complex.  2 +  stores in Jurkat T cells following  However,  higher concentrations  of  release from the ER in human PBTs. There is evidence  that high concentrations of DHP antagonists and other CCBs interfere with SR release and uptake of C a  2 +  in both cardiac and smooth muscle cells (197). Therefore it was  concluded that nifedipine is blocking L-type C a  channels found in the plasma  2 +  membrane, with minor inhibitory effects at higher concentrations on C a  2 +  release from  intracellular stores. Although the specificity of action of DHPs has been confirmed by determining the DHP binding sites on the channel-forming ai-subunit of L-type VDCCs (184, 185), there are studies demonstrating the non-specific inhibitory effects of high micromolar concentrations of DHPs on voltage-dependent  potassium (K ) channels and C a 2+  v  activated K channels (198-200). Even though these observations could be of concern in +  the present study, Fagni et al. reported that micromolar concentrations of both nifedipine and the stereospecific enantiomers of Bay K 8644 exhibited an inhibitory effect on K  +  current through Kv and Ca -activated K channels (199). This is in contradiction to the 2+  +  results in the present study, since it was shown that (+/-) Bay K 8644 has an agonistic effect while nifedipine antagonizes C a  2 +  influx in T lymphocytes. In addition, (+/-) Bay  K 8644 does not activate K channel currents (199). Therefore, the overall observed +  effect of the DHPs in this study was due to modulation of C a Ca  2 +  2 +  influx through L-type  channels. Minor inhibitory effects of DHPs on K current may occur at the higher +  141  micromolar concentrations, but are reportedly absent at the lower range of nifedipine used in this study where inhibition of C a  2 +  influx is still observed (198).  After confirming that (+/-) Bay K 8644 and nifedipine can modulate C a in T lymphocytes, the investigation of whether C a  2 +  2+  influx  influx through an L-type C a  2 +  channel can modulate early Ca -dependent signaling events, such as M A P kinase 2+  activity was conducted. It has been postulated that C a  2 +  can interact with the M A P kinase  signaling pathway in T lymphocytes by activating Lck and calmodulin-kinase, which are upstream of Erkl/2 and responsible for Ca -dependent Erkl/2 enzymatic activation 2+  (201). It was reported here that (+/-) Bay K 8644 could induce phosphorylation and activation of both Erkl and Erk2 in Jurkat T cells and weak activation of Erk2 in human PBTs. Furthermore, the activation of Erkl/2 by (+/-) Bay K 8644 could be blocked by pretreatment with nifedipine. These results support the hypothesis that C a through an L-type C a  2 +  2+  influx  channel mediates the MAP kinase signaling pathway during T  lymphocyte activation. The quantitative discrepancy between Erk activation in Jurkat T cells and primary T lymphocytes might occur if the human PBTs required a higher sustained level of intracellular C a  2 +  to induce Erk activity compared to transformed T  2_j_  lymphocytes. The Ca  ionophore, ionomycin, was observed to induce a 2.5-fold greater  increase in intracellular C a  2 +  in human PBTs compared to Jurkat T cells, which  corresponds with a more robust phosphorylation of Erkl/2 by ionomycin in human PBTs. In addition, Ca  entry induced by (+/-) Bay K 8644 in human PBTs was only transient  compared to the sustained C a amount of C a  2+  2+  influx in Jurkat T cells. Therefore the difference in the  influx could result in (+/-) Bay K 8644 strongly activating Erkl/2 in  Jurkat T cells, and only weakly inducing activation of Erk activity in human PBTs.  142  To examine whether an L-type Ca  channel can mediate more downstream Ca -  dependent signaling events during T lymphocyte activation, the effects of nifedipine on the transcriptional activity of NFAT, IL-2 secretion and IL-2R expression were studied. It was demonstrated that inhibiting C a NFAT in a dose-dependent  2+  influx with nifedipine could inhibit the activity of  manner in Jurkat T cells. Since NFAT regulates the  transcription of several cytokine genes, including IL-2, the effects of nifedipine on IL-2 production and IL-2R expression were then examined (196). In both Jurkat T cells and human PBTs, IL-2 production was blocked in the presence of nifedipine. The overall inhibition in IL-2 secretion mediated by nifedipine was confirmed to be due to a block in Ca  2+  influx and not cell death since the percentage of viable cells did not significantly  change with increasing nifedipine dose. These results are consistent with a previous report showing that inhibition of human T lymphocyte proliferation by 100 u M nifedipine was not due to drug cytotoxicity (186). It was also demonstrated that the block in IL-2 secretion by nifedipine can be reversed by the addition of ionomycin, confirming that low doses of nifedipine are inhibiting L-type C a  2+  channels and not suppressing the  function of other channels in T lymphocytes. IL-2R expression was only downregulated with high concentrations of nifedipine. This is consistent with the current understanding that the signaling requirements for expression of the IL-2R are less stringent than those for IL-2 production (202). Thus C a  2 +  entry through L-type C a  2 +  channels regulates N F A T  activity and IL-2 production but has a lesser effect on IL-2R expression. In addition to demonstrating that an L-type C a  2 +  channel mediates C a  during T lymphocyte activation, the investigation of whether an L-type C a  2+  2 +  influx  channel  would regulate the proliferation of T lymphocytes was addressed. To this end, the  143  proliferation of mouse splenocytes was assayed through an in vitro M L R and found that nifedipine markedly inhibited mouse splenocyte proliferation in a dose-dependent manner. These observations support the hypothesis that C a channels is required for sustained C a  2 +  2 +  influx through L-type C a  2 +  influx during T lymphocyte proliferation in vitro.  Taken together, the results in this study show that (+/-) Bay K 8644 and nifedipine partially modulate T lymphocyte activation and proliferation. Although evidence has been provided for the presence of a DHP sensitive L-type C a the plasma membrane of T lymphocytes, additional C a the C a  2+  2 +  channel in  2+  channels may also contribute to  influx pathway. This conclusion is in agreement with recent studies, describing  the involvement of TRP ion channels in regulating C a  2 +  influx in T lymphocytes. Cui et  al. showed that the TRPV subfamily member of ion channels, C a T l , is involved in generating  /CRAC  in Jurkat T cells, which is partially regulated through intracellular C a  store-depletion (105). Although CaTl plays a significant role in mediating C a  2 +  2 +  entry,  overexpression of a dominant negative pore-region mutant of CaTl did not completely abolish C a L-type C a  2+  2 +  influx in Jurkat T cells, leading to the possibility that other channels, such as channels are also involved in C a  modulated by intracellular C a  2+  2+  entry (105). TRP channels that are not  store-depletion have also been found in human T  lymphocytes. For instance, Sano et al. reported that the LTRPC2 protein is abundantly expressed in human peripheral blood and Jurkat T cells and mediates C a response to elevated levels of pyrimidine nucleotides, ADPR, and N A D mRNA and protein is also expressed in Jurkat T cells and PBTs and C a  2+  influx in  +  (108). TRPC6  2+  influx through  this channel is activated by D A G (110). In conjunction with the results from this study and the recent discovery of TRP protein expression in T lymphocytes, it is highly  144  probable Ca  entrance into T lymphocytes is mediated through multiple Ca  including both TRP and L-type C a Ca  2 +  2 +  channels,  channels. Given that the amplitude and duration of  signals in T lymphocytes are very diverse, a number of different channels may be  necessary to coordinate the different C a  2+  responses required for T lymphocyte  activation, proliferation and death.  145  CHAPTER 5: DETERMINING THE ROLE OF L-TYPE CALCIUM CHANNELS IN T LYMPHOCYTES IN VIVO  5.1 Introduction  In 2003, a study conducted by the World Health Organization reported that the cardiovascular disease, hypertension, is estimated to cause 4.5% of the global disease burden (203). In the United States, approximately 50 million American adults are affected  by hypertension (204). Recent surveys on antihypertensive  medications  documented that the short-acting first generation DHP, nifedipine (clinically known as Procardia™), and the long-acting third generation DHP, amlodipine (Norvasc™), are two of the most commonly prescribed drugs for the treatment of hypertension (205, 206). In patients with cardiovascular disease, nifedipine and amlodipine block the action of L-type VDCCs that are responsible for initiating the contraction of cardiac and vascular smooth muscles (157). Nifedipine and amlodipine are also frequently administered for other clinical ailments including angina, congestive heart failure, ischemia, migraine headaches and Raynaud's syndrome (205). Although DHP derivatives adequately control the symptoms of several diseases, there are many well-documented side effects associated with these medications, such as dizziness, lightheadedness, heat sensation, nausea, heartburn, palpation, nasal congestion and sore throat (205). Another troublesome side effect of both second and third generation DHP derivatives is peripheral edema (178).  146  In addition to these adverse reactions brought on by DHP administration, there are concerns that DHP derivatives might also affect the immune competence of patients receiving DHPs as an anti-hypertensive therapy. These concerns were initiated by the early demonstration that several clinically administered DHPs have marked inhibitory effects on T cell function in vitro (186, 187). Subsequently, the DHP C a  2+  channel  antagonist, nifedipine, was also found to augment both early and late Ca -dependent 2+  signaling events during T lymphocyte activation and proliferation in vitro (140). In conjunction with these studies, several investigators have shown that channel-forming ocisubunits of L-type C a  2 +  channels, which contain DHP binding sites, are expressed in both  human and mouse T lymphocytes (119, 120, 140). The findings in the previous studies collectively raise the question of whether long-term treatment with DHPs may act as an immunosuppressant, even though the serum concentrations of DHPs in patients (see Table 5-1) are 10 to 100-fold lower than the minimal inhibitory dose of nifedipine used in vitro. The concern that DHP administration may compromise the immune system of patients is heightened by the fact that a few studies have reported that CCBs have immunosuppressive effects in humans. It has been shown that administration of one 10 mg  oral dose of nifedipine to healthy humans transiently downregulated immune  responses in these individuals (207). In this study, Morgano et al. demonstrated that four hours after oral administration of nifedipine, PHA-induced peripheral blood lymphocyte proliferation was significantly reduced relative to the proliferation of lymphocytes isolated from the same individual prior to drug  administration (207). The defect in  proliferation was attributed to impaired production of IL-2 by lymphocytes shortly after  147  Chemical Structure  1,4Dihydropyridine  Nifedipine (Procardia™)  MeOOG s y l /  COOMe  Me'^U'^Me  Amlodipine (Norvasc™)  EtOOC \ Me  31 \\  /  COOMe CH OCH CH NH 2  2  2  Serum Concentration  Mean Plasma HalfLife (Single dose-studies)  -80-170 nM (20 mg oral dose over 7 days) (208)  ~6h (40 mg oral dose) (209)  -14 nM (5 mg oral dose over 7 days) (210)  35 h (15 mg oral dose) (211)  2  Table 5-1: Summary of pharmacokinetic parameters of nifedipine and amlodipine in humans. Nifedipine and amlodipine are two structurally similar DHP derivatives that are commonly prescribed for the treatment of hypertension. The clinical name for nifedipine is Procardia™, whereas amlodipine is clinically known as Norvasc™. The serum concentration and mean plasma half-life of both nifedipine and amlodipine were measured in several volunteers by high performance liquid chromatography and the average values were determined. Higher doses of nifedipine are typically administered to hypertensive patients compared to the amlodipine dose due to the short plasma half-life of nifedipine.  148  nifedipine treatment (207). Administration of the DHP derivative, nilvadipine, for a six month period has also shown to adversely affect several immunological parameters of hypertensive patients (212). The patients treated with nilvadipine not only showed a reduced frequency in various PBT subsets including CD4 CD8", CD4 CD45RA" and +  +  CD4 CD45RA T cells, but also had decreased concentrations of soluble IL-2R in the +  +  peripheral blood (212). In addition to the studies detailing the inhibitory effects of DHPs in patients, a single study has shown that the first generation phenylalkylamine, verapamil, also diminished the immune response of a patient receiving this drug (213). Administration of verapamil caused the patient to experience repeated and prolonged viral infections that was associated with suppressed lymphocyte proliferation in response to mitogen stimulation (213). Although the immunosuppressive properties of CCBs are not well defined, these previous studies suggest that CCBs may bind and modulate the function of L-type C a  2 +  channels in circulating lymphocytes, which may directly affect  the function of T cells, and therefore the immune competence of these patients. The investigation presented here began by exploring the effects of nifedipine treatment on the C D 8 T cell response to a defined histocompatibility antigen in vivo. +  DHP inhibition of T cell function in vivo would provide additional support that functional L-type C a  2 +  channels are expressed in T lymphocytes, and that DHPs may have  immunosuppressive effects in patients. The in vivo proliferation assay quantified the response of female T cells proliferating to the male-specific minor histocompatibility (H) antigen encoded on the Y chromosome (H-Y antigen) in male recipient mice treated with nifedipine. Only the female C D 8 T cell response was measured in this assay since the +  female mice were bearing a Tg TCRcq3 receptor that is M H C Class I restricted and  149  specific for the male H-Y antigen. The CD8 T cell proliferative response was quantified by loading the female H-Y specific TCR-Tg T cells with the fluorescent dye, CFSE, prior to female cell injection and nifedipine treatment of the male mice. CFSE labeling marked each successive cell division among proliferating female T cells since the fluorescence intensity of the dye is halved and distributed equally in the daughter cells. As a result of this analysis, the first direct evidence that nifedipine can inhibit T cell proliferation in vivo was demonstrated. Since  millions  of people  are administered  DHPs  for the  treatment  of  cardiovascular diseases and other clinical disorders, it was important to further investigate the potential immunosuppressive risks associated with long-term DHP administration. The investigation was continued by examining T cell function in renal disease patients that had received DHP treatment. Renal disease patients from St. Paul's Hospital in Vancouver, B.C. were chosen for this analysis since there is a high prevalence of hypertension in these patients, and a large cohort receiving either nifedipine or amlodipine treatment.  Additionally, at St. Paul's Hospital, the demographic and  medication data, as well as the infection rate is well recorded for renal disease patients with chronic kidney disease, on hemodialysis, on peritoneal dialysis and post-renal transplant. Taken together, the results from this study provide further support for the hypothesis that long-term treatment with DHPs may affect the immune competence of hypertensive patients.  150  5.2 Results  5.2.1 Nifedipine Inhibits T Cell Proliferation in Mice Since nifedipine clearly inhibited M L R induced splenocyte proliferation in vitro (140), it was evaluated whether nifedipine could block the proliferation of an antigen specific T cell response in vivo. To address this question the proliferative response of female H-Y-specific TCR-Tg C D 8 C F S E thymocytes transferred into male C57B1/6 +  +  mice was examined. In male recipients, H-Y-specific C D 8  +  CFSE  activated upon T C R engagement, eliciting a small persistent C a  2+  +  T cells become  influx and clonal  expansion that proceeds for several days (214). This clonal expansion can be easily monitored in cells derived from recipient spleens (214). This study is the first reported investigation examining the effects of nifedipine on T cell proliferation in vivo, therefore preliminary experiments were conducted to establish an effective treatment concentration of nifedipine in mice. Male mice injected with CFSE-labeled female H-Y-specific TCR-Tg T cells were treated with either the vehicle control (PBS containing 5% ethanol and 1% Tween-80) or nifedipine in concentrations ranging from 10, 15, 20, to 50 mg/kg. These nifedipine concentrations were chosen since previous investigations examining the in vivo pharmacokinetics of nifedipine and other DHP derivatives in mice used similar concentration ranges (125, 215, 216). In this study, the preliminary experiments showed that 10 mg/kg nifedipine had no inhibitory effects on T cell proliferation, whereas 15 mg/kg nifedipine retarded proliferation of H-Y-specific CD8  +  CFSE  +  T cells (data not shown). Male mice treated either 20 or 50 mg/kg  nifedipine demonstrated significant morbidity; in fact 50 mg/kg caused lethality in all  151  mice shortly after the i.p. nifedipine injection. As a result of this preliminary analysis, a nifedipine concentration of 15 mg/kg was used in the remaining in vivo proliferation assays. The in vivo anti-proliferative effects of nifedipine were confirmed by treating male mice with either four doses of 15 mg/kg nifedipine or vehicle control after the i.v. injection of 20-30x10 female H-Y-specific TCR-Tg C F S E 6  +  T cells. Four doses of  nifedipine were administered to the male mice at 6 h intervals over a period of 40 h since it was previously determined that the half-life of nifedipine in mouse peripheral blood is very short. Larkin et al. demonstrated that the half-life of a single i.p. dose of 6 mg/kg nifedipine is 11 min and 60 mg/kg nifedipine is 30 min in mouse blood (215). As illustrated in Figure 5-1 A , treating male mice with four doses of 15 mg/kg nifedipine inhibited, but did not completely abrogate H-Y-specific C D 8  +  T cell proliferation.  Nifedipine treatment resulted in an increased number of H-Y-specific C D 8 T cells not +  dividing or undergoing only 1 cell division, and significantly fewer cells transiting to 2 divisions compared to the vehicle control. The proliferative response of H-Y-specific C D 8 T cells in female C57J31/6 mice was also assayed as a control for no proliferation. +  As expected, the H-Y-specific C D 8 T cells present in the spleen did not proliferate in the +  female mice due to the absence of the male H - Y antigen (Figure 5-1 A, Control, open bar). The total number of H-Y-specific TCR-Tg C D 8 C F S E +  +  T cells present in the  spleen of female control, vehicle, and 15 mg/kg nifedipine treated male mice was also determined. It was found that significantly fewer proliferating T cells reached the spleen in nifedipine treated mice compared to the vehicle control (Figure 5-1B). Additionally,  152  Figure 5-1: T cell proliferative response to H - Y male antigen is decreased in mice following repeated nifedipine treatment. CFSE loaded thymocytes from C57B1/6 female mice with Tg TCRap H-Y receptor were i.v. injected into female (Control, n=\) or male recipients. Male mice received one i.p. dose of vehicle (n=5) or 15 mg/kg nifedipine (n=5) 1 h after the cell injection followed by three i.p. injections of either vehicle or nifedipine at 6 h intervals. Splenocytes were harvested 40 h after the initial i.v. injection and proliferation of C F S E , C D 8 and Tg TCR splenocytes was quantified by flow cytometry. (A) Bars represent % of total cells exhibiting a discrete CFSE (FL1) intensity reflecting an equal number of cell divisions: no divisions (open bar), 1 division (solid bar), and 2 divisions (hatched bar). *, PO.01, as comparing cell divisions between vehicle control and nifedipine treated male mice. (B) Total number of viable, H-Y-specific TCR-Tg C D 8 C F S E T cells present in the spleen of female control, vehicle, and nifedipine treated male recipients. *, P<0.01, when comparing total T cell number in vehicle control to nifedipine treated male mice. Each bar represents the mean and SD of assays from five mice. The results depicted are representative of two independent experiments. +  +  h i g h  +  +  153  the number of proliferating H-Y-specific CD8 T cells in the spleen of nifedipine treated mice was similar to the number of non-proliferating T cells found in the spleen of the female mouse. It should be noted that in each of the following experiments there was a variable number of T cells recovered in the female controls and male treated mice. These inconsistencies were probably due to slight differences in the number of female H - Y specific TCR-Tg C F S E T cells initially i.v. injected into recipient animals. +  5.2.2 Increasing the Number of Nifedipine Doses Augments Anti-Proliferative Effect Initial  experiments  examining  the  anti-proliferative  effects  of  nifedipine  established that four doses of 15 mg/kg nifedipine considerably slowed down T cell proliferation in vivo. The investigation on the anti-proliferative effects of nifedipine was continued by determining the minimal number of 15 mg/kg doses of nifedipine that would significantly inhibit T cell proliferation in mice. Beginning 1 h after the i.v. injection of the H-Y-specific TCR-Tg T cells, male mice were injected i.p. with 1, 2, or 3 doses of 15 mg/kg nifedipine at 6 h intervals. Mice treated with either 1 or 2 doses of 15 mg/kg nifedipine showed no profound changes in the percentage of proliferating H - Y specific C D 8 T cells in each cell division compared to the vehicle control (Figure 5-2A). +  However, treatment of the mice with 3 doses of 15 mg/kg nifedipine resulted in an increase in the number of proliferating T cells that had divided once, and a decrease in the number of cells that had undergone 2 divisions compared to the vehicle control (Figure 5-2A).  When comparing the degree of inhibition by either 3 or 4 doses of 15  mg/kg nifedipine, it was found that 4 doses of 15 mg/kg nifedipine inhibited T cell proliferation to a greater extent than 3 doses. In summary, these  experiments  154  CD  o ,o  g '&  2 ct  B  Female Control  Vehicle  Female Control  Vehicle  1 Dose  2 Doses 15 mg/kg Nifedipine  12 in o  10  X  CD  o I-  <D  8 6  T Dose  2 Doses 3 Doses 15 mg/kg . Nifedipine  Figure 5-2: Anti-proliferative effect of nifedipine is dependent on the number of doses administered to mice. CFSE loaded thymocytes from female mice with Tg TCRocp H-Y receptor were i.v. injected into female (Control, n~\) or male recipients. Male mice received 3 i.p. doses of vehicle (n=2) or 1, 2 or 3 doses of 15 mg/kg nifedipine (n=2). The first dose of either vehicle or nifedipine was administered 1 h after the cell injection followed by subsequent doses at 6 h intervals. Splenocytes were harvested 40 h after the initial i.v. injection and proliferation of C F S E , C D 8 and Tg T C R splenocytes was quantified. (A) Bars represent % of total cells exhibiting no divisions (open bar), 1 division (solid bar), 2 divisions (hatched bar), and 3 divisions (dotted bar). (B) Total number of viable, H-Yspecific TCR-Tg C D 8 C F S E T cells isolated from spleen of female control, vehicle, and nifedipine treated male recipients. Each bar represents the mean and SD of assays from two mice. +  +  +  +  h l g h  155  demonstrated that a minimal number of 3 to 4 doses of 15 mg/kg nifedipine were necessary to reproducibly inhibit T cell proliferation in vivo. The total number of H-Y-specific TCR-Tg C D 8 C F S E T cells that reached the +  +  spleen of both female and male treated mice was also evaluated. In comparison to the vehicle control, only mice treated with 3 doses of 15 mg/kg nifedipine showed a decrease in the number of proliferating T cells that could be harvested from the spleen (Figure 52B). In general, a decrease in T cell proliferation consistently correlated with fewer H - Y specific C D 8 T cells present in the spleen of nifedipine treated male mice. +  5.2.3 Anti-Proliferative Effect of Nifedipine is Dependent on the Timing of Administration Although the previous experiments demonstrated that 3 to 4 doses of 15 mg/kg nifedipine were necessary to inhibit T cell proliferation in vivo, it was important to determine whether the timing of the dose was more crucial to the anti-proliferative effect of nifedipine than the number of doses administered. To address this question, male mice were i.v. injected with H-Y-specific TCR-Tg T cells and then treated with one dose of 15 mg/kg nifedipine 21 h after the cell injection. It should be noted that in all of the previous experiments the first dose of 15 mg/kg nifedipine was administered only 1 h after the H Y-specific TCR-Tg T cells were injected. In this experiment, nifedipine was injected at 21 h since the H-Y-specific TCR-Tg T cells require approximately 24 h to home to peripheral lymphoid organs and proliferate in response to the male H-Y antigen (214). Even though the half-life of nifedipine is short-lived in mouse blood, treating male mice with one dose of 15 mg/kg nifedipine at a later time point inhibited H-Y-  156  specific C D 8 T cell proliferation to a similar extent as four doses of 15 mg/kg nifedipine +  (Figure 5-3A). A single dose of nifedipine resulted in an increased number of H-Yspecific C D 8 T cells undergoing only 1 -2 cell divisions and significantly fewer T cells +  transiting to 3 divisions compared to the vehicle control. Treatment with only one dose of 15 mg/kg nifedipine also resulted in a decrease in the absolute number of female H - Y specific TCR-Tg C D 8 C F S E T cells that were isolated from the spleen (Figure 5-3B). +  +  In the previous experiment, male mice treated with one dose of 15 mg/kg nifedipine only 1 h after the i.v. cell injection showed no decrease in T cell proliferation or the total number of H-Y-specific C D 8 T cells in the spleen (Figure 5-2). Therefore timing of +  nifedipine administration is critical for the anti-proliferative behavior ofthe drug.  5.2.4 Nifedipine Specifically Blocks T Cell Proliferation The previously observed decrease in T cell proliferation brought on by nifedipine treatment may have been caused by nifedipine inducing a non-specific change in the mice that lead to retarded T cell proliferation. To address this concern, the specificity of nifedipine was then investigated by determining whether 3 doses of 15 mg/kg nifedipine with the final dose administered 1 h (elimination of nifedipine half-life in mice) prior to i.v. injection of the H-Y-specific thymocytes would lead to a difference in the T cell proliferation profile compared to vehicle control (215). It was important to wait for the drug concentration to diminish so that there was no effective drug remaining in the bloodstream once the female T cells were i.v. injected. When examining the proliferative response of the H-Y-specific C D 8  +  T cells, it was found that the T cells from the  nifedipine treated mice proliferated to the same extent as the vehicle control, suggesting  157  l^\|  Female Control  B  Vehicle  15 mq/kq Nifedipine  12 in 10 o  O  £ 3  Female Control  Vehicle  15 mg/kg . Nifedipine  Figure 5-3: One later dose of nifedipine suppresses T cell proliferation in response to the H-Y male antigen in mice. CFSE loaded thymocytes from female mice with Tg TCRocp H-Y receptor were i.v. injected into female (Control, n=\) or male recipients. After 20-24 h, male mice received one i.p. dose of vehicle (n-4) or 15 mg/kg nifedipine (n=4). Splenocytes were harvested 40 h after the initial i.v. injection and proliferation of C F S E , C D 8 and Tg T C R splenocytes was quantified. (A) Bars represent % of total cells exhibiting no divisions (open bar), 1 division (solid bar), 2 divisions (hatched bar), and 3 divisions (dotted bar). *, P<0.01, as comparing cell divisions between vehicle control and nifedipine treated male mice. (B) Total number of viable, H-Y-specific TCR-Tg C D 8 C F S E T cells present in the spleen of female control, vehicle, and nifedipine treated male recipients. Each bar represents the mean and SD of assays from four mice. Results are representative of three separate experiments. +  +  h l g h  +  +  158  that nifedipine is specifically blocking L-type Ca  channels, and not causing a non-  specific effect (Figure 5-4). The absolute number of H-Y-specific TCR-Tg C D 8  +  CFSE  +  T cells present in the spleen was not determined in this experiment since there was no change in proliferation in the vehicle versus nifedipine treated mice.  5.2.5 Nifedipine  Reduces  T  Cell  Recovery  Through  an  Antigen-Dependent  Mechanism The final step in this investigation was to assess whether nifedipine treatment was directly inhibiting antigen-dependent T cell recovery in spleens of the male mice. In all previous experiments, a significant reduction in T cell proliferation by nifedipine administration was associated with a decrease in the total number of H-Y-specific C D 8  +  T cells that reached the spleen. It was important to determine whether the decrease in total T cell numbers in the spleen was simply due to reduced T cell proliferation or to specific inhibition of antigen-dependent T cell recovery by nifedipine. This question was addressed by examining the effects of nifedipine treatment on antigen-independent recovery of the H-Y-specific C D 8 T cells in the spleens of female C57B1/6 mice. In this +  experiment, female mice were treated with either 1 dose of vehicle or 15 mg/kg nifedipine approximately 20 h after the i.v. injection of the H-Y-specific TCR-Tg C F S E  +  thymocytes. In the female recipients, there was no significant difference observed between the percentage of non-dividing T cells (Figure 5-5 A) or the total number of H-Yspecific C D 8 T cells that reached the spleens (Figure 5-5B) when comparing vehicle to +  nifedipine treated mice. The results from this experiment demonstrate that H-Y-specific C D 8 T cell recovery from the spleen in an antigen-independent manner was not effected +  159  Figure 5-4: Anti-proliferative effect of nifedipine is not due to non-specific drug toxicity. Male mice received 3 i.p. doses of vehicle or 15 mg/kg nifedipine at 6 h intervals. 1 h after the last dose of either vehicle or nifedipine, CFSE loaded thymocytes from female mice with Tg TCRcq3 H-Y receptor were i.v. injected into female (Control, n-\) or male (n=4) recipients. Splenocytes were harvested 40 h after the initial i.v. injection and proliferation of C F S E , C D 8 and Tg T C R splenocytes was quantified. Bars represent % of total cells exhibiting no divisions (open bar), 1 division (solid bar), and 2 divisions (hatched bar). P>0.01, as comparing cell divisions between vehicle control and nifedipine treated male mice. Each bar represents the mean and SD of assays from four mice. +  +  h l g h  160  A  1Q0-I  ,/..  f-—,  90  •:. 1$ .80-  ••«••• J . "o  60  .  ^  50 :  | «  40  |j o o~  30 20 10- .  -••or '  I  —-—- . I  '  B  —•  15 mg/kg Nifedipine  Vehicle  •. : i 2 i in o  10  X  SH  o hO <D .Q  8 6  £ .4  ,o  2  IJi  —  1  :  "  •  '  1  —  15 mg/kg Nifedipine  Vehicle  Figure 5-5: Nifedipine treatment does not suppress antigen-independent T cell recovery in female mice. CFSE loaded thymocytes from female mice with Tg TCRa(3 H - Y receptor were i.v. injected into female recipients. After 20-24 h, female mice received one i.p. dose of vehicle (n=4) or 15 mg/kg nifedipine (n=4). Splenocytes were harvested 40 h after the initial i.v. injection and proliferation of C F S E , C D 8 and Tg T C R splenocytes was quantified. (A) Bars represent % of total cells exhibiting no divisions (open bar). The % of total cells in 1,2, and 3 divisions are not shown since the values are negligible. P>0.01, as comparing cell divisions between vehicle control and nifedipine treated mice. (B) Total number of viable, H-Y-specific TCR-Tg C D 8 C F S E T cells isolated from the spleen of vehicle and nifedipine treated female recipients. P>0.01, when comparing total T cell number in vehicle control to nifedipine treated female mice. Each bar represents the mean and SD of assays from four mice. +  +  h l g h  +  +  161  by nifedipine treatment. Instead, the data presented in Figure 5-5 confirms that nifedipine is inhibiting T cell recovery in the male mice through an antigen-dependent mechanism.  5.2.6 Examination of Human PBT Function from Renal Disease Patients Following DHP Administration Thus far it has been established that CCBs, such as nifedipine partially block L type C a  2 +  channel function during T cell activation and proliferation in vitro and in vivo.  The documented inhibitory effects of nifedipine raise the question of whether long-term administration of CCBs to patients for the treatment of cardiovascular diseases has deleterious side effects on circulating T lymphocytes. To further explore this question, PBT function from renal disease patients administered either first generation (i.e. nifedipine) or third generation (i.e. amlodipine) DHP derivatives for the treatment of hypertension was evaluated. Table 5-2 summarizes the DHP dosage, creatinine levels and T cell characteristics of the healthy female volunteer and three renal disease patients on hemodialysis and DHP therapy used in this study. Although a small cohort of renal disease patients were analyzed, previous studies examining the effects of DHPs on immune responses have used a similar number human subjects (207, 213). In the renal disease patients examined here, abnormal kidney function causes the levels of creatinine (a breakdown product of muscle creatine) to increase in the blood due to decreased excretion of creatinine in the urine (217). It should be noted that the sex, age and duration of DHP therapy of the renal disease patients was not available. The quantity of IL-2 secreted by anti-CD3 stimulated PBTs isolated from the healthy volunteer and the renal disease patients was compared in order to determine  162  Human Subjects:  Medication:  Creatinine level: Number of T cells per ml peripheral blood:  Patient A  Patient B  Patient C  None  60 mg nifedipine, twice daily  10 mg amlodipine, twice daily  10 mg amlodipine, twice daily  60  1012  900  833  Healthy Volunteer  8.1 x 10  2.5 x 10  6.9 x 10  4.1 x 10  % CD3 T cells:  78  88  93  91  % CD3 CD4 CD8 T cells:  38  32  45  70  % CD3 CD4 CD8 T cells:  33  51  27  16  5  +  +  +  +  +  5  5  5  Table 5-2: Summary of the DHP dosage, creatinine levels and T cell characteristics of the human subjects used in the study. The participants of the study were one healthy human female donor receiving no DHP therapy, and one renal disease patient administered nifedipine (patient A) and two renal disease patients receiving amlodipine (patients B and C) for the treatment of hypertension. T cell populations from all four individuals differed in the number of T cells isolated from peripheral blood, the percentage of C D 3 T cells, as well as the percentage of C D 4 and C D 8 T cells in the peripheral blood of each individual. Peripheral blood creatinine levels are also shown as an indicator of kidney function. +  +  +  163  whether DHP administration affected T cell function. The production of IL-2 was used as an indicator of T cell function since IL-2 gene expression is dependent upon extracellular Ca  2 +  influx (126), and blockage of L-type C a  2+  channels by nifedipine in vitro prevents  IL-2 secretion from human PBTs (140). As illustrated in Figure 5-6A, IL-2 secreted by PBTs from patient A receiving nifedipine and patient B administered amlodipine was impaired. However, the PBTs from patient C (also receiving a similar dose of amlodipine as patient B) showed no reduction in the amount of IL-2 secreted compared to IL-2 produced by the normal PBT control. The data presented in Figure 5-6A was normalized for the number of C D 3 T cells since each population of isolated PBTs had significantly +  different C D 3 T cell numbers (Table 5-2). +  Since CD3 CD4 CD8" T cells produce the majority of IL-2 compared to other +  +  lymphocytes, the same data was then normalized for the number of CD3 CD4 CD8" T +  +  cells. Under these new parameters, all three patients administered DHPs showed a subtle defect in IL-2 secretion compared to the normal PBT control (Figure 5-6B). Finally, the surface expression of the IL-2R and the early T cell activation marker, CD69 were also analyzed on the viable (PI negative) human PBTs after culture supernatants were removed for the IL-2 assay. In comparison to the expression on normal PBTs, IL-2R and CD69 receptor expression on the renal patients' PBTs was not downregulated following anti-CD3 stimulation (data not shown).  164  .0.0151  Amlodipine  co  O  . .  Amlodipine  >7 0.0101 CO  Q O .  Nifedipine  "&> 0.0051 Q.  Normal Control  B  Patient A  Patient B  Patient C  0.030  CO  0:025 O I- • + 0.020 Q O + co D  O .  Nifedipine Amlodipine Amlodipine  0.015  . CO Q.  0:010 OT . CL  c>i  0.005  Normal Control  Patient A  Patient B  Patient C  Figure 5-6: DHP administration may decrease IL-2 secretion from PBTs of renal disease patients. Human PBTs isolated from a healthy donor and three renal disease patients (patients A , B and C) were stimulated with immobilized OKT3 (10 Ug/ml) and 10 nM TPA. After 24 h, IL-2 secreted in the supernatants was measured by standard sandwich ELISA techniques. The results are normalized for either the quantity of IL-2 secreted by C D 3 T cells (A) or by CD3 CD4 CD8" T cells alone (B). The normal control is PBTs isolated from a healthy human female not administered DHPs. The DHP administered to each renal patient is indicated above the bars. Results depicted are representative of three independent experiments. Each bar represents the mean and SD of assays from triplicate wells. +  +  +  165  5.2.7 Uremic Serum from Renal Disease Patients Inhibits IL-2 Secretion from Normal Human PBTs One concern with the observed immunosuppressive effects of DHP administration was that uremic serum in renal disease patients may be partially responsible for the defectin IL-2 secretion, and therefore may mask any potential inhibitory effects of the DHP derivatives. This concern was warranted since an early report by Donati et al. demonstrated that 20% uremic serum from renal disease patients receiving hemodialysis inhibited PHA-induced proliferation of PBMCs and purified T cells, which coincided with downregulation of IL-2 production (217). The inhibitory effects of uremic serum were assessed in this study by treating normal human PBTs with either 10% normal serum from a healthy individual or 10% uremic serum from the three renal disease patients previously described. The PBTs were then stimulated with OKT3 and TPA, and IL-2 secretion was measured. A serum concentration of 10% was used in this analysis since a preliminary experiment demonstrated that both 15% and 20% normal serum completely abolished IL-2 secretion from normal human PBTs. As shown in Figure 5-7, treatment of normal PBTs with 10% normal and uremic serum slightly inhibited IL-2 secretion compared to preincubation of PBTs with medium alone. In addition, it was observed that only uremic serum from the two patients administered amlodipine reduced IL-2 secretion compared to treatment with normal serum  or  uremic  serum  from  the  patient  administered  nifedipine,  whereas  CD3 CD4 CD8" T cells from all three renal patients showed impairment in IL-2 +  +  secretion. IL-2R and CD69 surface expression were not downregulated on normal, viable PBTs preincubated with either normal or uremic serum following anti-CD3 stimulation  166  45 40 35 CO  o  •I.'. Q.  Nifedipine  30 25  Amlodipine Amlodipine  20 15 10  Medium Alone  Normal Serum  U r e m i c T —:—' U ic Serum S reerm um A B 1  r-  mm ic SUer er u C  Figure 5-7: Uremic serum from renal disease patients reduces IL-2 secretion from human PBTs. Human PBTs (donor, n-\) were incubated with 10% normal serum from a healthy donor or 10% uremic serum isolated from renal disease patients A , B and C. PBTs were stimulated with immobilized OKT3 (10 tig/ml) and 10 nM TPA, and IL-2 secreted in the supernatants was measured by standard sandwich ELISA techniques. The control is normal human PBTs treated with medium alone. The DHP administered to each renal patient is indicated above the bars. The human PBTs contained 10% CD3 CD4 CD8", 85% C D 3 C D 4 C D 8 , 4.0% CD3 CD4"CD8" and 1.0% CD3CD4CD8" cells. Similar data was obtained in two independent experiments. Each bar represents the mean and SD of assays from triplicate wells. +  +  +  +  +  167  compared to the media controls (data not shown). These results are in agreement with the study by Donati et al. that showed no alteration in IL-2R surface expression following normal or uremic serum treatment of PBMCs stimulated in the presence of PHA (217).  5.3 Discussion  Sustained C a  2+  influx for 1 to 2 h through plasma membrane C a  2 +  channels is  necessary to promote and maintain T cell proliferation in response to peptide-MHC 2"t_  complexes (10). To investigate whether L-type VDCCs contribute to sustained Ca influx during T cell proliferation in vivo, the effects of nifedipine on H-Y-specific TCRTg C D 8 T cell proliferation in male recipient mice were examined. Through a series of +  experiments it was demonstrated that one dose of 15 mg/kg nifedipine is sufficient to slow down the proliferation of T cells in vivo, even though the half-life of a single 15 mg/kg dose of nifedipine is less than 30 min in mouse blood (215). T cell proliferation was effectively inhibited by a single dose of nifedipine only when drug administration occurred at approximately 24 h after the H-Y-specific TCR-Tg thymocyte injection. Since the bulk of the H-Y-specific TCR-Tg T cells have reached the spleen and are beginning to proliferate in response to the male H-Y antigen at 24 h (214), nifedipine treatment at this time point may block L-type C a  2+  channels involved in sustained C a  2 +  mobilization, which is crucial for the initiation IL-2 gene transcription and T cell proliferation (10). The specificity of nifedipine in the in vivo proliferation assay was also addressed by demonstrating that treatment of the male mice with nifedipine prior to H-Y-  168  specific thymocyte injection did not interfere with the proliferation of the responding HY-specific C D 8 T cells. +  Overall, the observed anti-proliferative effects of nifedipine in mice paralleled the inhibitory action of nifedipine on mouse splenocyte proliferation in vitro. Low doses of nifedipine (1-10 jlM) used in the in vitro M L R and the single dose of 15 mg/kg nifedipine in vivo only lead to a partial block of T cell proliferation. Even following the application of multiple doses of nifedipine, the proliferation of H-Y-specific C D 8 T cells +  was not completely abolished. Therefore, the in vivo data also supports the hypothesis that in addition to L-type C a  2 +  may contribute to the C a  influx pathway involved in T cell proliferation in vivo. In  2 +  channels, other C a  2+  channels, such as TRP C a  2+  channels,  summary, the data collectively demonstrates that blocking the function of L-type C a  2 +  channels by nifedipine treatment significantly effects T cell proliferation both in vitro and in vivo. Nifedipine treatment not only caused a decrease in T cell proliferation, but it also resulted in fewer H-Y-specific C D 8  +  T cells reaching the spleen compared to mice  administered vehicle alone. Even though there are several plausible explanations for the reduced T cell numbers in nifedipine treated mice, the H - Y antigen model had its limitations and therefore, there was no clear method in determining how nifedipine treatment reduced the total number of H-Y-specific C D 8  +  T cells recovered from the  spleen of male mice. One possible explanation is that the decrease in T cell proliferation led to fewer cells existing in the nifedipine treated male mice, and consequently fewer cells reaching the spleens of these animals. In an attempt to understand how nifedipine reduced total H-Y-specific T cell numbers, it was found that nifedipine treatment of  169  female mice did not reduce the total number of H-Y-specific CD8 T cells present in the spleen. This data suggested that nifedipine exerts its inhibitory action through an antigendependent mechanism, such as antigen-dependent cell death or T cell homing to the spleen. The homing and extravasation of naive and effector T cells to secondary lymphoid organs, such as the peripheral and mesenteric lymph nodes and the spleen, is a complex, multiple step process involving the interaction between homing receptors on T cells and corresponding ligands on endothelial cells (218). Recent studies have established that the selective homing receptor-ligand interactions induce Ca -dependent 2+  signaling at different stages in the T cell homing process. Consequently, treatment of male mice with nifedipine may disrupt C a  mobilization triggered by ligated homing  2+  receptors in the H-Y-specific C D 8 T cells, directly effecting Ca -dependent signaling in +  2+  these cells. In addition to demonstrating the anti-proliferative effects of nifedipine in mice, the effects of nifedipine and amlodipine administration on circulating T lymphocytes in renal disease patients was examined. It was found that CD3 CD4 CD8" T cells from renal patients secreted less IL-2 compared to CD3 CD4 CD8" T cells isolated from a healthy +  individual when stimulated in vitro,  +  suggesting that DHP therapy may act an  immunosuppressant. However, conflicting data has demonstrated that renal disease patients not receiving DHPs also have impaired T cell responses (217). In the 1980's, studies showed an altered number of T cell subsets (219), and decreased lymphocyte responses to mitogenic stimulation in patients with end-stage renal disease (220). Uremic serum in these patients is partially responsible for the dampened immune responses (217). The inhibitory effects of uremic serum were confirmed in this study, by  170  demonstrating that treatment of normal PBTs with uremic serum from patients receiving amlodipine resulted in decreased IL-2 production. It should be noted that uremic serum from the patient administered nifedipine did not inhibit IL-2 secretion from PBTs to a greater extent than treatment with normal serum. Although uremic serum may contribute to impaired immune responses in renal disease patients, exacerbation of this defect by DHP administration cannot be ruled out. The present findings reported here should encourage further research into determining whether patients receiving DHPs are immunosuppressed. Future experiments examining T cell function of patients administered DHPs should be expanded to include a larger cohort of healthy controls as well as renal disease patients. By comparing T cell function in renal disease patients with or without DHP administration, the additive side effects of DHPs could be evaluated. Furthermore, it would be beneficial to examine T cell responses in renal patients immediately before and after DHP administration in order to pinpoint the potential immunosuppressive effects of DHPs. Examining whether the incidence of infection and cancer is higher in patients receiving DHPs would also lend support that these patients have compromised immune responses. In fact, recent studies have reported that patients receiving CCBs, including nifedipine, for the treatment of cardiovascular diseases may have increased risk of cancer (221, 222). Given the profound effects DHPs have on T cell responses in vitro and in vivo, the possibility that long-term treatment with DHPs may act as an immunosuppressant cannot be overlooked.  171  C H A P T E R 6: DISCUSSION  6.1 General Conclusions  From the  results  presented  here, evidence  was  provided that human T  lymphocytes, although non-excitable, express voltage-dependent-like C a  2 +  channels that  share some of the structural properties of conventional L-type VDCCs found in electrically excitable cells. A novel feature of both of the ai F-subunit splice isoforms is the presence of exonic sequence with amino acid identity to the human skeletal muscle ais-subunit. This is the first reported example of "splice conversion" of an L-type 0 C i subunit. The cDNA cloning of one of the alternatively spliced oci -subunit isoforms is the F  first direct evidence that a full-length ai -subunit mRNA of L-type VDCCs is expressed in T lymphocytes. Even though it was demonstrated that the mRNA and protein of  CCIF-  subunit exist in T lymphocytes, the functional role of L-type VDCCs in non-excitable cells still remains under debate. It is well established that in electrically excitable cells, such as neurons and muscle cells, L-type VDCCs are activated in response to membrane depolarization, which is a large shift in electrical potential across the plasma membrane to a less negative state. In resting T cells, the membrane potential across the plasma membrane is reported to be in the range of -50 to -70 mV (223). This membrane potential is primarily generated by K diffusion potential through Kv, and Ca -activated K +  2+  +  channels, and the sodium-K ATPase-pump (224). Unlike excitable cells, only small +  depolarizations and hyperpolarizations of the plasma membrane are measured in T cells when activated with the mitogens, Con A and PHA (223). Furthermore, depolarizing the  172  plasma membrane with high concentrations of K does not stimulate or inhibit T cell mitogenesis (223). Therefore, it is generally considered that membrane potential changes do not play a role in initiating signal transduction pathways during T cell activation (223). Based on these observations, many investigators have questioned how an L-type V D C C that is typically gated by membrane depolarization is capable of functioning in T lymphocytes when these cells do not exhibit large changes in membrane potential. One plausible explanation is that voltage-dependent-like  Ca  2 +  channels expressed in T  lymphocytes and other non-excitable cells may not be gated by changes in membrane potential. Interestingly, this hypothesis is supported by the identification of the  OCIF-  subunit splice isoforms that contain unique exon usages distinct from the ai F-subunit isolated from human retina that may render these channel variants insensitive to changes in membrane depolarization. The deletion of the IVS4 voltage sensor domain in the voltage negative variant, and the IVS3-S4 interlinker in the voltage positive variant cause the splice isoforms to lack important voltage sensing domains. Instead of being activated by depolarization, the otiF-subunit splice isoforms may be gated by an alternative mechanism, such as ER store-depletion or a direct signal from the TCR. In support of this hypothesis, it was demonstrated that the expression of the ai F-subunit splice isoforms is increased following TCR engagement in Jurkat T cells and to a lesser extent in human PBTs, suggesting that at least in Jurkat T cells, C a  2 +  influx through ai F-subunit splice  isoforms may be regulated through the TCR/CD3 complex. In summary, it appears that through alternative splicing, T lymphocytes have adopted structurally unique ai F-subunit proteins that in all probability are not gated by membrane depolarization.  173  The presence of an L-type channel-forming ai-subunit in T lymphocytes and its contribution to C a  2 +  influx during the T lymphocyte activation process was further  established by the results from the in vitro study with the DHP derivatives, (+/-) Bay K 8644 and nifedipine. Both (+/-) Bay K 8644 and nifedipine modulated early and late Ca -dependent signaling events in Jurkat T cells and human PBTs, strengthening the 2+  hypothesis that a DHP sensitive L-type V D C C is present in the plasma membrane of T lymphocytes. Furthermore, nifedipine inhibition of T cell proliferation in vivo provided additional support that functional L-type C a  2+  channels are expressed in T lymphocytes.  The mode of action of (+/-) Bay K 8644 and nifedipine may have been through binding to, and mediating the function of the alternatively spliced a i F-subunit isoforms. However, several studies have demonstrated that partial transcripts of two other channel-forming ai-subunits of L-type VDCCs, the aic-subunit and ais-subunit, exist in T lymphocytes (119, 135). The aic-subunit and ais-subunit also contain amino acids in the II1S5, 1I1S6 and IVS6 transmembrane domains that are capable of selectively binding to DHP derivatives. Due to the possible expression of more than one ai-subunit protein in T lymphocytes, it is plausible that (+/-) Bay K 8644 and nifedipine were mediating C a  2 +  influx and Ca -dependent T cell responses not only through the ai -subunit splice 2+  F  isoforms, but also through aic-subunit and ais-subunit. Regardless if (+/-) Bay K 8644 and nifedipine were binding to more than one ai-subunit subtype, it is clear that DHP sensitive L-type C a  2+  channels play a significant role in contributing to C a  2 +  entry into T  lymphocytes. Although the molecular and pharmacological studies point to a role for L-type VDCCs in mediating C a  2 +  influx pathways in T cells, at the present time, it is unclear  174  whether the aiF-subunits splice isoforms or other ai-subunits present in T cells contribute to  /CRAC-  It has been well established through patch-clamp recordings that the sole Ca  current induced by TCR engagement and by passive store-depletion in T lymphocytes is /CRAC  (10, 79). As previously mentioned, many of the biophysical characteristics of  /CRAC  are closely shared with L-type VDCCs. Similar to L-type VDCCs, C R A C channels exhibit a high selectivity for C a  2 +  over monovalent cations, that can be ascribed to C a  2 +  binding with micromolar affinity to sites within these channels (133). C R A C channels and VDCCs both exhibit a loss in C a  2 +  selectivity when the external [Ca ] is lowered to 2+  micromolar levels (127, 133). Kerschbaum et al. also demonstrated through probing C R A C channels with various organic monovalent cations of differing sizes that the physical diameter of the channel pore is ~0.6 nm for both C R A C channels and L-type V D C C s (128). Even though C R A C channels and L-type VDCCs differ in their gating mechanisms and single-channel conductance (VDCC in excitable cells is 300 times larger than the C R A C channel), they do share many biophysical characteristics, indicating that these channels may have a similar structure (128). Based on this analogous biophysical "fingerprint", and the possibility that the 0CiF-subunit splice isoforms may not be gated by depolarization, the argument that L-type VDCCs may be responsible for conducting part of  /CRAC  is plausible.  Over the past several decades, the continual discovery of novel channels proteins that mediate either C a  2 +  release from intracellular stores or C a  2 +  influx across the plasma  membrane is constantly reshaping the proposed models for Ca  mobilization in T  2+ lymphocytes. For example, it was initially reported that IP binding to the 3  channel was the central mechanism for C a  2+  IP3R  release from intracellular C a  2 +  Ca  stores.  175  However, the identification of RyR3 Ca  channel in T lymphocytes, together with the  second messenger, cADPR, has led investigators to reinterpret earlier models. Presently, it is believed that IP is necessary for the initial C a  2 +  3  while cADPR association with RyR C a phase of C a  2+  2 +  release from IP3R C a  2+  channels,  channels is essential for a prolonged second  release (75). The discovery of novel C a  across the plasma membrane is also redefining C a  2+  2+  channels that mediate C a  2 +  entry  influx models. The data obtained  from the in vitro and in vivo DHP studies demonstrated that (+/-) Bay K. 8644 did not maximally activate C a  influx, while TCR-induced C a  2 +  2+  influx was not completely  inhibited by nifedipine in Jurkat T cells and human PBTs. These results suggest that, although L-type VDCCs contribute to C a Ca  2 +  2 +  influx, additional C a  entry during T cell activation. When the TRP C a  2 +  2 +  channels also mediate  channel, C a T l , was originally  identified in T lymphocytes, it was proposed that this single channel protein was responsible for generating solely responsible for  /CRAC-  /CRAC  Further work by Cui et al. revealed that CaTl was not  since overexpression of a dominant negative CaTl pore  mutant did not completely abolish endogenous C R A C channel activity in Jurkat T cells (105). Since CaTl does not recapitulate all the properties of  /CRAC,  it has been suggested  that CaTl multimerizes with the TRPV5 channel protein and together, the CaTl/TRPV5 complex forms endogenous channels, other C a  2 +  /CRAC  (105). In addition to L-type VDCCs and TRP Ca  channels that are store-independent, such as LTRPC2 and TRPC6,  also may shape the C a  2 +  signal during sustained T cell activation. Since C a  lasts for 1 to 2 h in stimulated T cells, distinct C a  2 +  2+  signaling  channels may be involved in different  phases of the response, thus illustrating the complexity of C a  2 +  mobilization.  176  Based on these studies and the recent data presented on the oci F-subunit splice isoforms, a model of immediate and sustained C a  2+  signaling in T lymphocytes has been composed  (Figure 6-1). The immediate phase of C a  2 +  mobilization involves the C a  2 +  channels that  are activated shortly after the engagement of the TCR/CD3 complex by peptide-MHC. During this phase, the emptying of intracellular C a Ca  2 +  2 +  stores by C a  2 +  release through IP R 3  channels causes the opening of the C R A C channel tetrameric complex, potentially  comprised of CaTl and TRPV5. The voltage negative splice variant of the ai F-subunit may be involved in this phase since the mRNA expression of this channel isoform rapidly increases following T C R engagement. The mRNA expression of LTRPC2 was also shown to increase promptly after anti-CD3 treatment, so this channel may also contribute to the early phase of C a  2 +  signaling. Finally, D A G production may activate the TRPC6  channel immediately following ligation of the TCR. During the sustained phase of C a mobilization, RyR C a  2 +  channels are essential for a prolonged release of C a  2 +  2+  from  intracellular stores. The mRNA expression profiles of the voltage negative and positive splice variants in Jurkat T cells indicate that both channel isoforms may participate in Ca  2+  influx in this second phase. The observed increase in oci -subunit protein expression F  after 1 to 2 days of activation also supports the hypothesis for a role for both splice variants in contributing to sustained C a  2 +  channels coordinate a stable and dynamic C a  influx. In summary, together these C a 2+  2 +  signal that is necessary to promote T cell  activation and clonal expansion.  177  Figure 6-1: Proposed model for C a signaling in T lymphocytes.  2+  channels involved in distinct phases of C a  2+  During T cell activation, intracellular C a mobilization can be divided into immediate and sustained phases of C a signaling. In the early phase, it appears that the IP R C a channel is responsible for C a release from the ER, whereas C a T l , TRPC6, LTRPC2 and possibly the voltage negative splice variant ofthe aiF-subunit may regulate C a influx pathways. RyR C a channels participate in C a release from intracellular C a stores in the sustained phase of C a signaling. C a T l continues to provide C a influx during this later phase of the C a response. Both a| -subunit splice isoforms may also contribute to C a delivery across the plasma membrane. This figure was adapted from Lewis et al. (10). 2 +  2 +  2+  3  2 +  2 +  2 +  2 +  2+  2+  2+  2+  F  2 +  178  6.2 Future Directions  The ultimate goal of the research presented here is to understand the physiological role of L-type VDCCs in the C a  2+  entry process during T lymphocyte activation. Through  this research it has been established that novel channel-forming ociF-subunits of the Ltype V D C C are expressed  in T lymphocytes. Since it was determined that the  alternatively spliced (XiF-subunits lack several exons compared to the known otiF-subunit from human retina, it is important to demonstrate that the T lymphocyte aiF-subunits form functional C a contribute to C a  2 +  2 +  channels, and definitely determine whether these channel isoforms  influx pathways during T cell activation.  One method that is routinely used to establish whether a Ca  channel is  functional is to overexpress the cloned channel in a heterologous expression system, such as Xenopus oocytes. The C a  2+  currents gated by the overexpressed channel can then be  measured through patch-clamp recordings. These experiments can be performed with the voltage negative splice variant of the ai p-subunit and the human retina cti F-subunit since the full-length cDNA sequences of both channels have been cloned. By comparing the Ca  2 +  currents gated by the different C a  2 +  channels through patch-clamping, it will be  established whether the voltage negative splice variant of the ai F-subunit is actually insensitive to membrane depolarization. It may also be possible to determine whether the Ca  2 +  current gated by the voltage negative isoform can recapitulate the biophysical  properties of /CRAC- If it is not feasible to measure the current gated by the cloned a i F subunits in Xenopus oocytes, alternative approaches will be considered. For instance, the voltage negative splice variant may be overexpressed in Jurkat T cells. C a  2+  influx in  179  untransfected and transfected Jurkat T cells can be compared when the cells are loaded with the C a  2+  sensitive dye, indo-1, and activated with various stimuli, such as anti-CD3  mAb, thapsigargin (to deplete ER stores), or high concentrations of K  +  (to induce  membrane depolarization). These experiments will determine whether the exons that have been removed through alternative splicing in the voltage negative splice variant are required for a functioning C a  2 +  channel.  In addition to overexpressing  the  cloned  voltage negative variant in T  lymphocytes, experiments will also be conducted to produce aiF-subunit knock-out mice that are deficient in both ocip-subunit splice isoforms. The generation of ai F-subunit knock-out mice will allow the determination of whether the lack of 0 C i F-subunit expression leads to deficiencies in C a  2+  influx in the T cells of these animals. The  OCIF-  subunit knock-out mice will be generated by injecting embryonic stem cells (129 background) with the pPNT vector, containing 2 kb of the 5'-untranslated region and exon 1 of the C A C N A 1 F gene from human retina in the short arm, and 6 kb of exons 714 in the long arm. This will lead to an out-of-frame C A C N A 1 F gene sequence due to neomycin insertion in exons 2-6, converting an intracellular loop of the channel to an extracellular loop, disrupting ai F-subunit channel function. Splenocytes and thymocytes from these knock-out animals will be assayed for T cell function, including C a  2 +  influx,  N F A T activation and IL-2 secretion. It will also be assessed whether T lymphocytes from the knock-out mice proliferate in a MLR. These assays will determine whether the a i F  subunit splice isoforms directly contribute to Ca  influx during T cell activation and  proliferation. Furthermore, these experiments should clearly address the function of the (Xi F-subunit channel isoforms in T lymphocytes.  180  Although substantial efforts have been made over the past few years to elucidate the mechanisms controlling C a precise C a  2+  2 +  influx pathways in T lymphocytes, identifying the  channels that coordinate C a  2 +  entry remains a central goal for investigators.  The identification of novel voltage-dependent-like C a with TRP C a  2+  channels, implies that C a  2 +  2 +  channels in T lymphocytes, along  influx may be gated by several distinct C a  2 +  channels during the T cell activation process. An important long-range goal will be establishing the contribution of each C a Ca  2 +  2 +  channel that shape the immediate and sustained  signals. This is especially imperative in light of the fact that T lymphocytes from  severe-combined immunodeficiency patients have a principal defect in transmembrane Ca  2 +  influx (225). Although the patients' T cells are currently being used as a tool to  understand Ca -dependent signaling pathways, elucidation of the C a 2+  mediating C a  2+  influx is paramount to providing an effective  individuals. In conclusion, understanding the mode of C a  2 +  2+  channels  treatment to these  entry will eventually lead to  the development of novel therapeutic agents that could mediate T cell activation or inactivation states during an immune response.  181  REFERENCES  1. 2. 3. 4. 5. 6.  7.  Berridge, M. J., M . D. Bootman, and P. Lipp. 1998. Calcium—a life and death signal. Nature 395:645. Berridge, M. J., P. Lipp, and M . D. Bootman. 2000. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1:11. Bootman, M . D., and M. J. Berridge. 1995. The elemental principles of calcium signaling. Cell 83:675. Berridge, M . J., M . D. Bootman, and H. L. Roderick. 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4:517. Berridge, M . J. 1998. Neuronal calcium signaling. Neuron 21:13. Miyazaki, S., H. Shirakawa, K. Nakada, and Y. Honda. 1993. Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs. Dev Biol 158:62. Wang, S. Q., L. S. Song, E. G. Lakatta, and H. Cheng. 2001. Ca2+ signalling between single L-type Ca2+ channels and ryanodine receptors in heart cells.  Nature 410:592. 8. 9. 10.  Robb-Gaspers, L. D., and A. P. Thomas. 1995. Coordination of Ca2+ signaling by intercellular propagation of Ca2+ waves in the intact liver. J Biol Chem 270:8102. Dolmetsch, R. E., K. Xu, and R. S. Lewis. 1998. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392:933. Lewis, R. S. 2001. Calcium signaling mechanisms in t lymphocytes. Annu Rev  Immunol 19:497. 11. 12.  Orrenius, S., B. Zhivotovsky, and P. Nicotera. 2003. Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4:552. van Leeuwen, J. E., and L. E. Samelson. 1999. T cell antigen-receptor signal  transduction. Curr Opin Immunol 11:242. 13.  Rubin, B., L. Alibaud, A. Huchenq-Champagne, J. Arnaud, M. L. Toribio, and J. Constans. 2002. Some hints concerning the shape of T-cell receptor structures.  Scand JImmunol 55:111. 14. 15.  16.  Exley, M . , C. Terhorst, and T. Wileman. 1991. Structure, assembly and intracellular transport of the T cell receptor for antigen. Semin Immunol 3:283. Punt, J. A., J. L. Roberts, K. P. Kearse, and A. Singer. 1994. Stoichiometry of the T cell antigen receptor (TCR) complex: each TCR/CD3 complex contains one TCR alpha, one TCR beta, and two CD3 epsilon chains. J Exp Med 180:587. Thibault, G., and P. Bardos. 1995. Compared TCR and CD3 epsilon expression on alpha beta and gamma delta T cells. Evidence for the association of two TCR heterodimers with three CD3 epsilon chains in the TCR/CD3 complex. J Immunol  154:3814. 17. 18.  Weiss, A., and D. R. Liftman. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263. Appleman, L. J., and V. A. Boussiotis. 2003. T cell anergy and costimulation.  Immunol Rev 192:161.  182  19. 20. 21.  22. 23.  24. 25.  26. 27.  28.  29.  30. 31.  32.  33. 34.  35.  Winslow, M . M . , J. R. Neilson, and G. R. Crabtree. 2003. Calcium signalling in lymphocytes. Curr Opin Immunol 15:299. Hermiston, M . L., Z. Xu, and A. Weiss. 2003. CD45: a critical regulator of signaling thresholds in immune cells. Annu Rev Immunol 21:107. Holdorf, A. D., K. H. Lee, W. R. Burack, P. M . Allen, and A. S. Shaw. 2002. Regulation of Lck activity by CD4 and CD28 in the immunological synapse. Nat Immunol 3:259. Samelson, L. E. 2002. Signal transduction mediated by the T cell antigen receptor: the role of adapter proteins. Annu Rev Immunol 20:371. Di Bartolo, V., D. Mege, V . Germain, M . Pelosi, E. Dufour, F. Michel, G. Magistrelli, A. Isacchi, and O. Acuto. 1999. Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling. J Biol Chem 274:6285. Kane, L. P., and A. Weiss. 2003. The PI-3 kinase/Akt pathway and T cell activation: pleiotropic pathways downstream of PIP3. Immunol Rev 192:7. Bromley, S. K , W. R. Burack, K. G. Johnson, K. Somersalo, T. N . Sims, C. Sumen, M . M . Davis, A. S. Shaw, P. M . Allen, and M . L. Dustin. 2001. The immunological synapse. Annu Rev Immunol 19:375. Lanzavecchia, A., and F. Sallusto. 2000. From synapses to immunological memory: the role of sustained T cell stimulation. Curr Opin Immunol 12:92. Grakoui, A., S. K. Bromley, C. Sumen, M . M . Davis, A. S. Shaw, P. M . Allen, and M . L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221. Krogsgaard, M . , J. B. Huppa, M . A. Purbhoo, and M . M. Davis. 2003. Linking molecular and cellular events in T-cell activation and synapse formation. Semin Immunol 15:307. Davis, D. M . , T. Igakura, F. E . McCann, L. M. Carlin, K. Andersson, B. Vanherberghen, A. Sjostrom, C. R. Bangham, and P. Hoglund. 2003. The protean immune cell synapse: a supramolecular structure with many functions. Semin Immunol 15:317. Kupfer, A., and H. Kupfer. 2003. Imaging immune cell interactions and functions: SMACs and the Immunological Synapse. Semin Immunol 15:295. Sun, Z., C. W. Arendt, W. Ellmeier, E. M . Schaeffer, M . J. Sunshine, L. Gandhi, J. Annes, D. Petrzilka, A. Kupfer, P. L. Schwartzberg, and D. R. Littman. 2000. PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes. Nature 404:402. Freiberg, B. A., H. Kupfer, W. Maslanik, J. Delli, J. Kappler, D. M. Zaller, and A. Kupfer. 2002. Staging and resetting T cell activation in SMACs. Nat Immunol 3:911. Janes, P. W., S. C. Ley, A. I. Magee, and P. S. Kabouridis. 2000. The role of lipid rafts in T cell antigen receptor (TCR) signalling. Semin Immunol 12:23. Neilson, J., K. Stankunas, and G. R. Crabtree. 2001. Monitoring the duration of antigen-receptor occupancy by calcineurin/glycogen-synthase-kinase-3 control of N F - A T nuclear shuttling. Curr Opin Immunol 13:346. Randriamampita, C , and A. Trautmann. 2004. Ca2+ signals and T lymphocytes; "New mechanisms and functions in Ca2+ signalling". Biol Cell 96:69.  183  36.  37. 38.  39.  40.  41. 42. 43. 44.  45.  46.  47.  48.  49.  50.  51.  Venkatachalam, K., D. B. van Rossum, R. L. Patterson, H. T. Ma, and D. L. Gill. 2002. The cellular and molecular basis of store-operated calcium entry. Nat Cell Biol 4:E263. Anderson, K. A., and C. D. Kane. 1998. Ca2+/calmodulin-dependent protein kinase IV and calcium signaling. Biometals 11:331. Feske, S., H. Okamura, P. G. Hogan, and A. Rao. 2003. Ca2+/calcineurin signalling in cells of the immune system. Biochem Biophys Res Commun 311:1117. Bueno, O. F., E. B. Brandt, M . E. Rothenberg, and J. D. Molkentin. 2002. Defective T cell development and function in calcineurin A beta -deficient mice. Proc Natl Acad Sci USA 99:9398. Zhang, B. W., G. Zimmer, J. Chen, D. Ladd, E. Li, F. W. Alt, G. Wiederrecht, J. Cryan, E. A. O'Neill, C. E. Seidman, A. K. Abbas, and J. G. Seidman. 1996. T cell responses in calcineurin A alpha-deficient mice. J Exp Med 183:413. Kiani, A., A. Rao, and J. Aramburu. 2000. Manipulating immune responses with immunosuppressive agents that target NFAT. Immunity 12:359. Crabtree, G. R., and E. N. Olson. 2002. N F A T signaling: choreographing the social lives of cells. Cell 109 Suppl:S67. Rao, A., C. Luo, and P. G. Hogan. 1997. Transcription factors of the N F A T family: regulation and function. Annu Rev Immunol 15:707. Flanagan, W. M . , B. Corthesy, R. J. Bram, and G. R. Crabtree. 1991. Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352:803. Ranger, A. M . , M. R. Hodge, E. M. Gravallese, M . Oukka, L. Davidson, F. W. Alt, F. C. de la Brousse, T. Hoey, M. Grusby, and L. H. Glimcher. 1998. Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc. Immunity 8:125. Ohteki, T., M . Parsons, A. Zakarian, R. G. Jones, L. T. Nguyen, J. R. Woodgett, and P. S. Ohashi. 2000. Negative regulation of T cell proliferation and interleukin 2 production by the serine threonine kinase GSK-3. J Exp Med 192:99. Welsh, G. I., S. Miyamoto, N. T. Price, B. Safer, and C. G. Proud. 1996. T-cell activation leads to rapid stimulation of translation initiation factor eIF2B and inactivation of glycogen synthase kinase-3. J Biol Chem 271:11410. Mattila, P. S., K. S. Ullman, S. Fiering, E. A. Emmel, M. McCutcheon, G. R. Crabtree, and L. A. Herzenberg. 1990. The actions of cyclosporin A and FK506 suggest a novel step in the activation of T lymphocytes. Embo J 9:4425. Frantz, B., E. C. Nordby, G. Bren, N. Steffan, C. V. Paya, R. L. Kincaid, M. J. Tocci, S. J. O'Keefe, and E. A. O'Neill. 1994. Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B. Embo J 13:861. Kanno, T., and U. Siebenlist. 1996. Activation of nuclear factor-kappaB via T cell receptor requires a Raf kinase and Ca2+ influx. Functional synergy between Raf and calcineurin. J Immunol 157:5277. Pan, F., Z. Ye, L. Cheng, and J. O. Liu. 2004. Myocyte enhancer factor 2 mediates calcium-dependent transcription of the interleukin-2 gene in T lymphocytes: a calcium signaling module that is distinct from but collaborates with the nuclear factor of activated T cells (NFAT). J Biol Chem 279:14477.  184  52. 53. 54. 55. 56.  57.  58.  59.  60. 61.  62.  63.  64. 65.  66.  Blaeser, F., N. Ho, R. Prywes, and T. A. Chatila. 2000. Ca(2+)-dependent gene expression mediated by MEF2 transcription factors. J Biol Chem 275:197. Patel, S., S. K. Joseph, and A. P. Thomas. 1999. Molecular properties of inositol 1,4,5-trisphosphate receptors. Cell Calcium 25:247. Taylor, C. W., and A. J. Laude. 2002. IP3 receptors and their regulation by calmodulin and cytosolic Ca2+. Cell Calcium 32:321. Marks, A. R. 1997. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol 272:H597. Harnick, D. J., T. Jayaraman, Y. Ma, P. Mulieri, L. O. Go, and A. R. Marks. 1995. The human type 1 inositol 1,4,5-trisphosphate receptor from T lymphocytes. Structure, localization, and tyrosine phosphorylation. J Biol Chem 270:2833. Sugiyama, T., M . Yamamoto-Hino, A. Miyawaki, T. Furuichi, K. Mikoshiba, and M. Hasegawa. 1994. Subtypes of inositol 1,4,5-trisphosphate receptor in human hematopoietic cell lines: dynamic aspects of their cell-type specific expression. FEBS Lett 349:191. Sugiyama, T., A. Furuya, T. Monkawa, M. Yamamoto-Hino, S. Satoh, K. Ohmori, A. Miyawaki, N. Hanai, K. Mikoshiba, and M . Hasegawa. 1994. Monoclonal antibodies distinctively recognizing the subtypes of inositol 1,4,5trisphosphate receptor: application to the studies on inflammatory cells. FEBS Lett 354:149. Yamamoto-Hino, M . , T. Sugiyama, K. Hikichi, M. G. Mattei, K. Hasegawa, S. Sekine, K. Sakurada, A. Miyawaki, T. Furuichi, M . Hasegawa, and et al. 1994. Cloning and characterization of human type 2 and type 3 inositol 1,4,5trisphosphate receptors. Receptors Channels 2:9. Berridge, M . J. 1990. Calcium oscillations. J Biol Chem 265:9583. Jayaraman, T., K. Ondrias, E. Ondriasova, and A. R. Marks. 1996. Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science 272:1492. Maeda, N., T. Kawasaki, S. Nakade, N. Yokota, T. Taguchi, M. Kasai, and K. Mikoshiba. 1991. Structural and functional characterization of inositol 1,4,5trisphosphate receptor channel from mouse cerebellum. J Biol Chem 266:1109. Jayaraman, T., E. Ondriasova, K. Ondrias, D. J. Harnick, and A. R. Marks. 1995. The inositol 1,4,5-trisphosphate receptor is essential for T-cell receptor signaling. Proc Natl Acad Sci USA 92:6007. Jayaraman, T., and A. R. Marks. 1997. T cells deficient in inositol 1,4,5trisphosphate receptor are resistant to apoptosis. Mol Cell Biol 17:3005. Hirota, J., M . Baba, M . Matsumoto, T. Furuichi, K. Takatsu, and K. Mikoshiba. 1998. T-cell-receptor signalling in inositol 1,4,5-trisphosphate receptor (IP3R) type-1-deficient mice: is 1P3R type 1 essential for T-cell-receptor signalling? Biochem J 333 (Pt 3):615. Sugawara, H., M . Kurosaki, M . Takata, and T. Kurosaki. 1997. Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. Embo J 16:3078.  185  67.  68.  69. 70. 71. 72.  Guse, A. H., C. P. da Silva, F. Emmrich, G. A. Ashamu, B. V. Potter, and G. W. Mayr. 1995. Characterization of cyclic adenosine diphosphate-ribose-induced Ca2+ release in T lymphocyte cell lines. J Immunol 155:3353. Waldron, R. T., A. D. Short, and D. L. Gill. 1995. Thapsigargin-resistant intracellular calcium pumps. Role in calcium pool function and growth of thapsigargin-resistant cells. J Biol Chem 270:11955. MacKrill, J. J. 1999. Protein-protein interactions in intracellular Ca2+-release channel function. Biochem J 337 (Pt 3):345. Rossi, D., and V. Sorrentino. 2002. Molecular genetics of ryanodine receptors Ca2+-release channels. Cell Calcium 32:307. Berridge, M . J. 1993. Inositol trisphosphate and calcium signalling. Nature  361:315.  Hakamata, Y., S. Nishimura, J. Nakai, Y. Nakashima, T. Kita, and K. Imoto. 1994. Involvement of the brain type of ryanodine receptor in T-cell proliferation.  FEBS Lett 352:206. 73.  74.  Schwarzmann, N., S. Kunerth, K. Weber, G. W. Mayr, and A. H. Guse. 2002. Knock-down of the type 3 ryanodine receptor impairs sustained Ca2+ signaling via the T cell receptor/CD3 complex. J Biol Chem 277:50636. Takeshima, H., T. Ikemoto, M . Nishi, N. Nishiyama, M. Shimuta, Y. Sugitani, J. Kuno, I. Saito, H. Saito, M . Endo, M . lino, and T. Noda. 1996. Generation and characterization of mutant mice lacking ryanodine receptor type 3. J Biol Chem  271:19649. 75. 76.  11. 78. 19. 80.  Galione, A., and G. C. Churchill. 2002. Interactions between calcium release pathways: multiple messengers and multiple stores. Cell Calcium 32:343. Guse, A. H., C. P. da Silva, I. Berg, A. L. Skapenko, K. Weber, P. Heyer, M . Hohenegger, G. A. Ashamu, H. Schulze-Koops, B. V. Potter, and G. W. Mayr. 1999. Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose. Nature 398:70. De Flora, A., L. Guida, L. Franco, and E. Zocchi. 1997. The CD38/cyclic ADPribose system: a topological paradox. Int J Biochem Cell Biol 29:1149. Guse, A. H. 1998. Ca2+ signaling in T-lymphocytes. Crit Rev Immunol 18:419. Prakriya, M . , and R. S. Lewis. 2003. C R A C channels: activation, permeation, and the search for a molecular identity. Cell Calcium 33:311. Randriamampita, C , and R. Y. Tsien. 1993. Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature  364:809.  81.  82.  83.  Thomas, D., H. Y. Kim, and M . R. Hanley. 1996. Regulation of inositol trisphosphate-induced membrane currents in Xenopus oocytes by a Jurkat cell calcium influx factor. Biochem J 318 (Pt 2):649. Trepakova, E. S., P. Csutora, D. L. Hunton, R. B. Marchase, R. A. Cohen, and V. M . Bolotina. 2000. Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells. J Biol Chem 275:26158. Itagaki, K., and C. J. Hauser. 2003. Sphingosine 1-phosphate, a diffusible calcium influx factor mediating store-operated calcium entry. J Biol Chem 278:27540.  186  84.  85. 86.  87.  88. 89.  90.  91. 92.  93.  94. 95.  96. 91. 98. 99. 100. 101.  An, S., T. Bleu, and Y. Zheng. 1999. Transduction of intracellular calcium signals through G protein-mediated activation of phospholipase C by recombinant sphingosine 1-phosphate receptors. Mol Pharmacol 55:787. Patterson, R. L., D. B. van Rossum, and D. L. Gill. 1999. Store-operated Ca2+ entry: evidence for a secretion-like coupling model. Cell 98:487. Boulay, G., D. M . Brown, N. Qin, M . Jiang, A. Dietrich, M . X. Zhu, Z. Chen, M . Birnbaumer, K. Mikoshiba, and L. Birnbaumer. 1999. Modulation of Ca(2+) entry by polypeptides of the inositol 1,4, 5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca(2+) entry. Proc Natl Acad Sci USA 96:14955. Kiselyov, K., D. M . Shin, N. Shcheynikov, T. Kurosaki, and S. Muallem. 2001. Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells. Biochem J 360:17. Fasolato, C , M . Hoth, and R. Penner. 1993. A GTP-dependent step in the activation mechanism of capacitative calcium influx. J Biol Chem 268:20737. Somasundaram, B., J. C. Norman, and M . P. Mahaut-Smith. 1995. Primaquine, an inhibitor of vesicular transport, blocks the calcium-release-activated current in rat megakaryocytes. Biochem J 309 (Pt 3):725. Yao, Y., A. V. Ferrer-Montiel, M. Montal, and R. Y . Tsien. 1999. Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell 98:475. Putney, J. W., Jr. 1999. "Kissin' cousins": intimate plasma membrane-ER interactions underlie capacitative calcium entry. Cell 99:5. Khan, A. A., J. P. Steiner, M . G. Klein, M . F. Schneider, and S. H. Snyder. 1992. IP3 receptor: localization to plasma membrane of T cells and cocapping with the T cell receptor. Science 257:815. Tanimura, A., Y. Tojyo, and R. J. Turner. 2000. Evidence that type I, II, and III inositol 1,4,5-trisphosphate receptors can occur as integral plasma membrane proteins. J Biol Chem 275:27488. Montell, C , K. Jones, E. Hafen, and G. Rubin. 1985. Rescue of the Drosophila phototransduction mutation trp by germline transformation. Science 230:1040. Montell, C , and G. M . Rubin. 1989. Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron 2:1313. Xu, X. Z., H. S. Li, W. B. Guggino, and C. Montell. 1997. Coassembly of TRP and TRPL produces a distinct store-operated conductance. Cell 89:1155. Nilius, B. 2003. From TRPs to SOCs, CCEs, and CRACs: consensus and controversies. Cell Calcium 33:293. Chyb, S., P. Raghu, and R. C. Hardie. 1999. Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Nature 397:255. Montell, C , L. Birnbaumer, and V. Flockerzi. 2002. The TRP channels, a remarkably functional family. Cell 108:595. Roderick, H. L., and M. D. Bootman. 2003. Calcium influx: is Homer the missing link? Curr Biol 13.R976. Strubing, C , G. Krapivinsky, L. Krapivinsky, and D. E . Clapham. 2001. TRPC1 and TRPC5 form a novel cation channel in mammalian brain. Neuron 29:645.  187  102.  103. 104.  105. 106.  107. 108.  109.  110.  111. 112. 113. 114. 115.  116. 117. 118.  Hofmann, T., M . Schaefer, G. Schultz, and T. Gudermann. 2002. Subunit composition of mammalian transient receptor potential channels in living cells. Proc Natl Acad Sci USA 99:7461. Yue, L., J. B. Peng, M . A. Hediger, and D. E. Clapham. 2001. CaTl manifests the pore properties of the calcium-release-activated calcium channel. Nature 410:705. Cahalan, M . D., H. Wulff, and K. G. Chandy. 2001. Molecular properties and physiological roles of ion channels in the immune system. J Clin Immunol 21:235. Cui, J., J. S. Bian, A. Kagan, and T. V. McDonald. 2002. CaTl contributes to the stores-operated calcium current in Jurkat T-lymphocytes. J Biol Chem 277:47175. Voets, T., J. Prenen, A. Fleig, R. Vennekens, H. Watanabe, J. G. Hoenderop, R. J. Bindels, G. Droogmans, R. Penner, and B. Nilius. 2001. CaTl and the calcium release-activated calcium channel manifest distinct pore properties. J Biol Chem 276:47767. Kozak, J. A., H. H. Kerschbaum, and M. D. Cahalan. 2002. Distinct properties of C R A C and MIC channels in R B L cells. J Gen Physiol 120:221. Sano, Y . , K. Inamura, A. Miyake, S. Mochizuki, H. Yokoi, H. Matsushime, and K. Furuichi. 2001. Immunocyte ca2+ influx system mediated by ltrpc2. Science 293:1327. Hofmann, T., A. G. Obukhov, M. Schaefer, C. Harteneck, T. Gudermann, and G. Schultz. 1999. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397:259. Gamberucci, A., E. Giurisato, P. Pizzo, M. Tassi, R. Giunti, D. P. Mcintosh, and A. Benedetti. 2002. Diacylglycerol activates the influx of extracellular cations in T-lymphocytes independently of intracellular calcium-store depletion and possibly involving endogenous TRP6 gene products. Biochem J 364:245. Hofmann, F., L. Lacinova, and N. Klugbauer. 1999. Voltage-dependent calcium channels: from structure to function. Rev Physiol Biochem Pharmacol 139:33. Catterall, W. A. 2000. Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16:521. Doering, C. J., and G. W. Zamponi. 2003. Molecular pharmacology of high voltage-activated calcium channels. J Bioenerg Biomembr 35:491. Jurkat-Rott, K , and F. Lehmann-Horn. 2004. The impact of splice isoforms on voltage-gated calcium channel alphal subunits. J Physiol 554:609. Ertel, E. A., K. P. Campbell, M . M . Harpold, F. Hofmann, Y. Mori, E. PerezReyes, A. Schwartz, T. P. Snutch, T. Tanabe, L. Birnbaumer, R. W. Tsien, and W. A. Catterall. 2000. Nomenclature of voltage-gated calcium channels. Neuron 25:533. Chin, H. 1998. Molecular biology of neuronal voltage-gated calcium channels. Exp Mol Med 30:123. Densmore, J. J., G. Szabo, and L. S. Gray. 1992. A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes. FEBS Lett 312:161. Densmore, J. J., D. M . Haverstick, G. Szabo, and L. S. Gray. 1996. A voltageoperable current is involved in Ca2+ entry in human lymphocytes whereas ICRAC has no apparent role. Am J Physiol 27EC1494.  188  119.  120.  121.  122.  123. 124. 125.  126.  127.  128.  129. 130.  131. 132. 133. 134.  Brereton, H. M . , M . L. Harland, M . Froscio, T. Petronijevic, and G. J. Barritt. 1997. Novel variants of voltage-operated calcium channel alpha 1-subunit transcripts in a rat liver-derived cell line: deletion in the IVS4 voltage sensing region. Cell Calcium 22:39. Savignac, M . , A. Badou, M . Moreau, C. Leclerc, J. C. Guery, P. Paulet, P. Druet, J. Ragab-Thomas, and L. Pelletier. 2001. Protein kinase C-mediated calcium entry dependent upon dihydropyridine sensitive channels: a T cell receptor-coupled signaling pathway involved in IL-4 synthesis. Faseb J 15:1577. Kitagawa, ML, S. Takasawa, N. Kikuchi, T. Itoh, H. Teraoka, H. Yamamoto, and H. Okamoto. 1991. rig encodes ribosomal protein SI 5. The primary structure of mammalian ribosomal protein SI 5. FEBSLett 283:210. Moise, A. R., J. R. Grant, R. Lippe, R. Gabathuler, and W. A. Jefferies. 2004. The adenovirus E3-6.7K. protein adopts diverse membrane topologies following posttranslational translocation. J Virol 78:454. Brummelkamp, T. R., R. Bernards, and R. Agami. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550. Carlow, D. A., S. Y. Corbel, and H. J. Ziltener. 2001. Absence of CD43 fails to alter T cell development and responsiveness. J Immunol 166:256. Jinnah, H. A., J. P. Sepkuty, T. Ho, S. Yitta, T. Drew, J. D. Rothstein, and E. J. Hess. 2000. Calcium channel agonists and dystonia in the mouse. Mov Disord 15:542. Negulescu, P. A., N. Shastri, and M . D. Cahalan. 1994. intracellular calcium dependence of gene expression in single T lymphocytes. Proc Natl Acad Sci U S A 91:2873. Lepple-Wienhues, A., and M . D. Cahalan. 1996. Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells. Biophys J 71:787. Kerschbaum, H. H., and M . D. Cahalan. 1998. Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes. J Gen Physiol 111:521. Kerschbaum, H. H., and M . D. Cahalan. 1999. Single-channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes. Science 283:836. Fomina, A. F., C. M . Fanger, J. A. Kozak, and M . D. Cahalan. 2000. Single channel properties and regulated expression of Ca(2+) release- activated Ca(2+) (CRAC) channels in human T cells. J Cell Biol 150:1435. Parekh, A. B., and R. Penner. 1997. Store depletion and calcium influx. Physiol Rev 77:901. Grafton, G., and L. Thwaite. 2001. Calcium channels in lymphocytes. Immunology 104:119. Hoth, M . , and R. Penner. 1993. Calcium release-activated calcium current in rat mast cells. J Physiol 465:359. Rogge, L., E. Bianchi, M . Biffi, E. Bono, S. Y . Chang, H. Alexander, C. Santini, G. Ferrari, L. Sinigaglia, M . Seiler, M. Neeb, J. Mous, F. Sinigaglia, and U. Certa. 2000. Transcript imaging of the development of human T helper cells using oligonucleotide arrays. Nat Genet 25:96.  189  135.  136.  137.  138.  139.  140.  141. 142.  143.  144.  145.  146.  147.  148.  Grafton, G., L. Stokes, K. M. Toellner, and J. Gordon. 2003. A non-voltage-gated calcium channel with L-type characteristics activated by B cell receptor ligation. Biochem Pharmacol 66:2001. Fisher, S. E., A. Ciccodicola, K. Tanaka, A. Curci, S. Desicato, M . D'Urso, and I. W. Craig. 1997. Sequence-based exon prediction around the synaptophysin locus reveals a gene-rich area containing novel genes in human proximal Xp. Genomics 45:340. Strom, T. M . , G. Nyakatura, E. Apfelstedt-Sylla, H. Hellebrand, B. Lorenz, B. H. Weber, K. Wutz, N. Gutwillinger, K. Ruther, B. Drescher, C. Sauer, E. Zrenner, T. Meitinger, A. Rosenthal, and A. Meindl. 1998. An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness. Nat Genet 19:260. Boycott, K. M . , W. G. Pearce, and N. T. Bech-Hansen. 2000. Clinical variability among patients with incomplete X-linked congenital stationary night blindness and a founder mutation in CACNA1F. Can J Ophthalmol 35:204. Nakamura, M . , S. Ito, H. Terasaki, and Y . Miyake. 2001. Novel C A C N A 1 F mutations in Japanese patients with incomplete congenital stationary night blindness. Invest Ophthalmol Vis Sci 42:1610. Kotturi, M . F., D. A. Carlow, J. C. Lee, H. J. Ziltener, and W. A. Jefferies. 2003. Identification and functional characterization of voltage-dependent calcium channels in T lymphocytes. J Biol Chem 278:46949. Morgans, C. W., P. Gaughwin, and R. Maleszka. 2001. Expression of the alphalF calcium channel subunit by photoreceptors in the rat retina. Mol Vis 7:202. Naylor, M . J., D. E. Rancourt, and N. T. Bech-Hansen. 2000. Isolation and characterization of a calcium channel gene, Cacnal f, the murine orthologue of the gene for incomplete X-linked congenital stationary night blindness. Genomics 66:324. McRory, J. E., J. Hamid, C. J. Doering, E. Garcia, R. Parker, K. Hamming, L. Chen, M . Hildebrand, A. M . Beedle, L. Feldcamp, G. W. Zamponi, and T. P. Snutch. 2004. The C A C N A 1 F gene encodes an L-type calcium channel with unique biophysical properties and tissue distribution. J Neurosci 24:1707. Gerster, U., B. Neuhuber, K. Groschner, J. Striessnig, and B. E. Flucher. 1999. Current modulation and membrane targeting ofthe calcium channel alphalC subunit are independent functions of the beta subunit. J Physiol 517 (Pt 2):353. Donnadieu, E., D. Cefai, Y. P. Tan, G. Paresys, G. Bismuth, and A. Trautmann. 1992. Imaging early steps of human T cell activation by antigen-presenting cells. J Immunol 148:2643. Gray, L. S., J. R. Gnarra, J. A. Sullivan, G. L. Mandell, and V. H. Engelhard. 1988. Spatial and temporal characteristics of the increase in intracellular Ca2+ induced in cytotoxic T lymphocytes by cellular antigen. J Immunol 141:2424. Fanger, C. M . , A. L. Neben, and M. D. Cahalan. 2000. Differential Ca2+ influx, KCa channel activity, and Ca2+ clearance distinguish Thl and Th2 lymphocytes. J Immunol 164:1153. Healy, J. I., and C. C. Goodnow. 1998. Positive versus negative signaling by lymphocyte antigen receptors. Annu Rev Immunol 16:645.  190  149.  150.  151. 152.  153. 154. 155. 156.  157.  158.  159.  160.  161.  162.  163.  164.  Karttunen, J., and N. Shastri. 1991. Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene. Proc Natl Acad Sci USA 88:3972. Yang, Y., J. F. Chang, J. R. Parnes, and C. G. Fathman. 1998. T cell receptor (TCR) engagement leads to activation-induced splicing of tumor necrosis factor (TNF) nuclear pre-mRNA. J Exp Med 188:247. Ma, Y., E. Kobrinsky, and A. R. Marks. 1995. Cloning and expression of a novel truncated calcium channel from non- excitable cells. J Biol Chem 270:483. Stokes, L., J. Gordon, and G. Grafton. 2004. Non-voltage-gated L-type Ca2+ channels in human T cells: pharmacology and molecular characterization of the major alpha pore-forming and auxiliary beta-subunits. J Biol Chem 279:19566. Seoh, S. A., D. Sigg, D. M. Papazian, and F. Bezanilla. 1996. Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron 16:1159. Aggarwal, S. K., and R. MacKinnon. 1996. Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron 16:1169. Bezanilla, F. 2002. Voltage sensor movements. J Gen Physiol 120:465. Barry, E. L., F. A. Gesek, S. C. Froehner, and P. A. Friedman. 1995. Multiple calcium channel transcripts in rat osteosarcoma cells: selective activation of alpha ID isoform by parathyroid hormone. Proc Natl Acad Sci USA 92:10914. Hockerman, G. FL, B. Z. Peterson, B. D. Johnson, and W. A. Catterall. 1997. Molecular determinants of drug binding and action on L-type calcium channels. Annu Rev Pharmacol Toxicol 37:361. Schuster, A., L. Lacinova, N . Klugbauer, H. Ito, L. Birnbaumer, and F. Hofmann. 1996. The IVS6 segment of the L-type calcium channel is critical for the action of dihydropyridines and phenylalkylamines. Embo J 15:2365. Soldatov, N . M . , A. Bouron, and H. Reuter. 1995. Different voltage-dependent inhibition by dihydropyridines of human Ca2+ channel splice variants. J Biol Chem 270:10540. Dupuis, G., F. Aoudjit, I. Ricard, and M. D. Payet. 1993. Effects of modulators of cytosolic Ca2+ on phytohemagglutin-dependent Ca2+ response and interleukin-2 production in Jurkat cells. JLeukoc Biol 53:66. Peterson, B. Z., J. S. Lee, J. G. Mulle, Y . Wang, M . de Leon, and D. T. Yue. 2000. Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels. Biophys J 78:1906. Zuhlke, R. D., G. S. Pitt, K. Deisseroth, R. W. Tsien, and H. Reuter. 1999. Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399:159. Wielowieyski, P. A., J. T. Wigle, M . Salih, P. Hum, and B. S. Tuana. 2001. Alternative splicing in intracellular loop connecting domains II and III of the alpha 1 subunit of Cavl .2 Ca2+ channels predicts two-domain polypeptides with unique C-terminal tails. J Biol Chem 276:1398. Malouf, N. N . , D. K. McMahon, C. N. Hainsworth, and B. K. Kay. 1992. A twomotif isoform of the major calcium channel subunit in skeletal muscle. Neuron 8:899.  191  165.  166.  167.  168.  169.  170. 171. 172. 173. 174. 175.  176. 177.  178. 179.  180. 181.  Willmott, N. J., Q. Choudhury, and R. J. Flower. 1996. Functional importance of the dihydropyridine-sensitive, yet voltage-insensitive store-operated Ca2+ influx of U937 cells. FEES Lett 394:159. Ullman, K. S., J. P. Northrop, C. L. Verweij, and G. R. Crabtree. 1990. Transmission of signals from the T lymphocyte antigen receptor to the genes responsible for cell proliferation and immune function: the missing link. Annu Rev Immunol 8:421. Zacharias, D. A., and E. E . Strehler. 1996. Change in plasma membrane Ca2(+)ATPase splice-variant expression in response to a rise in intracellular Ca2+. Curr Biol 6:1642. Liu, H., M. Rhodes, D. L. Wiest, and D. A. Vignali. 2000. On the dynamics of TCR:CD3 complex cell surface expression and downmodulation. Immunity 13:665. Miyamoto, S., J. Qin, and B. Safer. 2001. Detection of early gene expression changes during activation of human primary lymphocytes by in vitro synthesis of proteins from polysome-associated mRNAs. Protein Sci 10:423. Fleckenstein, A. 1983. History of calcium antagonists. Circ Res 52:13. Nayler, W. G., and J. S. Dillon. 1986. Calcium antagonists and their mode of action: an historical overview. Br J Clin Pharmacol 21 Suppl 2:97S. Lewis, G. R., K. D. Morley, B. M . Lewis, and P. J. Bones. 1978. The treatment of hypertension with verapamil. N Z Med J 87:351. Schamroth, L., D. M. Krikler, and C. Garrett. 1972. Immediate effects of intravenous verapamil in cardiac arrhythmias. Br Med J 1:660. Krikler, D. M . , and R. A. Spurrell. 1974. Verapamil in the treatment of paroxysmal supraventricular tachycardia. Postgrad Med J 50:447. Livesley, B., P. F. Catley, R. C. Campbell, and S. Oram. 1973. Double-blind evaluation of verapamil, propranolol, and isosorbide dinitrate against a placebo in the treatment of angina pectoris. Br Med J 1:375. Triggle, D. J. 1990. Calcium antagonists. History and perspective. Stroke 21.PV49. Mitterdorfer, J., M . Grabner, R. L. Kraus, S. Hering, H. Prinz, H. Glossmann, and J. Striessnig. 1998. Molecular basis of drug interaction with L-type Ca2+ channels. J Bioenerg Biomembr 30:319. Messerli, F. H. 2003. Evolution of calcium antagonists: past, present, and future. Clin Cardiol 26:1112. Striessnig, J., B. J. Murphy, and W. A. Catterall. 1991. Dihydropyridine receptor of L-type Ca2+ channels: identification of binding domains for [3H](+)-PN200110 and [3H]azidopine within the alpha 1 subunit. Proc Natl Acad Sci USA 88:10769. Triggle, D. J. 2003. 1,4-Dihydropyridines as calcium channel ligands and privileged structures. Cell Mol Neurobiol 23:293. Nakayama, H., M. Taki, J. Striessnig, H. Glossmann, W. A. Catterall, and Y. Kanaoka. 1991. Identification of 1,4-dihydropyridine binding regions within the alpha 1 subunit of skeletal muscle Ca2+ channels by photoaffinity labeling with diazipine. Proc Natl Acad Sci USA 88:9203.  192  182.  183.  184.  185.  186. 187.  188.  189.  190.  191. 192.  193. 194.  195.  196. 197.  Tang, S., A. Yatani, A. Bahinski, Y. Mori, and A. Schwartz. 1993. Molecular localization of regions in the L-type calcium channel critical for dihydropyridine action. Neuron 11:1013. Peterson, B. Z., T. N . Tanada, and W. A. Catterall. 1996. Molecular determinants of high affinity dihydropyridine binding in L- type calcium channels. J Biol Chem 271:5293. Mitterdorfer, J., Z. Wang, M . J. Sinnegger, S. Hering, J. Striessnig, M. Grabner, and H. Glossmann. 1996. Two amino acid residues in the 1IIS5 segment of L-type calcium channels differentially contribute to 1,4-dihydropyridine sensitivity. J Biol Chem 271:30330. Peterson, B. Z., B. D. Johnson, G. H. Hockerman, M. Acheson, T. Scheuer, and W. A. Catterall. 1997. Analysis of the dihydropyridine receptor site of L-type calcium channels by alanine-scanning mutagenesis. J Biol Chem 272:18752. Birx, D. L., M . Berger, and T. A. Fleisher. 1984. The interference of T cell activation by calcium channel blocking agents. J Immunol 133:2904. Gelfand, E. W., R. K. Cheung, S. Grinstein, and G. B. Mills. 1986. Characterization of the role for calcium influx in mitogen-induced triggering of human T cells. Identification of calcium-dependent and calcium-independent signals. Eur J Immunol 16:907. Marx, M . , M . Weber, F. Merkel, K. H. Meyer zum Buschenfelde, and H. Kohler. 1990. Additive effects of calcium antagonists on cyclosporin A-induced inhibition of T-cell proliferation. Nephrol Dial Transplant 5:1038. Padberg, W. M . , C. Bodewig, H. Schafer, K. H. Muhrer, and K. Schwemmle. 1990. Synergistic immunosuppressive effect of low-dose cyclosporine A and the calcium antagonist nifedipine, mediated by the generation of suppressor cells. Transplant Proc 22:2337. Ricci, A., A. Bisetti, E. Bronzetti, L. Felici, F. Ferrante, F. Veglio, and F. Amenta. 1996. Pharmacological characterisation of Ca2+ channels of the L-type in human peripheral blood lymphocytes. Eur J Pharmacol 301:189. Young, W., J. Chen, F. Jung, and P. Gardner. 1988. Dihydropyridine Bay K 8644 activates T lymphocyte calcium-permeable channels. Mol Pharmacol 34:239. Barger, S. W. 1999. Complex influence of the L-type calcium-channel agonist BayK8644(+/-) on N-methyl-D-aspartate responses and neuronal survival. Neuroscience 89:101. Lullmann, H., P. B. Timmermans, and A. Ziegler. 1979. Accumulation of drugs by resting or beating cardiac tissue. Eur J Pharmacol 60:277. Atherfold, P. A., M . S. Norris, P. J. Robinson, E. W. Gelfand, and R. A. Franklin. 1999. Calcium-induced ERK activation in human T lymphocytes. Mol Immunol 36:543. Dolmetsch, R. E., U. Pajvani, K. Fife, J. M . Sports, and M . E. Greenberg. 2001. Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the M A P kinase pathway. Science 294:333. Lyakh, L., P. Ghosh, and N. R. Rice. 1997. Expression of NFAT-family proteins in normal human T cells. Mol Cell Biol 17:2475. Wang, T., L. 1. Tsai, and A. Schwartz. 1984. Effects of verapamil, diltiazem, nisoldipine and felodipine on sarcoplasmic reticulum. Eur J Pharmacol 100:253.  193  198. 199.  200.  201.  202.  203.  204. 205. 206.  207.  208.  209.  210.  211.  212.  213.  Kaji, D. M . 1990. Nifedipine inhibits calcium-activated K transport in human erythrocytes. Am J Physiol 259.C332. Fagni, L., J. L. Bossu, and J. Bockaert. 1994. Inhibitory effects of dihydropyridines on macroscopic K+ currents and on the large-conductance Ca(2+)-activated K+ channel in cultured cerebellar granule cells. Pflugers Arch 429:176. Zhang, X., J. W. Anderson, and D. Fedida. 1997. Characterization of nifedipine block of the human heart delayed rectifier, hKvl .5. J Pharmacol Exp Ther 281:1247. Franklin, R. A., P. A. Atherfold, and J. A. McCubrey. 2000. Calcium-induced E R K activation in human T lymphocytes occurs via p56(Lck) and CaM-kinase. Mol Immunol 37:675. Fraser, J. D., B. A. Irving, G. R. Crabtree, and A. Weiss. 1991. Regulation of interleukin-2 gene enhancer activity by the T cell accessory molecule CD28. Science 251:313. 2003. 2003 World Health Organization (WHO)/International Society of Hypertension (ISH) statement on management of hypertension. J Hypertens 21:1983. Elliott, W. J. 2003. The economic impact of hypertension. J Clin Hypertens (Greenwich) 5:3. Claussen, D. W. 1995. The most commonly prescribed medications in the United States. Gastroenterol Nurs 18:71. Lentz, A. E., and D. G. Kerns. 1995. Twenty commonly dispensed medications at a United States military installation and their significance to dentists. Mil Med 160:513. Morgano, A., I. Pierri, R. Stagnaro, M. Setti, F. Puppo, and F. Indiveri. 1990. Decreased lymphocyte blastogenesis, IL2 production and NK activity following nifedipine administration to healthy humans. Eur J Clin Pharmacol 39:545. Porchet, H. C , F. Loew, L. Gauthey, and P. Dayer. 1992. Serum concentrationeffect relationship of (+/-)-nicardipine and nifedipine in elderly hypertensive patients. Eur J Clin Pharmacol 43:551. Ochs, H. R., K. D. Ramsch, B. Verburg-Ochs, D. J. Greenblatt, and J. Gerloff. 1984. Nifedipine: kinetics and dynamics after single oral doses. Klin Wochenschr 62:427. Watanabe, K., Y. Ochiai, T. Washizuka, T. Inomata, Y . Miyakita, M . Shiba, T. Izumi, A. Shibata, Y. L. Qu, and T. Nagatomo. 1996. Clinical evaluation of serum amlodipine level in patients with angina pectoris. Gen Pharmacol 27:205. Stopher, D. A., A. P. Beresford, P. V. Macrae, and M . J. Humphrey. 1988. The metabolism and pharmacokinetics of amlodipine in humans and animals. J Cardiovasc Pharmacol 12 Suppl 7:S55. Kagawa, H., S. Nomura, Y. Ozaki, M. Nagahama, and S. Fukuhara. 1999. Effects of nilvadipine on cytokine-levels and soluble factors in collagen disease complicated with essential hypertension. Clin Exp Hypertens 21:1177. Tietz, N. W., and J. Thompson. 1995. Possible concentration-dependent suppression of immune response by verapamil. Arch Fam Med 4:368.  194  214.  215.  216.  217.  218. 219.  220. 221.  222. 223. 224. 225. 226.  Tanchot, C , S. Guillaume, J. Delon, C. Bourgeois, A. Franzke, A. Sarukhan, A. Trautmann, and B. Rocha. 1998. Modifications of CD8+ T cell function during in vivo memory or tolerance induction. Immunity 8:581. Larkin, J. G., G. G. Thompson, G. Scobie, G. Forrest, J. E. Drennan, and M . J. Brodie. 1992. Dihydropyridine calcium antagonists in mice: blood and brain pharmacokinetics and efficacy against pentylenetetrazol seizures. Epilepsia 33:760. Quartermain, D., V . G. deSoria, and A. Kwan. 2001. Calcium channel antagonists enhance retention of passive avoidance and maze learning in mice. Neurobiol Learn Mem 75:77. Donati, D., D. Degiannis, J. Raskova, and K. Raska, Jr. 1992. Uremic serum effects on peripheral blood mononuclear cell and purified T lymphocyte responses. Kidney Int 42:681. Iparraguirre, A., and W. Weninger. 2003. Visualizing T cell migration in vivo. Int Arch Allergy Immunol 132:277. Raska, K., Jr., J. Raskova, S. M . Shea, R. M . Frankel, R. H. Wood, J. Lifter, I. Ghobrial, R. P. Eisinger, and L. Homer. 1983. T cell subsets and cellular immunity in end-stage renal disease. Am J Med 75:734. Kunori, T., I. Fehrman, O. Ringden, and E. Moller. 1980. In vitro characterization of immunological responsiveness of uremic patients. Nephron 26:234. Pahor, M . , J. M . Guralnik, M . E. Salive, M . C. Corti, P. Carbonin, and R. J. Havlik. 1996. Do calcium channel blockers increase the risk of cancer? Am J Hypertens 9:695. Pahor, M . , and C. D. Furberg. 1998. Is the use of some calcium antagonists linked to cancer? Evidence from recent observational studies. Drugs Aging 13:99. Grinstein, S., and S. J. Dixon. 1989. Ion transport, membrane potential, and cytoplasmic pH in lymphocytes: changes during activation. Physiol Rev 69:417. Panyi, G., Z. Varga, and R. Gaspar. 2004. Ion channels and lymphocyte activation. Immunol Lett 92:55. Feske, S., J. Giltnane, R. Dolmetsch, L. M . Staudt, and A. Rao. 2001. Gene regulation mediated by calcium signals in T lymphocytes. Nat Immunol 2:316. Bech-Hansen, N. T., M . J. Naylor, T. A. Maybaum, W. G. Pearce, B. Koop, G. A. Fishman, M . Mets, M . A. Musarella, and K. M . Boycott. 1998. Loss-of-function mutations in a calcium-channel alphal -subunit gene in X p l 1.23 cause incomplete X-linked congenital stationary night blindness. Nat Genet 19:264.  195  APPENDIX A ClustalW cDNA sequence alignment of the human retina (Xi F-subunit (GenBank accession number AF067227) with the voltage negative splice isoform (Variantl) and the voltage positive splice isoform (Variant2) of the 0Ci F-subunit isolated from human spleen. The asterisks represent identically aligned nucleotides, whereas dashes represent spliced exons. Retina ATGTCGGAATCTGAAGGCGGGAAAGGTGAGAGAATCCTTCCATCCCTGCAGACCCTTGGA 60 V a r i a n t l ATGTCGGAATCTGAAGGCGGGAAAGGTGAGAGAATCCTTCCATCCCTGCAGACCCTTGGA 60 V a r i a n t 2 ATGTCGGAATCTGAAGGCGGGAAAGGTGAGAGAATCCTTCCATCCCTGCAGACCCTTGGA 60 ******************************************************** Retina GCAAGCATCGTGGAGTGGAAGCCCTTCGACATCCTCATCCTGCTGACCATCTTTGCCAAC 120 V a r i a n t l GCAAGCATCGTGGAGTGGAAGCCCTTCGACATCCTCATCCTGCTGACCATCTTTGCCAAC 120 V a r i a n t 2 GCAAGCATCGTGGAGTGGAAGCCCTTCGACATCCTCATCCTGCTGACCATCTTTGCCAAC 120 ************************************************************ Retina TGCGTGGCCCTGGGAGTTTACATCCCCTTCCCTGAGGACGACTCCAACACTGCCAACCAC 180 VariantlTGCGTGGCCCTGGGAGTTTACATCCCCTTCCCTGAGGACGACTCCAACACTGCCAACCAC 180 V a r i a n t 2 TGCGTGGCCCTGGGAGTTTACATCCCCTTCCCTGAGGACGACTCCAACACTGCCAACCAC 180 ************************************************************ Re t i n a AACCTGGAGCAGGTGGAGTACGTATTCCTGGTGATTTTCACTGTGGAGACGGTGCTCAAG 2 40 VariantlAACCTGGAGCAGGTGGAGTACGTATTCCTGGTGATTTTCACTGTGGAGACGGTGCTCAAG 2 40 V a r i a n t 2 AACCTGGAGCAGGTGGAGTACGTATTCCTGGTGATTTTCACTGTGGAGACGGTGCTCAAG 2 4 0 **************************************************** ** ****** Re t i n a ATCGTGGCCTACGGGCTGGTGCTCCACCCCAGCGCCTACATCCGCAATGGCTGGAACCTA 3 0 0 V a r i a n t 1 ATCGTGGCCTACGGGCTGGTGCTCCACCCCAGCGCCTACATCCGCAATGGCTGGAACCTA 3 00 V a r i a n t 2 ATCGTGGCCTACGGGCTGGTGCTCCACCCCAGCGCCTACATCCGCAATGGCTGGAACCTA 3 00 ************************************************************ Retina CTCGACTTCATCATCGTCGTGGTCGGGCTGTTCAGCGTTCTGCTGGAGCAGGGCCCCGGA 3 60 VariantlCTCGACTTCATCATCGTCGTGGTCGGGCTGTTCAGCGTTCTGCTGGAGCAGGGCCCCGGA 3 60 V a r i a n t 2 CTCGACTTCATCATCGTCGTGGTCGGGCTGTTCAGCGTTCTGCTGGAGCAGGGCCCCGGA 3 60 ************************************************************ Retina CGGCCAGGCGACGCCCCGCACACCGGGGGAAAGCCAGGAGGCTTCGATGTGAAGGCATTG 420 VariantlCGGCCAGGCGACGCCCCGCACACCGGGGGAAAGCCAGGAGGCTTCGATGTGAAGGCATTG 42 0 V a r i a n t 2 CGGCCAGGCGACGCCCCGCACACCGGGGGAAAGCCAGGAGGCTTCGATGTGAAGGCATTG 420 ************************************************************ Retina AGGGCGTTTCGGGTGCTGCGGCCACTGAGGCTGGTGTCTGGGGTCCCGAGCCTGCACATA 480 V a r i a n t l AGGGCGTTTCGGGTGCTGCGGCCACTGAGGCTGGTGTCTGGGGTCCCGAGCCTGCACATA 4 80 V a r i a n t 2 AGGGCGTTTCGGGTGCTGCGGCCACTGAGGCTGGTGTCTGGGGTCCCGAGCCTGCACATA 480 ************************************************************ Retina GTGCTCAATTCCATCATGAAGGCTCTGGTGCCGCTGCTGCACATTGCACTGCTCGTGCTC 540 V a r i a n t l GTGCTCAATTCCATCATGAAGGCTCTGGTGCCGCTGCTGCACATTGCACTGCTCGTGCTC 54 0 Var i ant 2 GTGCTCAATTCCATCATGAAGGCTCTGGTGCCGCTGCTGCACATTGCACTGCTCGTGCTC 54 0 ************************************************************ Retina TTCGTCATCATCATTTATGCCATCATTGGGCTCGAGCTGTTCCTTGGACGAATGCACAAG 600 V a r i ant1 TTCGTCATCATCATTTATGCCATCATTGGGCTCGAGCTGTTCCTTGGACGAATGCACAAG 600 V a r i a n t 2 TTCGTCATCATCATTTATGCCATCATTGGGCTCGAGCTGTTCCTTGGACGAATGCACAAG 600 ************************************************************  196  Retina ACGTGCTACTTCCTGGGATCCGACATGGAAGCGGAGGAGGACCCATCGCCCTGTGCGTCT 660 Var i ant 1 ACGTGCTACTTCCTGGGATCCGACATGGAAGCGGAGGAGGACCCATCGCCCTGTGCGTCT 660 V a r i a n t 2 ACGTGCTACTTCCTGGGATCCGACATGGAAGCGGAGGAGGACCCATCGCCCTGTGCGTCT 660 ************************************************************ Re t i n a TCGGGATCAGGGCGTGCGTGCACGCTGAACCAGACTGAGTGCCGCGGGCGCTGGCCAGGG 7 20 VariantlTCGGGATCAGGGCGTGCGTGCACGCTGAACCAGACTGAGTGCCGCGGGCGCTGGCCAGGG 7 20 V a r i a n t 2 TCGGGATCAGGGCGTGCGTGCACGCTGAACCAGACTGAGTGCCGCGGGCGCTGGCCAGGG 72 0 ************************************************************ Retina CCCAATGGAGGCATCACCAACTTTGACAACTTCTTCTTCGCCATGCTGACAGTCTTCCAG 7 80 VariantlCCCAATGGAGGCATCACCAACTTTGACAACTTCTTCTTCGCCATGCTGACAGTCTTCCAG 7 80 V a r i a n t 2 CCCAATGGAGGCATCACCAACTTTGACAACTTCTTCTTCGCCATGCTGACAGTCTTCCAG 7 80 ************************************************************ Retina TGTGTCACCATGGAAGGCTGGACCGATGTGCTCTACTGGATGCAAGATGCCATGGGGTAT 840 Variant1TGTGTCACCATGGAAGGCTGGACCGATGTGCTCTACTGGATGCAAGATGCCATGGGGTAT 840 V a r i a n t 2 TGTGTCACCATGGAAGGCTGGACCGATGTGCTCTACTGGATGCAAGATGCCATGGGGTAT 840 ************************************************************ Retina GAACTGCCCTGGGTGTACTTTGTGAGCCTTGTCATCTTTGGGTCCTTCTTCGTCCTCAAC 9 00 VariantlGAACTGCCCTGGGTGTACTTTGTGAGCCTTGTCATCTTTGGGTCCTTCTTCGTCCTCAAC 9 00 V a r i a n t 2 GAACTGCCCTGGGTGTACTTTGTGAGCCTTGTCATCTTTGGGTCCTTCTTCGTCCTCAAC 9 00 ************************************************************ Retina CTTGTGCTTGGCGTCCTGAGTGGGGAGTTCTCCAAGGAGAGAGAGAAAGCGAAAGCTCGC 9 60 VariantlCTTGTGCTTGGCGTCCTGAGTGGGGAGTTCTCCAAGGAGAGAGAGAAAGCGAAAGCTCGC 9 60 V a r i a n t 2 CTTGTGCTTGGCGTCCTGAGTGGGGAGTTCTCCAAGGAGAGAGAGAAAGCGAAAGCTCGC 9 60 ************************************************************ Retina GGGGACTTCCAGAAGCAGCGGGAGAAGCAGCAGATGGAGGAAGACCTGCGGGGCTACCTG 102 0 VariantlGGGGACTTCCAGAAGCAGCGGGAGAAGCAGCAGATGGAGGAAGACCTGCGGGGCTACCTG 1020 Var i ant 2 GGGGACTTCCAGAAGCAGCGGGAGAAGCAGCAGATGGAGGAAGACCTGCGGGGCTACCTG 1020 ************************************************************ Retina GACTGGATCACTCAAGCCGAAGAGCTGGACATGGAGGACCCCTCCGCCGATGACAACCTT 1080 VariantlGACTGGATCACTCAAGCCGAAGAGCTGGACATGGAGGACCCCTCCGCCGATGACAACCTT 1080 V a r i a n t 2 GACTGGATCACTCAAGCCGAAGAGCTGGACATGGAGGACCCCTCCGCCGATGACAACCTT 1080 ************************************************************ Re t i n a GGTTCTATGGCTGAAGAGGGCCGGGCGGGCCATCGGCCACAGCTGGCCGAGCTGACCAAT 114 0 VariantlGGTTCTATGGCTGAAGAGGGCCGGGCGGGCCATCGGCCACAGCTGGCCGAGCTGACCAAT 1140 V a r i a n t 2 GGTTCTATGGCTGAAGAGGGCCGGGCGGGCCATCGGCCACAGCTGGCCGAGCTGACCAAT 114 0 ************************************************************ Retina AGGAGGCGTGGACGTCTGCGCTGGTTCAGTCATTCTACTCGCTCCACACACTCCACCAGC 12 0 0 V a r i a n t l AGGAGGCGTGGACGTCTGCGCTGGTTCAGTCATTCTACTCGCTCCACACACTCCACCAGC 1200 V a r i a n t 2 AGGAGGCGTGGACGTCTGCGCTGGTTCAGTCATTCTACTCGCTCCACACACTCCACCAGC 1200 ************************************************************ Re t i n a AGCCATGCCAGCCTCCCAGCCAGTGACACCGGTTCCATGACAGAGACCCAAGGCGATGAG 1260 V a r i a n t l AGCCATGCCAGCCTCCCAGCCAGTGACACCGGTTCCATGACAGAGACCCAAGGCGATGAG 1260 V a r i a n t 2 AGCCATGCCAGCCTCCCAGCCAGTGACACCGGTTCCATGACAGAGACCCAAGGCGATGAG 1260 ************************************************************ Retina GATGAGGAGGAGGGGGCTCTGGCCAGCTGTACACGCTGCCTAAACAAGATCATGAAAACC V a r i a n t l GATGAGGAGGAGGGGGCTCTGGCCAGCTGTACACGCTGCCTGAACAAGATCATGAAAACC V a r i a n t 2 GATGAGGAGGAGGGGGCTCTGGCCAGCTGTACACGCTGCCTGAACAAGATCATGAAAACC  132 0 1320 132 0  Re t ina AGAGTCTGCCGCCGCCTCCGCCGAGCCAACCGGGTCCTTCGGGCACGCTGCCGTCGGGCA 1380 Var i ant 1 AGAGTCTGCCGCCGCCTCCGCCGAGCCAACCGGGTCCTTCAGGCACGCTGCCGTCGGGCA 1380 Vari ant 2 AGAGTCTGCCGCCGCCTCCGCCGAGCCAACCGGGTCCTTCAGGCACGCTGCCGTCGGGCA 1380 ****************************************  *******************  Retina GTGAAGTCCAATGCCTGCTACTGGGCTGTGCTGTTGCTCGTCTTCCTCAACACGTTGACC 1440 Variant1GTGAAGTCCAATGCCTGCTACTGGGCTGTGCTGTTGCTCGTCTTCCTCAACACGTTGACC 1440 Variant2 GTGAAGTCCAATGCCTGCTACTGGGCTGTGCTGTTGCTCGTCTTCCTCAACACGTTGACC 144 0 ************************************************************ Retina ATCGCCTCTGAGCACCACGGGCAGCCTGTGTGGCTCACCCAGATCCAGGAGTATGCCAAC 1500 V a r i a n t l ATCGCCTCTGAGCACCACGGGCAGCCTGTGTGGCTCACCCAGATCCAGGAGTATGCCAAC 1500 Variant2 ATCGCCTCTGAGCACCACGGGCAGCCTGTGTGGCTCACCCAGATCCAGGAGTATGCCAAC 1500 ************************************************************ Retina AAAGTGTTGCTCTGTCTGTTCACGGTGGAGATGCTTCTCAAATTGTACGGTCTGGGCCCC 1560 V a r i a n t l AAAGTGTTGCTCTGTCTGTTCACGGTGGAGATGCTTCTCAAATTGTACGGTCTGGGCCCC 1560 Variant2 AAAGTGTTGCTCTGTCTGTTCACGGTGGAGATGCTTCTCAAATTGTACGGTCTGGGCCCC 1560 ************************************************************ Retina TCTGCCTATGTGTCTTCCTTCTTCAACCGCTTTGACTGCTTTGTGGTCTGTGGGGGCATC 162 0 VariantlTCTGCCTATGTGTCTTCCCTCTTCAACCGCTTTGACTGCTTTGTGGTCTGTGGGGGCATC 162 0 Variant2 TCTGCCTATGTGTCTTCCCTCTTCAACCGCTTTGACTGCTTTGTGGTCTGTGGGGGCATC 162 0 ****************** ***************************************** Retina CTAGAGACCACCTTGGTGGAGGTGGGCGCCATGCAGCCCTTGGGCATCTCAGTGCTCCGA 1680 VariantlCTAGAGACCACCTTGGTGGAGGTGGGTGCCATGCAGCCCTTGGGCATCTCAGTGCTCCGA 1680 Variant2 CTAGAGACCACCTTGGTGGAGGTGGGTGCCATGCAGCCCTTGGGCATCTCAGTGCTCCGA 1680 ************************** ********************************* Re t ina TGTGTGCGCCTCCTCAGGATCTTTAAGGTCACCAGACACTGGGCTTCTCTGAGCAATCTG 174 0 Variant1TGTGTGCGCCTCCTCAGGATCTTTAAGGTCACCAGACACTGGGCTTCTCTGAGCAATCTG 174 0 Vari ant 2 TGTGTGCGCCTCCTCAGGATCTTTAAGGTCACCAGACACTGGGCTTCTCTGAGCAATCTG 174 0 ************************************************************  Re t ina GTGGCATCCCTGCTCAATTCAATGAAATCCATCGCATCCTTGCTGCTTCTCCTCTTCCTC 180 0 Variant1GTGGCATCCCTGCTCAATTCAATGAAATCCATCGCATCCTTGCTGCTTCTCCTCTACCTC 180 0 Vari ant 2 GTGGCATCCCTGCTCAATTCAATGAAATCCATCGCATCCTTGCTGCTTCTCCTCTACCTC 180 0 ******************************************************* **** Retina TTCATCATTATCTTCTCCCTGCTTGGCATGCAGCTGTTTGGGGGCAAGTTCAACTTTGAC 1860 VariantlTTCATCATTATCTTCTCCCTGCTTGGCATGCAGCTGTTTGGGGGCAAGTTCAACTTTGAC 1860 Variant2 TTCATCATTATCTTCTCCCTGCTTGGCATGCAGCTGTTTGGGGGCAAGTTCAACTTTGAC 1860 ************************************************************  Retina CAGACCCACACCAAGCGAAGCACCTTTGACACGTTCCCCCAGGCCCTCCTCACTGTCTTT V a r i a n t l CAGACCCACACCAAGCGAAGCACCTTTGACACGTTCCCCCAGGCCCTCCTCACTGTCTTT Variant2 CAGACCCACACCAAGCGAAGCACCTTTGACACGTTCCCCCAGGCCCTCCTCACTGTCTTT *********************************  192 0 192 0 192 0  Retina CAGATCCTGACAGGTGAGGACTGGAACGTGGTCATGTATGATGGTATCATGGCATATGGT 1980 Variant1CAGGTCCTGACAGGTGAGGACTGGAACGTGGTCATGTATGATGGTATCATGGCATATGGT 1980 Variant2 CAGGTCCTGACAGGTGAGGACTGGAACGTGGTCATGTATGATGGTATCATGGCATATGGT 1980 *** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Re t ina GGCCCCTTCTTCCCAGGAATGTTGGTGTGCATCTATTTCATCATTCTCTTCATCTGTGGC 2 040 VariantlGGCCCCTTCTTCCCAGGAATGTTGGTGTGCATCTATTTCAACATTCTCTTCATCTGTGGC 2 040 Variant2 GGCCCCTTCTTCCCAGGAATGTTGGTGTGCATCTATTTCAACATTCTCTTCATCTGTGGC 2 040  Retina AACTACATCCTGTTGAACGTGTTTCTTGCCATTGCTGTGGACAACCTGGCCAGTGGAGAT V a r i a n t l AACTACATCCTGTTGAACGTGTTTCTTGCCATTGCTGTGGACAACCTGGCCAGTGGAGAT V a r i a n t 2 AACTACATCCTGTTGAACGTGTTTCTTGCCATTGCTGTGGACAACCTGGCCAGTGGAGAT  210 0 210 0 210 0  * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  GCAGGCACTGCCAAGGACAAGGGCGGGGAGAAGAGCAATGAGAAGGATCTCCCACAGGAG  2160  VariantlGCAGGCACTGCCAAGGGCAAGGGCGGGGAGAAGAGCAATGAGAAGGATCTCCCACAGGAG 2160 V a r i a n t 2 GCAGGCACTGCCAAGGGCAAGGGCGGGGAGAAGAGCAATGAGAAGGATCTCCCACAGGAG 2160 **************** ******************************************* Retina AATGAAGGCCTGGTGCCTGGTGTGGAGAAAGAGGAAGAGGAGGGTGCAAGGAGGGAAGGA V a r i a n t l AATGAAGGCCTGGTGCCTGGTGTGGAGAAAGAGGAAGAGGAGGGTGCAAGGAGGGAAGGA V a r i a n t 2 AATGAAGGCCTGGTGCCTGGTGTGGAGAAAGAGGAAGAGGAGGGTGCAAGGAGGGAAGGA ************************************************************  222 0 222 0 222 0  Retina GCAGACATGGAGGAGGAGGAGGAGGAGGAAGAAGAGGAAGAAGAGGAAGAAGAGGAAGAG VariantlGCAGACATGGAGGAGGAGGAGGAGGAGGAAGAAGAGGAAGAAGAGGAAGAAGAGGAAGAG V a r i a n t 2 GCAGACATGGAGGAGGAGGAGGAGGAGGAAGAAGAGGAAGAAGAGGAAGAAGAGGAAGAG ************************************************************  2280 2280 2280  Re t i n a GGTGCAGGGGGTGTGGAACTCCTGCAGGAAGTTGTACCCAAGGAGAAGGTGGTACCCATC V a r i a n t l GGTGCAGGGGGTGTGGAACTCCTGCAGGAAGTTGTACCCAAGGAGAAGGTGGTACCCATC V a r i a n t 2 GGTGCAGGGGGTGTGGAACTCCTGCAGGAAGTTGTACCCAAGGAGAAGGTGGTACCCATC ************************************************************  2340 2 34 0 2 34 0  Retina CCTGAGGGCAGCGCCTTCTTCTGCCTCAGCCAAACCAACCCGCTGAGGAAGGGCTGCCAC 2 40 0 V a r i a n t l C C T G A G G G C A G C G C C T T C C T C T G C C T C A G C C A A A C C A A C C C G C T G A G G A A G G G C T G C C A C 240 0 V a r i a n t 2 CCTGAGGGCAGCGCCTTCCTCTGCCTCAGCCAAACCAACCCGCTGAGGAAGGGCTGCCAC 2 40 0 ****************** ***************************************** Retina A C C C T C A T C C A C C A T C A T G T C T T C A C C A A T C T T A T C C T G G T G T T C A T C A T C C T C A G C A G T 2 460 V a r i a n t l A C C C T C A T C C A C C A T C A T G T C T T C A C C A A T C T T A T C C T G G T G T T C A T C A T C C T C A G C A G T 2460 V a r i a n t 2 A C C C T C A T C C A C C A T C A T G T C T T C A C C A A T C T T A T C C T G G T G T T C A T C A T C C T C A G C A G T 2460 ************************************************************ Retina  GTGTCCCTGGCCGCTGAGGACCCCATCCGAGCCCACTCCTTCCGCAACCATATTCTGGGT  2 52 0  V a r i a n t l GTGTCCCTGGCCGCTGAAGACCCCATCCGAGCCCACTCCTTCCGCAACCACATTCTGGGT 2 52 0 V a r i a n t 2 GTGTCCCTGGCCGCTGAAGACCCCATCCGAGCCCACTCCTTCCGCAACCACATTCTGGGT 2 52 0 ***************** ******************************** ********* Retina T A C T T C G A T T A T G C C T T C A C C T C C A T T T T C A C T G T G G A G A T T C T A C T A A A G A T G A C A G T G 2 580 V a r i a n t l T A C T T C G A T T A T G C C T T C A C C T C C A T T T T C A C T G T G G A G A T T C T A C T A A A G A T G A C A G T G 2 580 V a r i a n t 2 T A C T T C G A T T A T G C C T T C A C C T C C A T T T T C A C T G T G G A G A T T C T A C T A A A G A T G A C A G T G 2 580 ************************************************************ Re t i n a TTTGGGGCCTTCCTGCACCGCGGCTCCTTCTGCCGTAGCTGGTTTAATATGTTGGATCTG 2 64 0 V a r i a n t l T T T G G G G C C T T C C T G C A C C G C G G C T C C T T C T G C C G T A G C T G G T T T A A T A T G T T G G A T C T G 2 640 V a r i a n t 2 TTTGGGGCCTTCCTGCACCGCGGCTCCTTCTGCCGTAGCTGGTTTAATATGTTGGATCTG 2 64 0 ************************************************************ Retina CTGGTGGTCAGTGTGTCCCTCATCTCCTTTGGCATCCACTCCAGCGCCATCTCGGTGGTG 270 0 V a r i a n t l C T G G T G G T C A G T G T G T C C C T C A T C T C C T T T G G C A T C C A C T C C A G C G C C A T C T C G G T G G T G 270 0 V a r i a n t 2 CTGGTGGTCAGTGTGTCCCTCATCTCCTTTGGCATCCACTCCAGCGCCATCTCGGTGGTG 270 0 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina AAGATTCTGCGAGTACTCCGAGTACTGCGGCCCCTCCGAGCCATCAACAGGGCCAAGGGA V a r i a n t 1 AAGATTCTGCGAGTACTCCGAGTACTGCGGCCCCTCCGAGCCATCAACAGGGCCAAGGGA V a r i a n t 2 AAGATTCTGCGAGTACTCCGAGTACTGCGGCCCCTCCGAGCCATCAACAGGGCCAAGGGA  2 7 60 2 7 60 27 60  Retina CTCAAGCATGTGGTGCAGTGTGTATTTGTGGCCATCCGGACCATCGGAAACATCATGATT 282 0 V a r i a n t l C T C A A G C A C G T G G T G C A G T G T G T A T T T G T G G C C A T C C G G A C C A T C G G A A A C A T C A T G A T T 2 82 0 V a r i a n t 2 CTCAAGCACGTGGTGCAGTGTGTATTTGTGGCCATCCGGACCATCGGAAACATCATGATT 2 82 0 ******** *************************************************** Retina GTCACCACACTTCTGCAATTTATGTTCGCCTGCATCGGGGTGCAGCTCTTCAAGGGGAAA VariantlGTCACCACACTTCTGCAATTTATGTTCGCCTGCATCGGGGTGCAGCTCTTCAAGGGGAAA V a r i a n t 2 GTCACCACACTTCTGCAATTTATGTTCGCCTGCATCGGGGTGCAGCTCTTCAAGGGGAAA ************************************************************  2880 2880 2880  Retina TTCTACACCTGCACGGACGAGGCCAAACACACCCCTCAAGAATGCAAGGGCTCCTTCCTG VariantlTTCTACACCTGCACGGACGAGGCCAAACACACCCCTCAAGAATGCAAGGGCTCCTTCCTG V a r i a n t 2 TTCTACACCTGCACGGACGAGGCCAAACACACCCCTCAAGAATGCAAGGGCTCCTTCCTG ************************************************************  2 94 0 2 94 0 2 94 0  Re t i n a GTATACCCAGATGGAGACGTGTCACGGCCCCTGGTCCGGGAGCGGCTCTGGGTCAACAGT Variant1GTATACCCAGATGGAGACGTGTCACGGCCCCTGGTCCGGGAGCGGCTCTGGGTCAACAGT V a r i a n t 2 GTATACCCAGATGGAGACGTGTCACGGCCCCTGGTCCGGGAGCGGCTCTGGGTCAACAGT ************************************************************  3 00 0 3 00 0 300 0  Re t i n a G A T T T C A A C T T T G A C A A T G T C C T T T C A G C C A T G A T G G C C C T G T T C A C T G T C T C C A C C T T T 3 060 V a r i a n t l G A T T T C A A C T T T G A C A A T G T C C T T T C A G C C A T G A T G G C C C T G T T C A C T G T C T C C A C C T T T 3 060 V a r i a n t 2 G A T T T C A A C T T T G A C A A T G T C C T T T C A G C C A T G A T G G C C C T G T T C A C T G T C T C C A C C T T T 3 060 ************************************************************ Retina GAAGGCTGGCCTGCACTGCTATACAAGGCCATCGATGCATATGCAGAGGACCATGGCCCC 312 0 V a r i a n t l G A A G G C T G G C C A G C A C T G C T A T A C A A G G C C A T C G A T G C A T A T G C A G A G G A C C A C G G C C C C 312 0 V a r i a n t 2 GAAGGCTGGCCAGCACTGCTATACAAGGCCATCGATGCATATGCAGAGGACCACGGCCCC 312 0 *********** ***************************************** ****** Retina A T C T A T A A T T A C C G T G T G G A G A T C T C A G T G T T C T T C A T T G T C T A C A T C A T C A T C A T T G C G 318 0 V a r i a n t l A T C T A T A A T T A C C G T G T G G A G A T C T C A G T G T C C T T C A T T G T C T A C A T C A T C A T C A T T G C G 3180 V a r i a n t 2 A T C T A T A A T T A C C G T G T G G A G A T C T C A G T G T C C T T C A T T G T C T A C A T C A T C A T C A T T G C G 3180 ******************************* **************************** Retina TTCTTCATGATGAACATCTTCGTGGGCTTCGTCATCATCACTTTCCGTGCCCAGGGCGAG V a r i a n t 1 TTCTTCATGATGAACATCTTCGTGGGCTTCGTCATCATCACTTTCCGTGCCCAGGGCGAG V a r i a n t 2 TTCTTCATGATGAACATCTTCGTGGGCTTCGTCATCATCACTTTCCGTGCCCAGGGCGAG ************************************************************  324 0 324 0 3240  Retina CAGGAGTACCAAAACTGTGAGCTGGACAAGAACCAGCGTCAATGTGTGGAATATGCCCTC VariantlCAGGAGTACCAAAACTGTGAGCTGGACAAGAACCAGCGTCAATGTGTGGAATATGCCCTC V a r i a n t 2 CAGGAGTACCAAAACTGTGAGCTGGACAAGAACCAGCGTCAATGTGTGGAATATGCCCTC  33 0 0 33 00 330 0  * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina AAGGCCCAGCCACTCCGCCGTTACATCCCCAAGAACCCGCATCAGTATCGTGTGTGGGCC V a r i a n t l AAGGCCCAGCCACTCCGCCGTTACATCCCCAAGAACCCGCATCAGTATCGTGTGTGGGCC  3360 3360  V a r i a n t 2 AAGGCCCAGCCACTCCGCCGTTACATCCCCAAGAACCCGCATCAGTATCGTGTGTGGGCC ************************************************************  3360  Retina ACTGTGAACTCTGCTGCCTTTGAGTACCTGATGTTCCTGCTCATCCTGCTCAACACAGTT V a r i a n t l ACTGTGAACTCTGCTGCCTTTGAGTACCTGATGTTCCTGCTCATCCTGCTCAACACAGTT V a r i a n t 2 ACTGTGAACTCTGCTGCCTTTGAGTACCTGATGTTCCTGCTCATCCTGCTCAACACAGTT ************************************************************  342 0 342 0 342 0  Retina GCCCTAGCCATGCAGCACTATGAGCAGACTGCTCCCTTCAACTATGCCATGGACATCCTC VariantlGCCCTAGCCATGCAGCACTATGAGCAGACTGCTCCCTTCAACTATGCCATGGACATCCTC V a r i a n t 2 GCCCTAGCCATGCAGCACTATGAGCAGACTGCTCCCTTCAACTATGCCATGGACATCCTC  3480 3480 3480  Retina  AACATGGTCTTCACTGGCCTCTTCACTATTGAGATGGTGCTCAAAATCATCGCCTTCAAG  3 540  Variantl  AACATGGTCTTCACTGGCCTCTTCACTATTGAGATGGTACTCAAAATCATCGCCTTCAAG  3 540  V a r i a n t 2 AACATGGTCTTCACTGGCCTCTTCACTATTGAGATGGTACTCAAAATCATCGCCTTCAAG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  3540  Retina  CCCAAGCATTACTTCACTGATGCCTGGAACACGTTTGACGCTCTTATTGTGGTGGGCAGC  3 60 0  Variantl  CCCAAG  3546  V a r i a n t 2 CCCAAGCATTACTTCACTGATGCCTGGAACACGTTTGACGCTCTTATTGTGGTGGGCAGC * * * * * *  3 60 0  Retina  3 660  ATAGTGGATATTGCCGTCACTGAAGTCAATAATGGTGGCCACCTTGGCGAGAGCTCTGAG  Variantl V a r i a n t 2 ATAGTGGATATTGCCGTCACTGAAGTCAAT  Retina  AGCTCTGAG  3 639  GACAGCTCCCGCATTTCCATTACCTTCTTTCGCCTCTTCCGAGTTATGCGGCTGGTCAAG  372 0  V a r i an12 GACAGCTCCCGCATTTCCATTACCTTCTTTCGCCTCTTCCGAGTTATGCGGCTGGTCAAG  3 699  Retina  CTTCTCAGTAAGGGTGAAGGGATCCGCACATTGCTCTGGACATTCATCAAGTCCTTCCAG  3780  V a r i a n t 2 CTTCTCAGTAAGGGTGAAGGGATCCGCACATTGCTCTGGACATTCATCAAGTCCTTCCAG  37 59  Retina  GCCTTGCCCTATGTGGCTCTTCTCATCGCAATGATATTCTTCATCTATGCCGTCATTGGC  3840  V a r i a n t 2 GCCTTGCCCTATGTGGCTCTTCTCATCGCAATGATATTCTTCATCTATGCCGTCATTGGC  3819  Re t i n a  39 0 0  Variantl  Variantl  Variantl  Variantl  ATGCAGATGTTCGGCAAGGTGGCTCTTCAGGATGGCACACAGATAAACCGAAACAACAAC ATGTTCGGCAAGGTGGCTCTTCAGGATGGCACACAGATAAACCGAAACAACAAC  3 60 0  V a r i a n t 2 ATGCAGATGTTCGGCAAGGTGGCTCTTCAGGATGGCACACAGATAAACCGAAACAACAAC * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  387 9  Retina  TTCCAGACCTTTCCACAGGCTGTGCTGCTTCTGTTCAGGTGTGCCACTGGTGAGGCATGG  3960  VariantlTTCCAGACCTTTCCACAGGCTGTGCTGCTTCTGTTCAGGTGTGCCACTGGTGAGGCATGG  3 660  V a r i a n t 2 TTCCAGACCTTTCCACAGGCTGTGCTGCTTCTGTTCAGGTGTGCCACTGGTGAGGCATGG  3939  * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  CAGGAGATAATGCTTGCCAGCCTTCCCGGAAATCGGTGTGATCCTGAGTCTGACTTCGGC  4 02 0  VariantlCAGGAGATAATGCTTGCCAGCCTTCCCGGAAATCGATGTGATCCTGAGTCTGACTTCGGC  372 0  V a r i a n t 2 CAGGAGATAATGCTTGCCAGCCTTCCCGGAAATCGATGTGATCCTGAGTCTGACTTCGGC * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  3999  Retina  4 080  CCTGGTGAAGAGTTTACCTGTGGTAGCAATTTTGCCATCGCCTATTTCATCAGCTTCTTC  V a r i ant1 CCTGGTGAAGAGTTTACCTGTGGTAGCAATTTTGCCATCGCCTATTTCATCAGCTTCTTC  3780  V a r i a n t 2 CCTGGTGAAGAGTTTACCTGTGGTAGCAATTTTGCCATCGCCTATTTCATCAGCTTCTTC  4 059  * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  ATGCTCTGTGCCTTCCTGATCATAAATCTCTTTGTGGCTGTGATCATGGACAACTTTGAT  Variantl  ATGCTCTGTGCCTTCCTG  414 0 3798  V a r i a n t 2 ATGCTCTGTGCCTTCCTG  4077  ****************** Retina Variantl Variant2  TATCTCACCAGAGATTGGTCCATCCTGGGCCCCCATCACCTTGATGAATTCAAGAGGATC  4200  Re t i n a TGGTCTGAATATGACCCTGGGGCCAAGGGCCGCATCAAACACTTGGATGTGGTTGCCCTG 4260 Variantl GGGCCGCATCAAACACTTGGATGTGGTTGCCCTG 3832 Variant2 GGGCCGCATCAAACACTTGGATGTGGTTGCCCTG 4111  **********************************  Retina CTGAGACGTATCCAGCCCCCTCTGGGATTTGGGAAGCTGTGCCCACACCGAGTGGCCTGC 4 320 VariantlCTGAGACGTATCCAGCCCCCTCTGGGATTTGGGAAGCTGTGCCCACACCGAGTGGCCTGC 3892 V a r i a n t 2 CTGAGACGTATCCAGCCCCCTCTGGGATTTGGGAAGCTGTGCCCACACCGAGTGGCCTGC 4172 ************************************************************ Retina AAGAGACTTGTGGCAATGAACATGCCCCTCAACTCAGATGGGACGGTGACATTCAACGCC 4380 V a r i a n t l AAGAGACTTGTGGCAATGAACATGCCCCTCAACTCAGATGGGACGGTGACATTCAACGCC 3952 V a r i ant 2 AAGAGACTTGTGGCAATGAACATGCCCCTCAACTCAGATGGGACGGTGACATTCAACGCC 4231 ************************************************************ Re t i n a ACACTCTTTGCCCTGGTCCGGACATCCCTGAAGATCAAAACAGAAGGGAACCTGGAGCAA 4440 V a r i ant 1 ACACTCTTTGCCCTGGTCCGGACATCCCTGAAGATCAAAACAGAAGGGAACCTGGAGCAA 4 012 V a r i a n t 2 ACACTCTTTGCCCTGGTCCGGACATCCCTGAAGATCAAAACAGAAGGGAACCTGGAGCAA 4291 ************************************************************ Retina GCCAACCAGGAGCTGCGGATTGTCATCAAAAAGATCTGGAAGCGGATGAAACAGAAGCTG 4500 VariantlGCCAACCAGGAGCTGCGGATTGTCATCAAAAAGATCTGGAAGCGGATGAAACAGAAGCTG 4072 V a r i a n t 2 GCCAACCAGGAGCTGCGGATTGTCATCAAAAAGATCTGGAAGCGGATGAAACAGAAGCTG 4351 ************************************************************ Retina CTAGATGAGGTCATCCCCCCACCAGACGAGGAGGAGGTCACCGTGGGCAAATTCTACGCC 4 560 VariantlCTAGATGAGGTCATCCCCCCACCAGACGAGGAGGAGGTCACCGTGGGCAAATTCTACGCC 4132 V a r i ant 2 CTAGATGAGGTCATCCCCCCACCAGACGAGGAGGAGGTCACCGTGGGCAAATTCTACGCC 4411 ************************************************************ Retina ACATTTCTGATCCAGGACTATTTCCGCAAATTCCGGCGGAGGAAAGAAAAAGGGCTACTA 4 62 0 V a r i a n t l ACATTTCTGATCCAGGACTATTTCCGCAAATTCCGGCGGAGGAAAGAAAAAGGGCTACTA 4192 V a r i a n t 2 ACATTTCTGATCCAGGACTATTTCCGCAAATTCCGGCGGAGGAAAGAAAAAGGGCTACTA 4471 ************************************************************ Retina GGCAACGACGCCGCCCCTAGCACCTCTTCCGCCCTTCAGGCTGGTCTGCGGAGCCTGCAG 4 680 V a r i ant 1 GGCAACGACGCCGCCCCTAGCACCTCTTCCGCCCTTCAGGCTGGTCTGCGGAGCCTGCAG 4252 V a r i a n t 2 GGCAACGACGCCGCCCCTAGCACCTCTTCCGCCCTTCAGGCTGGTCTGCGGAGCCTGCAG 4 531 ************************************************************ Retina GACTTGGGTCCTGAGATGCGGCAGGCCCTCACCTGTGACACAGAGGAGGAGGAAGAAGAG 474 0 VariantlGACTTGGGTCGTGAGATGCGGCAGGCCCCCACCTGTGACACAGAGGAGGAGGAAGAAGAG 4312 V a r i a n t 2 GACTTGGGTCGTGAGATGCGGCAGGCCCCCACCTGTGACACAGAGGAGGAGGAAGAAGAG 4591 ********** ***************** ******************************* Retina GGGCAGGAGGGAGTGGAGGAGGAAGATGAAAAGGACTTGGAAACTAACAAAGCCACGATG 480 0 VariantlGGGCAGGAGGGAGTGGAGGAGGAAGATGAAAAGGACTTGGAAACTAACAAAGCCACGATG 437 2 V a r i a n t 2 GGGCAGGAGGGAGTGGAGGAGGAAGATGAAAAGGACTTGGAAACTAACAAAGCCACGATG 4651 ************************************************************ Retina GTCTCCCAGCCCTCAGCTCGCCGGGGCTCCGGGATTTCTGTGTCTCTGCCTGTCGGGGAC 4860 V a r i a n t l GTCTCCCAGCCCTCAGCTCGCCGGGGCTCCGGGATTTCTGTGTCTCTGCCTGTCGGGGAC 4 432 V a r i a n t 2 GTCTCCCAGCCCTCAGCTCGCCGGGGCTCCGGGATTTCTGTGTCTCTGCCTGTCGGGGAC 4711 ************************************************************ Retina AGACTTCCAGATTCACTCTCCTTTGGGCCCAGTGATGATGACAGGGGGACTCCCACCTCC 492 0 V a r i a n t l AGACTTCCAGATTCACTCTCCTTTGGGCCCAGTGACGATGACAGGGGGACTCCCACCTCC 4 492 V a r i a n t 2 AGACTTCCAGATTCACTCTCCTTTGGGCCCAGTGACGATGACAGGGGGACTCCCACCTCC 4771 *********************************** ************************  202  Retina  AGTCAGCCCAGTGTGCCCCAGGCTGGATCCAACACCCACAGGAGAGGCTCTGGGGCTCTC  4980  Variantl  AGTCAGCCCAGTGTGCCCCAGGCTGGATCCAACACCCACAGGAGAGGCTCTGGGGCTCTC  45 52  Variant2  AGTCAGCCCAGTGTGCCCCAGGCTGGATCCAACACCCACAGGAGAGGCTCTGGGGCTCTC  4831  * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  ATTTTCACCATCCCAGAAGAAGGAAATTCTCAGCCCAAGGGAACCAAAGGGCAAAACAAG  VariantlATTTTCACCATCCCAGAAGAAGGAAATTCTCAGCCCAAGGGAACCAAAGGGCAAAACAAG Variant2  ATTTTCACCATCCCAGAAGAAGGAAATTCTCAGCCCAAGGGAACCAAAGGGCAAAACAAG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  CAAGATGAGGATGAGGAAGTCCCTGATCGGCTTTCCTACCTAGATGAGCAGGCAGGGACT  5040 4 612 4891  5100  VariantlCAAGATGAGGATGAGGAAGTCCCTGATCGGCTTTCCTACCTAGATGAGCAGGCAGGGACT  4672  Variant2  4951  CAAGATGAGGATGAGGAAGTCCCTGATCGGCTTTCCTACCTAGATGAGCAGGCAGGGACT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  CCCCCGTGCTCAGTCCTTTTGCCACCTCACAGAGCTCAGAGATACATGGATGGGCACCTG  5160  VariantlCCCCCGTGCTCAGTCCTTTTGCCACCTCACAGAGCTCAGAGATACATGGATGGGCACCTG  4732  Variant2  5 011  CCCCCGTGCTCAGTCCTTTTGCCACCTCACAGAGCTCAGAGATACATGGATGGGCACCTG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  GTACCACGCCGCCGTCTGCTGCCCCCCACACCTGCAGGTCGGAAGCCCTCCTTCACCATC  522 0  Variantl  GTACCACGCCGCCGTCTGCTGCCCCCCACACCTGCAGGTCGGAAGCCCTCCTTCACCATC  4792  V a r i a n t 2 GTACCACGCCGCCGTCTGCTGCCCCCCACACCTGCAGGTCGGAAGCCCTCCTTCACCATC  5071  * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Retina  CAGTGTCTGCAGCGCCAGGGCAGTTGTGAGGATTTACCCATCCCAGGCACCTATCATCGT  5280  VariantlCAGTGTCTGCAGCGCCAGGGCAGTTGTGAGGATTTACCCATCCCAGGCACCTATCATCGT  4852  Variant2  5131  CAGTGTCTGCAGCGCCAGGGCAGTTGTGAGGATTTACCCATCCCAGGCACCTATCATCGT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * GGGCGAAATTCAGGGCCCAATAGGGCTCAGGGTTCCTGGGCAACACCACCTCAGCGGGGT  534 0  V a r i a n t 1 GGGCGAAATTCAGGGCCCAATAGGGCTCAGGGTTCCTGGGCAACACCACCTCAGCGGGGT  Retina  4912  Variant2  5191  GGGCGAAATTCAGGGCCCAATAGGGCTCAGGGTTCCTGGGCAACACCACCTCAGCGGGGT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  CGGCTCCTGTATGCCCCGCTGTTGTTGGTGGAAGAGGGCGCAGCGGGGGAGGGGTACCTC  540 0  Variant1CGGCTCCTGTATGCCCCGCTGTTGTTGGTGGAAGAGGGCGCGGCGGGGGAGGGGTACCTC  4972  Variant2  52 51  CGGCTCCTGTATGCCCCGCTGTTGTTGGTGGAAGAGGGCGCGGCGGGGGAGGGGTACCTC * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  * * * * * * * * * * * * * * * * * *  GGCAGATCCAGTGGCCCACTGCGCACCTTCACCTGTCTGCACGTGCCTGGAACCCACTCG  5460  V a r i ant 1 GGCAGATCCAGTGGCCCACTGCGCACCTTCACCTGTCTGCACGTGCCTGGAACCCACTCG  5032  Variant2  5311  GGCAGATCCAGTGGCCCACTGCGCACCTTCACCTGTCTGCACGTGCCTGGAACCCACTCG  ************************************ Retina  GACCCCAGCCATGGGAAGAGGGGCAGTGCCGACAGCTTGGTGGAGGCTGTGCTTATCTCA  552 0  VariantlGACCCCAGCCATGGGAAGAGGGGCAGTGCCGGCAGCTTGGTGGAGGCTGTGCTTATCTCA  5092  Variant2  5371  GACCCCAGCCATGGGAAGAGGGGCAGTGCCGGCAGCTTGGTGGAGGCTGTGCTTATCTCA * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  * * * * * * * * * * * * * * * * * * * * * * * * * * * *  GAGGGTCTGGGCCTCTTTGCTCGAGACCCACGTTTCGTGGCCCTGGCCAAGCAGGAGATT  5 580  Variant1GAGGGTCTGGGCCTCTTTGCTCGAGACCCACGTTTCGTGGCCCTGGCCAAGCAGGAGATT  5152  Variant2  5431  GAGGGTCTGGGCCTCTTTGCTCGAGACCCACGTTTCGTGGCCCTGGCCAAGCAGGAGATT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  Retina  GCAGATGCGTGTCGCCTGACGCTGGATGAGATGGACAATGCTGCCAGTGACCTGCTGGCA  5640  Variantl  GCAGATGCGTGTCGCCTGACGCTGGATGAGATGGACAATGCTGCCAGTGACCTGCTGGCA  5212  Variant2  GCAGATGCGTGTCGCCTGACGCTGGATGAGATGGACAATGCTGCCAGTGACCTGCTGGCA  5491  Retina  CAGGGAACCAGCTCTCTCTATAGCGACGAGGAGTCCATCCTCTCCCGCTTCGATGAGGAG  57 00  VariantlCAGGGAACCAGCTCTCTCTATAGCGACGAGGAGTCCATCCTCTCCCGCTTCGATGAGGAG  527 2  Variant2  5 551  CAGGGAACCAGCTCTCTCTATAGCGACGAGGAGTCCATCCTCTCCCGCTTCGATGAGGAG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * GACTTGGGAGACGAGATGGCCTGCGTCCACGCCCTCTGAATTCCCACCCCTCCCCAACTG  57 60  V a r i ant1 GACTTGGGAGACGAGATGGCCTGCGTCCACGCCCTCTGAATTCCCACCCCTCCCCAACTG  Retina  5332  V a r i a n t 2 GACTTGGGAGACGAGATGGCCTGCGTCCACGCCCTCTGAATTCCCACCCCTCCCCAACTG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  5611  CTCAATAAACCTCCTGCCCTCCCCTCCCCAGCAGGAGGCAGGCATGGACCACA  5813  V a r i a n t l CTCAATAAACCTCCCGCCCTCCCCTCCCCAGCAGGAGGCAGGCATGGACCACA  5385  V a r i a n t l CTCAATAAACCTCCCGCCCTCCCCTCCCCAGCAGGAGGCAGGCATGGACCACA  56 64  Retina  APPENDIX B ClustalW amino acid sequence alignment of the human retina a i F-subunit (GenBank accession number AF067227) with the voltage negative splice isoform (Variantl) and the voltage positive splice isoform (Variant2) of the ai F-subunit isolated from human spleen. Bech-Hansen et al. predicted the transmembrane domains in the human retina 0Ci F-subunit (226). The transmembrane sequences are underlined and highlighted in red, and the putative EF-hand C a binding motif in the carboxyl-terminus is underlined and highlighted in blue. The asterisks represent identically aligned amino acids. 2+  isi  Retina  MSESEGGKGERILPSLQTLGASIVEWKPFDILILLTIFAMCVALGVYIPFPEDDSNTANH  VariantlMSESEGGKGERILPSLQTLGASIVEWKPFDILILLTIFAMCVALGVYIPFPEDDSNTANH  60 60  V a r i a n t 2 M S E S E G G K G E R I L P S L Q T L G A S I V E W K P F D I L I L L T I F A N C V A L G V Y I P F P E D D S N T A N H 60 ************************************************************ IS2 IS3 Retina N L E Q V E Y V F L V I F T V E T V L K I V A Y G L V L H P S A Y I R N G W N L L D F I I V W G L F S V L L E Q G P G 12 0 V a r i a n t l N L E Q V E Y V F L V I F T V E T V L K I V A Y G L V L H P S A Y I R N G W N L L D F I I V W G L F S V L L E Q G P G 120 V a r i a n t 2 N L E Q V E Y V F L V I F T V E T V L K I V A Y G L V L H P S A Y I R H G W N L L D F I I V W G L F S V L L E Q G P G 120 ************************************************************  Retina  IS4 RPGDAPHTGGKPGGFDVKALRAFRVLRPLRLVSGVPSLHIVLNSIMKALVPLLHIALLVL  180  VariantlRPGDAPHTGGKPGGFDVKALRAFRVLRPLRLVSGVPSLHIVLNSIMKALVPLLHIALLVL  180  Variant2 RPGDAPHTGGKPGGFDVKALRAFRVLRPLRLVSGVPSLHIVLNSIMKALVPLLHIALLVL  180  ************************************************************  IS5 Retina FVIIIYAIIGLELFLGRMHKTCYFLGSDMEAEEDPSPCASSGSGRACTLNQTECRGRWPG 2 40 VariantlFVIIIYAIIGLELFLGRMHKTCYFLGSDMEAEEDPSPCASSGSGRACTLNQTECRGRWPG 2 40 V a r i a n t 2 FVIIIYAIIGLELFLGRMHKTCYFLGSDMEAEEDPSPCASSGSGRACTLNQTECRGRWPG 2 40 ************************************************************  Retina  IS6 PNGGITNFDNFFFAMLTVFQCVTMEGWTDVLYWMQDAMGYELPWVYFVSLVIFGSFFVLN  VariantlPNGGITNFDNFFFAMLTWQCVTMEGWTDVLYWMQDAMGYELPWVYFVSLVIFGSFFVLH  3 00 300  V a r i a n t 2 P M G G I T N F D N F F F A M L T V F Q C V T M E G W T D V L Y W M Q D A M G Y E L P W V Y F V S L V I F G S F F V L M 3 00 ************************************************************  Retina LVLGVLSGEFSKEREKAKARGDFQKQREKQQMEEDLRGYLDWITQAEELDMEDPSADDNL 3 60 VariantlLVLGVLSGEFSKEREKAKARGDFQKQREKQQMEEDLRGYLDWITQAEELDMEDPSADDNL 3 60 V a r i a n t 2 LVLGVLSGEFSKEREKAKARGDFQKQREKQQMEEDLRGYLDWITQAEELDMEDPSADDNL 3 60 ************************************************************  Retina GSMAEEGRAGHRPQLAELTNRRRGRLRWFSHSTRSTHSTSSHASLPASDTGSMTETQGDE 420 VariantlGSMAEEGRAGHRPQLAELTNRRRGRLRWFSHSTRSTHSTSSHASLPASDTGSMTETQGDE 42 0 V a r i a n t 2 GSMAEEGRAGHRPQLAELTNRRRGRLRWFSHSTRSTHSTSSHASLPASDTGSMTETQGDE 420 ************************************************************  205  IIS1 Retina DEEEGALASCTRCLNKIMKTRVCRRLRRANRVLRARCRRAVKSNACYWAVLLLVFLNTLT 480 VariantlDEEEGALASCTRCLMKIMKTRVCRRLRRANRVLQARCRRAVKSNACYWAVLLLVFLNTLT 480 V a r i a n t 2 DEEEGALASCTRCLNKIMKTRVCRRLRRANRVLQARCRRAVKSNACYWAVLLLVFLNTLT 480 ********************************* ************************** IIS2 Retina  IIS3  IASEHHGQPWLTQIQEYANKVLLCLFTVEMLLKLYGLGPSAWSSFFNRFDCFWCGGI  VariantlIASEHHGQPVWLTQIQEYAMKVLLCLFTVEMLLKLYGLGPSAYVSSLFNRFDCFWCGGI  Variant2  540  IASEHHGQPVWLTQIQEYANKVLLCLFTVEMLLKLYGLGPSAYVSSLFNRFDCFWCGGI  **********************************************  540 540  *************  IIS4 Retina  LETTLVEVGAMQPLGISVLRCVRLLRIFKVTRHWASLSNLVASLLNSMKSIASLLLLLFL  VariantlLETTLVEVGAMQPLGISVLRCVRLLRIFKVTRHWASLSNLVASLLNSMKSIASLLLLLYL  Variant2  600  LETTLVEVGAMQPLGISVLRCVRLLRIFKVTRHWASLSNLVASLLNSMKSIASLLLLLYL  ***************************************  600 600  *  IIS5 Retina  FIIIFSLLGMQLFGGKFNFDQTHTKRSTFDTFPQALLTWQILTGEDWNVVMYDGIMAYG  VariantlFIIIFSLLGMQLFGGKFNFDQTHTKRSTFDTFPQALLTVFQVLTGEDWNVVMYDGIMAYG  Variant2  FIIIFSLLGMQLFGGKFNFDQTHTKRSTFDTFPQALLTVFQVLTGEDWNWMYDGIMAYG  Retina  GPFFPGMLVCIYFIILFICGNYILLNVFLAIAVDNLASGDAGTAKDKGGEKSNEKDLPQE  *****************************************  ******************  660 660  660  IIS6 VariantlGPFFPGMLVCIYFNILFICGNYILLNVFLAIAVDNLASGDAGTAKGKGGEKSNEKDLPQE  Variant2  GPFFPGMLVCIYFNILFICGNYILLNVFLAIAVDNLASGDAGTAKGKGGEKSNEKDLPQE  *************  *******************************  **************  Retina NEGLVPGVEKEEEEGARREGADMEEEEEEEEEEEEEEEEEGAGGVELLQEWPKEKWPI V a r i a n t l NEGLVPGVEKEEEEGARREGADMEEEEEEEEEEEEEEEEEGAGGVELLQEWPKEKWPI V a r i a n t 2 NEGLVPGVEKEEEEGARREGADMEEEEEEEEEEEEEEEEEGAGGVELLQEWPKEKWPI ************************************************************  720 720  720  780 780 780  IIIS1 Retina  PEGSAFFCLSQTNPLRKGCHTLIHHHVFTNLILVFIILSSVSLAAEDPIRAHSFRNHILG  VariantlPEGSAFLCLSQTNPLRKGCHTLIHHHVFTNLILVFIILSSVSLAAEDPIRAHSFRNHILG  Variant2  840  PEGSAFLCLSQTNPLRKGCHTLIHHHVFTNLILVFIILSSVSLAAEDPIRAHSFRNHILG  ******  840 840  ***************************************************** IIIS2  IIIS3  Retina Variantl Variant2  YFDYAFTSIFTVEILLKMTVFGAFLHRGSFCRSWFNMLDLLWSVSLI SFGIH  Retina  KILRVLRVLRPLRAINRAKGLKHWQCVFVAIRTIGNIMIVTTLLQFMFACIGVQLFKGK  YFDYAFTSIFTVEILLKMTVFGAFLHRGSFCRSWFNMLDLLWSVSLISFGIHSSAISW  SSAISW  S SAI ************************************************************  YFDYAFTS IFTVEILLKMTVFGAF LHRGSFCRSWFNMLDLLWSVSLI SFGIH  IIIS4  S W  9 00 9 00 9 00  IIIS5 9 60  VariantlKILRVLRVLRPLRAINRAKGLKHWQCVFVAIRTIGNIMIVTTLLQFMFACIGVQLFKGK  9 60  Variant2  9 60  KILRVLRVLRPLRAINRAKGLKHWQCVFVAIRTIGNIMIVTTLLQFMFACIGVQLFKGK  ************************************************************  Retina FYTCTDEAKHTPQECKGSFLVYPDGDVSRPLVRERLWVNSDFNFDNVLSAMMALFTVSTF VariantlFYTCTDEAKHTPQECKGSFLVYPDGDVSRPLVRERLWVNSDFNFDNVLSAMMALFTVSTF V a r i a n t 2 FYTCTDEAKHTPQECKGSFLVYPDGDVSRPLVRERLWVNSDFNFDNVLSAMMALFTVSTF  102 0 102 0 102 0  IIIS6 Retina EGWPALLYKAIDAYAEDHGPIYNYRVEISVFFIVYIIIIAFFMMNIFVGFVIITFRAQGE 1080 V a r i a n t l EGWPALLYKAIDAYAEDHGPIYNYRVEISVSFIVYIIIIAFFMMNIFVGFVIITFRAQGE 1080 V a r i a n t 2 EGWPALLYKAIDAYAEDHGPIYNYRVEISVSFIVYIIIIAFFMMMIFVGFVIITFRAQGE 108 0  ******************************  *****************************  IVS1 Retina QEYQNCELDKNQRQCVEYALKAQPLRRYIPKNPHQYRVWATVNSAAFEYLMFLLILLNTV 1140 VariantlQEYQNCELDKNQRQCVEYALKAQPLRRYIPKNPHQYRVWATVNSAAFEYLMFLLILIJITV 1140 V a r i a n t 2 QEYQNCELDKNQRQCVEYALKAQPLRRYIPKNPHQYRVWATVNSAAFEYLMFLLILLMTV 1140 ********************************************************* IVS2  IVS3  Retina ALAMQHYEQTAPFNYAMDILNMVFTGLFTIEMVIjKIIAFKPKHYFTDAWNTFDALIWGS 120 0 V a r i a n t l ALAMQHYEQTAPFNYAMDILNMVFTGLFTIEMVLKIIAFKPK 1182 V a r i a n t 2 A I J ^ Q H Y E Q T A P F I S r Y A M D I I J I M V F T G L F T I E M V L K I I A F K P K H Y F T D A W N T F D A L I V V G S 1200 ****************************************** IVS4 Retina IVDIAVTEVNNGGHLGESSEDSSRISITFFRLFRVMRLVKLLSKGEGIRTLLWTFIKSFQ 1260 Variantl V a r i a n t 2 IVDIAVTEVN SSEDSSRISITFFRLFRVMRLVKLLSKGEGIRTLLWTFIKSFQ 1253  IVS5 Retina ALPYVALLIAMIFFIYAVIGMQMFGKVALQDGTQINRNNNFQTFPQAVLLLFRCATGEAW 1320 Variantl MFGKVALQDGTQINRMNNFQTFPQAVLLLFRCATGEAW 122 0 V a r i a n t 2 ALPYVALLIAMIFFIYAVIGMQMFGKVALQDGTQINRNNNFQTFPQAVLLLFRCATGEAW 1313  ************************************  IVS6 Retina QEIMLASLPGNRCDPESDFGPGEEFTCGSNFAIAYFISFFMLCAFLIINLFVAVIMDNFD 138 0 V a r i a n t l QEIMLASLPGNRCDPESDFGPGEEFTCGSNFAIAYFISFFMLCAFL 1266 V a r i a n t 2 QEIMLASL PGNRC DPES DFGPGEEFTCG SNFAI AYF I SFFMLCAFL 13 59 **********************************************  EF-hand m o t i f Retina YLTRDWSILGPHHLDEFKRIWSEYDPGAKGRIKHLDWALLRRIQPPLGFGKLCPHRVAC 1440 Variantl GPHQTLGCGCPAETYPAPSGIWEAVPTPSGL 1297 Variant2 GPHQTLGCGCPAETYPAPSGIWEAVPTPSGL 1390  Retina KRLVAMNMPLNSDGTVTFNATLFALVRTSLKIKTEGNLEQANQELRIVIKKIWKRMKQKL VariantlQETCGNEHAPQLRWDGDIQRHTLCPGPDIPEDQNRREPGASQPGAADCHQKDLEADETEA V a r i a n t 2 QETCGNEHAPQLRWDGDIQRHTLCPGPDIPEDQNRREPGASQPGAADCHQKDLEADETEA  150 0 13 57 14 50  Retina LDEVIPPPDEEEVTVGKFYATFLIQDYFRKFRRRKEKGLLGNDAAPSTSSALQAGLRSLQ 1560 V a r i a n t l AR 1359 V a r i a n t 2 AR 1452  Retina DLGPEMRQALTCDTEEEEEEGQEGVEEEDEKDLETNKATMVSQPSARRGSGISVSLPVGD 162 0 Variantl Variant2  Retina RLPDSLSFGPSDDDRGTPTSSQPSVPQAGSNTHRRGSGALIFTIPEEGNSQPKGTKGQNK 168 0 Variantl  Retina QDEDEEVPDRLSYLDEQAGTPPCSVLLPPHRAQRYMDGHLVPRRRLLPPTPAGRKPSFTI Variantl Variant2  1740  Retina QCLQRQGSCEDLPIPGTYHRGRNSGPNRAQGSWATPPQRGRLLYAPLLLVEEGAAGEGYL Variantl Variant2  180 0  Retina GRSSGPLRTFTCLHVPGTHSDPSHGKRGSADSLVEAVLISEGLGLFARDPRFVALAKQEI 1860 Variantl Variant2  Retina ADACRLTLDEMDNAASDLLAQGTSSLYSDEESILSRFDEEDLGDEMACVHAL Variantl Variant2  1912  208  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

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.831.1-0092362/manifest

Comment

Related Items