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The role of the dynein motor system on the trafficking of cardiac potassium ion channels Khurana, Anu 2006

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THE ROLE OF THE DYNEIN MOTOR S Y S T E M ON THE TRAFFICKING OF CARDIAC POTASSIUM ION C H A N N E L S By A N U K H U R A N A B.Sc, University of British Columbia (2004) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE F A C U L T Y OF G R A D U A T E STUDIES (Physiology) THE UNIVERSITY OF BRITISH C O L U M B I A August 2006 © Anu Khurana, 2006 11 Abstract In this thesis, we establish a role for the retrograde dynein motor in mediating the trafficking of cardiac potassium channels. Using dynamitin overexpression to disrupt the dynein motor complex, we demonstrate that dynein disruption doubles K v 1.5 currents and increases surface K v 1 .5 protein levels in HEK293 cells. Similar results were seen when these cells were treated with the endocytosis inhibitor, dynamin inhibitory peptide. Confocal images of HEK293 cells and rat atrial myocytes reveal a redistribution of K v 1 .5 staining to the plasma membrane when dynein is disrupted, either by dynamitin overexpression or in the presence of the microtubule depolymerizing agent, nocodazole (35 uM). Sustained currents from rat atrial myocytes were increased by 2-fold upon nocodazole pretreatment. These increases in sustained currents were inhibited by the K v l .5-specific blocker D M M (100 nM). We also demonstrate that K v 1 .5 colocalizes with the early endosomal marker, E E A 1 , suggesting that K v 1 .5 enters the early endoso'mal pathway. Taken together, these experiments suggest that K v l .5 surface expression is regulated by the dynein-mediated endocytosis. In addition, pretreatment of nocodazole at 37°C reduced T T 0 currents by one-half. Furthermore, dynamitin overexpression in H E K 2 9 3 cells reduced K v 4 . 2 and K v 4.3 channel currents and reduced Kv4.2 staining at the cell surface in confocal images. Dynein motility is coordinated by the anterograde motor, kinesin, and vice versa (Hamm-Alvarez et al., 1993; Valetti et al., 1999; Deacon et al., 2003). Given this and results from a previous report that suggest that the forward trafficking of K v 4 . 2 is mediated by kinesin, we suggest that dynein disruption impairs the anterograde trafficking of K v 4 .2 and K v 4.3 channels (Chu et al., 2006). Lastly, we found no effect on I K i - l i k e currents in nocodazole pretreated rat atrial myocytes at 37°C. Similarly, dynamitin overexpression and nocodazole did not alter the current density of the iKi-forming channel, Kj r 2.1, in Ilk- cells. This insensitivity of K; r2.1 may suggest an equal dependence for both the dynein and kinesin motor system in regulating the trafficking of Kj r2.1 to the cell surface. On the whole, this thesis suggests variable dependencies of the dynein motor in the trafficking of various cardiac potassium channels, and as such, may have important implications in our understanding of the mechanisms that determine potassium channel trafficking in the heart. Ill Table of Contents Abstract Table of Contents 1 1 1 List of Figures 1 V List of Symbols and Abbreviations v Acknowledgements v m Introduction 1 Methods 2 2 Results 3 0 Discussion D : > Bibliography 69 IV List of Figures Figure 1. p50/dynamitin coexpression significantly increases K v 1 .5 current levels and surface expression 32 - Contributions: Woo-Sung Choi (Fig. 2 A - C ) , Rajesh Mathur (Fig. D) Figure 2. K v l .5 is internalized via an endocytotic pathway 35 Figure 3. Internalization of K v l .5 is mediated by dynamin-dependent endocytosis internalized via an endocytotic pathway 36 - Contributions: Woo-Sung Choi Figure 4. Concentration-dependent depolymerization of p-tubulin with nocodazole in rat atrial myocytes 38 Figure 5. Nocodazole pretreatment increases I K S U S and I K I - but not I t 0 currents in rat atrial myocytes 40 - Contributions: Woo-Sung Choi Figure 6. Nocodazole pretreatment increases K v 1.5 surface expression in rat atrial myocytes 41 Figure 7. Interference with microtubule-based trafficking downregulates A-type currents, but does not alter I K I currents at 37°C 44 Figure 8. Kj r2.1 currents are insensitive to disruption of the dynein motor by p50 overexpression in mouse Itk- cells 46 Figure 9. K; r2.1 currents are insensitive to the microtubule depolymerizing drug, nocodazole, in mouse Itk- cells 47 Figure 10. Dynamitin co-expression reduces K v 4 .2 currents and decrease K v 4 . 2 staining at the cell surface in HEK293 cells 50 - Contributions: Woo-Sung Choi (Fig. 11 A ) Figure 11. Dynamitin co-expression reduces K v 4.3 currents in HEK293 cells 51 V List of Symbols and Abbreviations ~: Approximately a: Alpha AchR: Acetylcholine Receptor A M P A : alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor 4-AP: 4-Aminopyridine AP-2 : Clathrin Adaptor Protein-2 p: Beta C: Celsius, Cysteine, or Carboxyl-terminus Ca' -1": Calcium ion C H O : Chinese Hamster Ovary C1C2: Chloride channel 2 C O : : Carbon dioxide COPII: Coat protein complex type 1 C O S : African green monkey kidney cells °: Degree A : Delta DIP: Dynamin Inhibitory Peptide D M M : 2-(3,4-Dimethylphenyl)-3-(4-methoxyphenethyl) metathiazan-4-one D N A : Deoxyribonucleic acid E: Glutamate E H N A : Erythro-9-[3-(2-hydroxynonyl)] adenine E K : Nernst equilibrium potential for potassium ions ER: Endoplasmic Reticulum F: Phenylalanine Fig.: Figure GPy: G-coupled beta-gamma subunit G I R K : G-protein coupled inward rectifying potassium channel GluR2: Glutamate receptor subunit 2 GR1P1: Glutamate receptor interacting protein 1 H5: hydrophobic segment #5 HeLa: Immortalized cervical cancer cells taken from Henrietta Lacks H E K : Human Embryonic Kidney H E P E S : 2-[4-(2-Hydroxyethyl)-l-piperazinyl] ethanesulfonic acid Hsp40: Heat shock protein 40 Hsp70: Heat shock cognate 71 kDa protein Hz: Hertz I: Current I K : Potassium current I K i : Inward rectifying potassium c u i T e n t - 1 I K A C H : Acetylcholine-activated potassium current I K A T P : ATP-sensitive potassium current ]yir: Rapidly-activating delayed rectifying potassium current I K s : Slowly-activating delayed rectifying potassium current IK S U S : Cardiac sustained potassium current I t 0: Transient outward cardiac potassium current I K u r : Ultra-rapid activating cardiac potassium current K + : Potassium ion K C h i P : Potassium Channel Interacting Protein K„-: Inward rectifying potassium channel K v : Voltage-gated potassium channel L : Litre or Leucine Ilk-; Mouse tumor cell line u: Micro (It)"6) M : Molar M E M : Minimum essential media M g 2 + : Magnesium ion min: Minute mLin lO: Scaffolding Mint 1 protein m R N A : Messenger Ribonucleic Ac id ms: Milliseconds mV:Mil l ivo l t s N : Asparagine or Amino-terminus nA: Nanoampere N a + : Sodium ion Kif5b: Kinesin family member 5b K i f l 7 : Kinesin family member 17 N M D A : N-methyl-d-aspartate N R 2 B : N M D A channel subunit 2B Q: Ohm p: Pico (!()"-) p50: Interchangeable with Dynamitin pA: Picoampere p'F: Picofarad P B S : Phosphate buffer solution P D Z : Postsynaptic density-95/disc large/zona occludens-1 P1P 2: Phosphatidylinositol bisphosphate P-loop: Pore-loop domain QT: QT interval in electrocardiogram R: Arginine R M P : Resting membrane potential R O M K : Renal outer medullary potassium channel S: Serine S(#): Ion channel transmembrane segment number (#) S E M : Standard error of the mean S U R 1 : Sulfonylurea receptor type 1 SUR2: Sulfonylurea receptor type 2 ^ac t i va t ion : Activation time constant T l : Tetramerization T E A : Tetraethylammonium T M : Transmembrane vii V : Voltage, Valine, or numeral number 5 VT: Numeral number 6 X : Non-conserved amino acid Y : Tyrosine Vlll Acknowledgements It is often said, that you can define yourself by the people that you surround yourself with. In my graduate degree, I had the opportunity to collaborate with many unique individuals, all who have helped me mature in my academic career. 1 would like to especially thank Dr. David Fedida, my supervisor. In the +4 years that I have known him, he has given me full support such that I can become a better scientific researcher. Now what does that mean? Well , science I have learned is a competitive business. Y o u have to be tough, give criticism, take criticism, open-minded, hard working, you must have perseverance but know when to stop, and keen to learn new things and take risks. Thank you David, I believe I have developed these attributes or I am at least aware that I must continue to work diligently in these areas throughout my scientific career. Members of my supervisor committee (Dr. Eric A c c i l i and Dr. Hakima Mouhkles) have been supportive throughout the duration of my master's degree and I thank you for your dedication and you patience with me when I ask too many questions. I would also like to thank some of the members of the laboratory who I feel had a significant role in my academic career and have often lightened up my day especially when my experiments didn't turn out like I hoped: Grace Luu, Fif i Chu, Harley Kurata, Vijay Viswanathan, Jodene Eldstrom, Grace L u , Woo-Sung Choi, Saman Rezazadeh, Dave Steele and Andrew Home. I would also like to especially thank Martina Jochova. I know very well that our friendship wi l l continue on after this. Y o u have no idea how much I cherish your friendship. Y o u know me sometimes better then I know myself. Also, I would like to give special thanks to Zhuren Wang. I have known you for a long time and I think of you as a mentor. I appreciate your kindness and sincerity and most of all. your company at our trips to McDonalds! I would like to recognize all of my family who still has absolutely no idea what I do. but it really doesn't matter. M y parents are amazing individuals. I love you and I am grateful that 1 had, and still have your full undivided support throughout these years and for the years to come. To my little brother, Arjun, I love you. Before you were born, I hated the thought of not being the youngest child anymore. But then when you were bom, I knew that you were one of the best things that have ever happened to me. I would like to thank my sister Priti, the twin 1 never had. Although you never understood what 1 actually did in my master's ("yah, in the heart"'), you supported me in whatever I did, as long as I did it well. There were also some friends that have helped me deal with some of the hard times I had to face in my masters. I just want to establish that I am sorry for being moody, tired, and frustrated. More then one of you have said that my mood for the day was determined by how my cells were. Thank you to Scott Widenmaier, Sophia Khan, Rachel Luu, and "ate'' K i m Rivera for being there for me. K i m . I love you like a sister. To Scott and Rachel, thank you for showing me the light of Grace. And to Sophia, thank you for just being there when I didn't even have to ask. 1 Introduction Altering potassium (K'") channel expression is a very complex and sensitive way in which cardiac cells can regulate their excitability. The extremely diverse family of K + channels assists in maintaining the resting membrane potential, regulates cardiac contractility, and plays an important role in regulating cardiac action potential duration. rC channel activity is controlled by a variety of factors that include GPy (Clapham and Neer, 1997; Dascal, 1997; Kurachi and Ishii, 2004), A T P (Ashcroft and Ashcroft, 1990), calcium (Magleby. 2003), and also may be gated by membrane potential. Intrinsic regulators of these channels include phosphorylation, interaction with accessory subunits, and by proteins which are intimately involved in the trafficking of the ion channel to the cell surface. Several cardiac diseases have been linked to poor K + channel protein expression including atrial fibrillation (Van Wagoner et al., 1997; Grammer et al., 2000; Brundel et al., 2001; Van Wagoner, 2003), congenital long QT syndrome, and renovascular hypertension (Takimoto et al.. 1997; Kaab et al., 1998: Wakisaka et al.. 2004). The mechanisms by which K + channel surface expression are altered is not clearly understood, but it is believed that trafficking of FC channels plays a key role in the maintenance of these disease states. The function of IC channels, which govern neuronal and cardiac excitability, is ultimately determined by the relative abundance of functional channels expressed at the cell surface. Mechanisms which affect trafficking of K + channels have recently been emphasized as a method by which cardiac cells may regulate their excitability and contractility and furthermore, can also provide an explanation of the mechanism by which drugs may act to achieve their therapeutic actions (Ficker et al., 2000; Ficker et al., 2004). Previous work by several groups have studied the role of specific domains within K r channels that influence the efficiency of channel transport to cellular membranes. However, little is known about the specific trafficking machinery involved in this process in cardiac myocytes. Cytoplasmic motors like dynein and kinesin are responsible for the movement of organelles and vesicular cargo within the cell. This investigation is focused on understanding the complex mechanisms that contribute to the trafficking of cardiac K + channels, including voltage-gated (K v) and inward-rectilying K + channels (Kjr), and the role by which dynein dynamically regulates the steady-state cell surface expression levels in various mammalian cell lines and in cardiac myocytes. I) Structure and Function off? Channels in the Heart i. Cardiac IC Currents The numerous and diverse I K currents in the heart help shape the cardiac action potential by affecting the length of the plateau and termination phase. In the heart, these IK currents can account for the prolonged plateau phase, which is approximately a hundred-fold longer than action potentials recorded from nerve or skeletal muscle tissues. Substantial progress has been made in understanding the molecular correlates that correspond to various I K currents in the heart. Several distinct I K currents have been identified, such as I t 0, \ K W , kr, IKS> and IKI • These cardiac potassium currents each display unique properties that determine their functional role in the heart, possessing distinct activation and inactivation kinetics, voltage-dependency, rectification properties, pharmacology, and localization (Nerbonne, 2000). However, there is still some controversy surrounding the exact function of specific cloned ion channels in mediating various I C currents in the heart and these discrepancies may be explained by differences in myocyte isolation and culturing procedures, heterotetramerization of K + channel subunits, heteromultimerization of K + channels with accessory subunits, and specific primary antibodies used to detect specific clones of K+ channels. ii) Structure of Voltage-Gated iC Channels Many of these K + currents arise due to the expression and activity of K v channels in the heart. To date, mammalian K v channels, which are gated by changes in membrane potential, contain 12 subfamily members ( K v 1-12; K C N A 1 - K C N H 4 in modern nomenclature (Chandy. 1991)). The first four subfamilies ( K v l - 4 ) encode the pore-forming a-subunits of K v chaimels (Pongs, 1992; Barry and Nerbonne, 1996; Nerbonne, 2000), and were initially identified based on sequence homology and similarity to single gene orthologs in Drosophila; K v l . x (Shaker), K v 2 . x (Shab), K v 3 . x (Shaw), and K v 4 . x (Shal) (Kamb et al., 1987; Papazian et al., 1987; Jan and Jan, 1997). These subfamilies are further divided into distinct sub-family members (as denoted by "x"). K v channels are formed by the assembly of four individual a-subunits, with each a-subunit contributing to the ion-selective pore of the channel. a-Subunits of K v channels include six transmembrane domains which criss-cross the membrane such that the amino and carboxyl- terminal regions of the channel are exposed to the cytoplasmic environment. In addition, the hydrophobic S1-S4 regions form the 'voltage sensor" region (the S4 transmembrane domain forms the primary voltage sensor domain), while the extracellular and intracellular linkers, including the pore-forming loop between S5 and S6, are generally composed of hydrophilic residues (Li et al., 1992; Yellen, 1998). Also, 4 diversity can be achieved by heteromultimerization o f channels o f the same subfamily, often with functional properties intermediate between those of channels formed from homomultimers of the constituent subunits (Isacoff et al., 1990; Ruppersberg et al., 1990; L i et al., 1992; Hopkins et al., 1994). iii) Transient Outward tC Channels In both atrial and ventricular myocytes, considerable advances have been made in identifying the molecular correlates of I t 0 , a calcium-independent transiently-activating and fast-inactivating outward K + current which mediates the early-repolarization phase of the cardiac action potential. In rat and humans (Comer et al., 1994; Fiset et al., 1997; Johns et al., 1997; Barry et al., 1998: Brahmajothi et al., 1999; Guo et al., 1999; Wickenden et al., 1999; X u et al., 1999), I t 0 is attributed to membrane currents associated with voltage-gated K v 4 .2 and K v 4.3 channels. K v 4 . x channels are highly homologous, with strong sequence similarity in the transmembrane regions o f these channels. However, sequence divergence is greater at the amino and carboxy- termini. Expression of these channels in heterologous cell expression systems reveal that these channels activate at subthreshold levels, inactivate rapidly, and recover rapidly from inactivation relative to other fast inactivating K v channels, such as the Shaker-related K v 1 .4 channel (Birnbaum et al., 2004). Transgenic and knockout animals, and antisense oligonucleotide targeting of these channels have revealed a role for K v 4 . 2 and K v 4 .3 channels in mediating I t 0 currents .in various species (Fiset et al., 1997; Johns et al., 1997; Barry et al., 1998; Guo et al.. 2002). Except for rat atrial myocytes, there is a general consensus that I,0 is composed of the heteromultimerization of K v 4 .2 and K v 4.3 primary a-subunits (Birnbaum et al., 2004). 5 In rat atrial cells, antisense knockout studies show that K v 4 .2 is an important player in mediating I t 0 , as antisense oligonucleotide treatment of K v 4 .3 does not reduce I t 0 currents in rat atrial myocytes. However, the S7?a/-related potassium channel, K v 1 .4 , is also capable of forming I t 0 currents in the ventricles of transgenic animals expressing a dominant-negative form of K v 4 .2 (Barry et al., 1998). K v 1 .4 has been speculated to play a protective role in preventing arrhythmias, as there is evidence that K v 1 .4 is upregulated in cardiac disease when other 57?t7/-related l t 0 currents are downregulated (Wickenden et al., 2000; Bodi et a l , 2003). In addition, K v 4 . x channels oligomultimerize with accessory K C h i P subunits in the heart, which are thought to improve efficiency of transport of these channels to their final destination and to modulate their activation and inactivation kinetic properties (An et al., 2000; Beck et al., 2002; Birnbaum et al., 2004). iv) Ultra-rapid Delay ed-Rectifying iC Channel, KJ.S During the atrial action potential, repolarization is facilitated by an outward-rectifying, rapidly-activating K. + current ( r a c t j v i , t i o n < 10 ms) (Snyders et al., 1993; Wang et al., 1993; Nattel et al., 1999; Tamargo et al., 2004), termed I K l i r . In humans, the electrical recording of \Y.W has been found in atria but is absent in ventricular tissue, which suggests a primary role for this current in mediating atrial repolarization in humans (Fedida et al., 1993: Wang et al., 1993; Nattel et al., 1999). Sustained currents in human atrial myocytes were demonstrated to have similar pharmacological and biophysical properties as the rapidly-activating delayed-rectifying potassium channel, K v 1 . 5 , as these currents showed little inactivation, insensitivity to pharmacological agents such as T E A , barium ions, and dendrotoxin, and are strongly blocked by 4 -AP. K v 1 .5 is thought to underlie l K u r currents 6 in human, canine and rat atrium but is not believed to play a role in the ventricles of these species, even though K v l . 5 m R N A and protein have been found in both atrial and ventricle tissue (Fedida et al., 1993; Barry and Nerbonne, 1996; Feng et al., 1997; Van Wagoner et al., 1997; Fedida et al., 2003). It is widely recognized that K v 1 .5 downregulation can contribute to a variety of cardiac dysfunctions such as atrial fibrillation and pulmonary hypertension (Van Wagoner et al., 1997; Brandt et al., 2000; van der Velden et al., 2000; Archer et al., 2001; Brundel et al., 2001; Van Wagoner, 2003; Abdelhadi et al., 2004). v) Inward-Rectifying FC Chan nets Another major class of K + channels expressed in the heart is the inward-rectifier K r channels. The inward-rectifying K ^ current, I K I , plays a significant role in cardiac electrophysiology. I K I is a major contributor to the terminal phase of repolarization and is necessary for maintaining the resting membrane potential (Lopatin and Nichols, 2001; Miake et al., 2003). Near the resting membrane potential, K j r channels that contribute to I K I have a large conductance and as such, regulation of this channel can serve as a potential mechanism by which cardiac muscle cells can regulate their excitability. In heart failure, I K i has been found clinically to be downregulated in electrophysiological remodeling (Tomaselli and Marban, 1999). Like K v channels, they are extremely diverse, but in vast contrast to the classical voltage-gated K + channel family, are not gated by voltage, as they lack the putative voltage sensor S1-S4 region identified in voltage-gated ion channels. At potentials negative to E«, K | r channels display an ohmic conductance-voltage relationship. The rectification properties seen in K ] r channels, at membrane 7 potentials positive to E K , is the result of block by divalent cations, such as M g + , or caused by the block of outward K"'" current by intracellular polyamines upon cell depolarization. Ki,- channels form tetramers (Jan and Jan, 1997), with each channel subunit consisting of two transmembrane domains, M l and M2 and a putative ion selective pore region, the H5 loop. These domains correspond to the Shaker S5-P-loop-S6 segments of voltage-gated K + channels. Similar to K v channels, Kj r channels generally contain a large cytosolic domain, composed of roughly 50 amino acids at the amino-terminus and approximately 200 amino acids in the carboxy-terminal region. K; r channels contain seven subfamily members (Kj rl .x- Kj,-7.x: Where x denotes members of subfamilies (Nichols and Lopatin, 1997; Nishida and MacKinnon, 2002)) and are distinguished by their strength of rectification and their responses to intracellular factors, including P I P ? . Of these, Kj r l .x (ROMK) channel activity is regulated by YJ concentration and pH and controls K + secretion in the kidney, K; r2.x (IRK) channels encode a constitutively active strongly inward-rectifying (IKI) current in the skeletal and cardiac muscle tissue, the strongly rectifying Kjr3.x (GIRK) channels form the G-protein coupled K M * currents which are directly activated by muscarinic acetylcholine receptors, and Kj r6.x subunits encode K A T P channels (Nichols and Lopatin. 1997). To date, the precise molecular composition of I K I is uncertain. However, a growing body of evidence suggests that the heteromeric assembly of Kjr2.1 and Kjr2.2 channels underlies the main component of IKI in the heart (Liu et al., 2001; Zaritsky et al., 2001; Preisig-Muller et al., 2002; Zobel et al. 2003). In humans, mutations of K;r2.1 have been linked to the cardiac arrhythmia termed Anderson's syndrome (Plaster et al., 2001), a disease that is characterized by QT-prolongation, periodic paralysis, and dysmorphic features. II) Channel Assembly, Trafficking and Regulation i) General Overview After the biogenesis of ion channels, the cell dynamically regulates the fate of these proteins. Like most membrane proteins, ion channels are sorted by the cells secretory machinery. Recent work from Carol Deutsch's lab has demonstrated that the N -terminal tetramerization domain ( T l Domain) of nascent peptides self-associate with other monomeric K v channels alpha subunits, surprisingly'while nascent K v peptides are still attached to ribosomes (Lu et al., 2001; Kosolapov and Deutsch, 2003; Kosolapov et al.. 2004; Robinson and Deutsch. 2005). These early T l - T l interactions are thought to promote the assembly of channels that belong to the same K v subfamily. The T l domain in K v channels is a multipurpose domain that is present in K v l . x - K v 4 . x channels and mediates channel sorting and assembly (Shen et a l , 1993; Shen and Pfaffinger, 1995; Y u et al., 1996; Schulteis et al., 1998; L u et al., 2001; Strang et al., 2001; Kosolapov and Deutsch. 2003), interaction with accessory |3- and K C h l P s subunits (Rehm and Lazdunski, 1988; Rettig et al., 1994; Sewing et al., 1996; Pongs et al., 1999; Gulbis et al., 2000: Scannevin et al., 2004; Zhou et al., 2004), and has also been demonstrated to modify the gating kinetics of K v l . x channels (Cushman et al., 2000; Minor et al., 2000; Kurata et al., 2002). Unlike K v channels, K„- channels contain no functional N-terminal tetramerization domain. Rather, there is evidence suggesting that the C-terminal region of the M 2 transmembrane domain and the proximal region of the C-terminus controls heteromeric assembly of members of K„2.x channels (Tinker et al., 1996). It is generally believed that after the insertion of individual K + alpha subunits in the E R membrane, 9 channels interact with several ER-resident proteins to aid in the formation of their quaternary structure. Furthermore, glycosylating enzymes resident in the ER, mediate core N-linked glycosylation of a single consensus N-linked glycosylation site (NXT/S; with X being any amino acid except for proline) located between the extracellular S1-S2 linker on K v l . x alpha subunits. Calnexin, an ER-resident protein, which functions as a molecular chaperone to facilitate protein folding and assists in the assembly of immature proteins, was demonstrated to stabilize the potassium channel, K v1.2, in the ER and improve its transport to the cell surface (Manganas and Trimmer, 2004). However, not all K7 channels are core glycosylated. These channels include K v 4.x channels which do not possess a consensus core N-linked glycosylation site in their protein sequence. After synthesis and tetramerization in the ER. K"'" channels are forwarded to the Golgi complex from where they are finally exported to the plasma membrane. Many suggest that the rate limiting step of ion channel trafficking is ER export (Lodish et al., 1983) and export efficiency is determined by a number of quality control system parameters including proper folding and stoichiometry of channels, assembly, and specific ER retention and export signals (Pelham, 1989a; Pelham, 1989b: Deutsch, 2003). Also chaperone proteins, such as Hsp 70 and Hsp 40, detect improperly folded ion channel complexes by shielding ER export signals, thereby prevent their transport to ER exit sites (Ficker et al., 2003). Because ion channel maturation is not always completely effective and the production of improperly folded and assembled K T channels does arise, channels that fit in this category must be degraded. These channels are disposed of through the ER-associated degradation pathways, such as the cytoplasmic ubiquitin-proteosome system. In K A T P channels, both Kj r6.3 and accessory SUR1 and SUR2 binding partners possess a cytoplasmic R X R retention motif which inhibits early E R export (Zerangue et al., 1999). These R X R motifs may act as signals that 'communicate' to the cell that proper assembly has not occurred, as the masking of these retention signal may negate signals involved in retaining K i r 6.3 channels in the E R . Conversely, E R export in K,-r2.1 is mediated by the C-terminal F Y C E N E forward trafficking motif and can accelerate channel surface expression when inserted into several K + channels, including K j r l . l channels which lack this motif (Ma et al., 2001; Stockklausner et al., 2001). Furthermore, Golgi transport of K.j,2.1 is accelerated through an amino-terminal motif (Stockklausner et al., 2001). In K v channels, a forward trafficking determinant have been identified in the pore and in concert, the carboxyl-terminus of K v 1 . 4 channels, V X X S L (Manganas et al., 2001; Zhu et al., 2001). A cognate L X X S L in K v 1 .5 channels acts in a similar maimer as K v l .4, but has been shown to be less effective (L i et al., 2000). Once channels are trafficked to the Golgi complex, the sugar moieties that were added back in the E R are trimmed and glycan moieties are added. Currently, the physiological significance of glycosylation in determining the surface expression and function of ion channels is not understood, but many suggest glycosylation mediates protein folding (Lennarz, 1983), forward trafficking of ion channels to the plasma membrane and their half-lives once there (Zhu et al., 2003), and may even alter their biophysical properties (Watanabe et al., 11 2003). In a process that is less understood, channels are sorted in the Trans-Golgi Network where they are diverted into vesicles that eventually localize K + channels to the cell surface and perhaps to specific domains within the plasma membrane. Once at the cell surface, channels may be endocytosed and may be recycled back into the plasma membrane or alternatively, may be degraded through a lysosomal or proteosomic degradation pathway. Ill) Cytoplasmic Motors Molecular motors provide the transport machinery necessary to drive the transport of cargo-containing vesicles, such as neurotransmitters and integral membrane proteins, between the E R and the plasma membrane. These cytoplasmic motors can be divided into three distinct classes; all utilizing A T P hydrolysis to generate movement. Firstly, myosin motors transport their cargo along cortical actin filaments surrounding the periphery of cells and, with respect to myosin isoforms V and V I , mediate vesicle trafficking either to (Langford, 2002) or away from the plasma membrane (Hasson, 2003). After the vesicle is internalized, it is transferred over from the myosin motors to the dynein motor system. Less restricted in their localization, the microtubule-dependent motors kinesin and dynein generally traffic cargo in the anterograde (towards the plasma membrane) and retrograde (towards the nucleus) direction, respectively. Both microtubule-associated motors are vital in positioning cellular organelles (Howard and Hyman, 2003), maintaining Golgi complex integrity (Allan et al., 2002), driving trafficking of cargo to and from the E R and Golgi (Presley et al., 2002), and transport vesicles to and from the Trans-Golgi Network and cell periphery. Very diverse in their structure and function, all three classes of 12 cytoplasmic motors are associated with caveolar-dependent, clathrin-dependent and clatlirin-independent forms of endocytosis (Valetti et al., 1999; Goode et al., 2001; Uruno et al., 2001; Weaver et al., 2001). The dynein complex contains several proteins, including dynamins which are large GTPase proteins involved in the scission of nascent vesicles from the membrane (Schroer, 1994). Dynamin forms a helix around the. neck of newly forming endocytotic vesicles where cooperative GTPase hydrolysis allows the lengthwise extension of this helix, eventually collapsing the vesicle neck and freeing the vesicle into an early endosome (Kelly, 1999). Another dynein-associated protein, dynactin links the entire dynein complex, including the large dynactin protein, p l 5 0 g l u e d , with its vesicle cargo (Schroer, 2004). Currently, the exact structure of the dynein complex remains undetermined and is thought to be composed of several interacting components such as the actin-related protein, A r p l , and dynamitin (also known as p50). In order to elucidate the role of the dynein motor complex in vesicular trafficking, the complex is often disrupted by the over-expressing dynamitin (Dhani et al., 2003; Varadi et al., 2004; Choi et al., 2005). Consequently, this disrupts the dynein/dynactin interaction by disassociating the p l 5 0 g l u e d subunit that interacts with dynein, from the A r p l filament, thought to link the cargo to the dynein motor complex. This disruption renders the interaction between dynein and its vesicular cargo ineffective (Echeverri et al., 1996; Burkhardt et al., 1997; Valetti et al., 1999). 13 IV) Receptor-Mediated Endocytosis Cells may regulate the expression of membrane-associated proteins by altering their internalization rate by receptor-mediated endocytosis. Endocytosis of proteins ensures degradation of membrane proteins, which may be mediated by polyubiquitination or lysosomal-mediated degradation, and also regulates the recycling/reinsertion rates of these proteins back into the plasma membrane. Receptor-mediated endocytosis involves the recruitment of a plethora of cellular proteins. The majority of receptor-mediated endocytosis involves the formation of clathrin-coated pits. Clathrin oligomerization maintains the structural integrity of immature endosomes and is associated with various adaptor proteins, including AP -2 . Internalization of immature endosomes is predominantly mediated by dynamin. Dynamin plays a pivotal role in distorting cellular membranes to energetically drive the formation of newly endocytic vesicles. Myosin motors, mediate the movement of vesicles within the actin cytoskeleton and transfer their cargo, in the retrograde direction, to the dynein motors, where these early endosomes may either be recycled back into the plasma membrane or alternatively, sorted to lysosomes or proteosomes for protein degradation. As such, surface expression of ion channels may be regulated by altering internalization rates through receptor-mediated endocytosis. Since endocytosis of membrane proteins is regulated by a number of cellular components, including microtubule-dependent molecular motors kinesin and dynein, it is interesting to note that several groups have demonstrated a functional role for microtubule-dependent molecular motors in modulating endocytosis of several ligand-gated and voltage-dependent ion 14 channels (Setou et al., 2000; Setou et al., 2002; Dhani et al., 2003; Guillaud et al., 2003; Choi et al., 2005: Chu et al., 2006). Protein-protein interactions are likely determinants of internalization of surface resident ion channels. Mechanisms that may stimulate or inhibit internalization rates include phosphorylation/ dephosphorylation of channels via tyrosine kinases or accessory subunit interaction between endocytic machinery and ion channels. For example, polyubiquitination of membrane localized ion channels may act as a recruiting signal to localize cellular components required for endocytosis of channels and also may target the protein into a degradation pathway, rather then a recycling route (Katzmann et al., 2002). Below, is a general overview of these reports. V) Regulation of Surface Expression of Ion Channels by Microtubule-Dependent Motor Mediated Endocytosis Microtubule-dependent motors, such as dyneins and kinesins, drive the movement of transport vesicles within the cell. Dyneins and kinesins utilize A T P to power membrane vesicular movement along their microtubule tracks to traffic secretory proteins to their final destination. To date, there are several studies which have imposed a relationship of how these molecular motors may regulate the dynamic expression of a variety of membrane spanning proteins, including ion channels. One of the most effective cell models to investigate the role by which microtubule-dependent motors may regulate ion channel localization has been completed in neurons. Neurons are a great model, as specific localization (dendrite, cell body, and axon) of ion channels is critical for neuronal function and are relatively large compared to 15 other polarized cell types, including epithelial cells. In 2000, Setou et al. identified that the brain and dendritic specific anterograde kinesin motor complex, K i f l 7, was responsible for driving the forward trafficking of N R 2 B subunits of the N M D A receptor to dendrites in murine brains . The N R 2 B subunit co-assembles with its essential NR1 subunit to form the N M D A receptor at dendritic membranes. Knock out of K i f l 7 function, by the use of a dominant negative mutant, was demonstrated to attenuate N R 2 B synaptic expression. In addition, K i f l 7 vesicles were shown to colocalize with dendritic N.R2B synaptic clusters. A n interesting observation from Guillaud et al. (2003) showed that N M D A antagonists, which are known to increase the surface expression of the N R 2 A and N R 2 B subunits, resulted in a con-commitment increase of surface expression of the molecular motor K i f l 7 and suggested that the expression of N R 2 B and K i f l 7 are strongly co-regulated. Thus, this suggests that the co-regulation between N R 2 B and its molecular motor, K i f l 7 , may arise to effectively modulate synaptic events. Moreover, it has been reported that the N R 2 B subunit is specifically required for synaptic localization of the N M D A receptor and enhancement of learning and memory in mice, In addition, microtubule depolymerization causing drugs, which disrupt kinesin motility, are able to inhibit N M D A receptor-mediated ionic and synaptic currents in cortical pyramidal neurons (Yuen et al., 2005) supporting a role for kinesin-based motors in altering N M D A function in neurons. The specific interaction of K i f l 7 and the N R 2 B subunit may explain why the N R 2 B subunit is necessary for functional learning and may be critically important in generating plasticity within the post-synaptic terminal of neurons. 16 In contrast, the anterograde axonal-specific kinesin Kif5b transporter interacts with the carboxy-tenninus of the GluR2 subunit of the A M P A receptor thru the binding of a P D Z domain of glutamate-receptor-interacting protein, GR1P-1. In this study, Setou et al. (20.02) demonstrated a direct interaction between GluR2 and Kif5b and in addition, dominant negative mutations of Kif5b were shown to prevent GRIP-1 specific localization to Golgi-related structures, vesicles in dendrite shafts, and in post-synaptic densities. Together, this data suggest a possible role for the specific kinesin isoform, Kif5b, in the forward trafficking of GluR2 subunits of the A M P A receptor in post-synaptic dendrites, which is assisted by its anchorage to the GRIP-1 adaptor protein. To date, there has been a single report suggesting an interaction between a voltage-gated potassium channel and the anterograde molecular motor, kinesin. Recently, Chu et al. (2006) report in rat cortical neurons, K v 4 .2 is trafficked to dendritic synapses by the kinesin isoforms, K i f l 7. In addition, this interaction may be through an indirect or direct interaction with the carboxy-terminus of K v 4 .2 (Chu et al., 2006). In this report, dominant negative isoforms of several kinesins were used to investigate whether a specific isoform could regulate dendritic targeting of K v 4 .2 . Chu et al. (2006) showed, through immunocytochemistry, that only the K i f l 7 isoform was able to co-localize with Kv-4.2, in contrast to other kinesin isoforms including Kif5b and K i f 2 I B . This study also investigated domains within K v 4 .2 that could influence the K v 4 . 2 - K i f l 7 interaction. Data from various deletion constructs of K v 4 .2 pointed to a role for the carboxyl-terminus of Kv-4.2 in the binding of K i f l 7. However, deletion of the C-terminal dileucine motif, which previously has been implicated in mediating dendritic targeting of K v 4 . 2 , 17 surprisingly did not alter this interaction, studied by coimmunoprecipitation and immunocytochemistry, between the channel and its molecular motor K i l l 7. Overall, this investigation concluded that K i l l 7 is very likely to provide anterograde motility of K v 4 . 2 to dendrites but the authors suggested that localization of K v 4 .2 may not solely depend on K i l l 7 for K v 4 .2 localization to dendritic membranes. There are several studies which also demonstrate a role for the anterograde dynein motor complex in the trafficking and localization of ion channels. In 2003, Dhani et al. implicated a role for the retrograde motor, dynein, in mediating endosomal trafficking of the chloride conductance channel, C1C-2 channel. These channels, which form the hyperpolarization and swelling-activated inward rectifying chloride current in retinal pigment epithelial and Sertoli cells, were shown to directly interact with several molecules within the dynein motor complex, including the heavy and intermediate chains of dynein in both in vitro and in vivo binding assays. Furthermore, when Dhani et al. (2003) disrupted the dynein motor complex, using dynamitin overexpression and the drug E H N A (erytlii*o-9-[3-(2-hydroxynonyl)] adenine; an inhibitor of ATPase activity of the dynein motor), they found that this resulted in an increase of surface expressed C1C-2 channels. Using biotinylation assays and immunocytochemistry, the authors also concluded a functional role for the dynein motor system in mediating the endocytosis of C l C - 2 containing vesicles into the early endosomal secretory pathway. Disruption of the microtubule network, which impairs dynein motor function, by the microtubule depolymerizing agent nocodazole also resulted in a redistrubution of channels from its normal perinuclear localization to a more scattered distribution throughout the cytosol 18 and increased staining to the periphery of the cells in COS7 cells. Overexpression of dynamitin also resulted in a similar redistribution pattern of C1C-2 channels, showing more channel staining towards the edges of COS7 cells. Thus, cell surface expression may be regulated by dynein-mediated endocytosis in ClC-2 channels. Recently, tyrosine phosphorylation of Shaker IC channel K v1.2 was demonstrated to suppress channel surface expression by accelerating channel endocytosis (Nesti et al., 2004), a process that is mediated by the retrograde dynein motor complex. In this study, a single tyrosine residue within the C-terminus of K v1.2, Y132 was found to be necessary for channel suppression and dynamin- mediated endocytosis, upon stimulation of i phosphorylation by drugs such as Pervanadate and Carbachol in HEK293 cells. Inhibition of dynamin, a GTPase that is involved in the formation and pinching off of clathrin coated pits to form clathrin coated vesicles, by the usage of a dominant negative form (K44A dynamin) resulted in a loss of phosphorylation-induced suppression of surface labeled K v l .2 . This was a novel finding as it was the first to claim a role for dynamin, a protein intimately involved in the majority of clathrin mediated endocytosis, in the regulation of the trafficking of a voltage-gated ion channel and thus suggested that phosphorylation of K v l . x channels may serve to regulate channel surface expression via the endocytic pathway. The authors suggested a possible role for a SH3 binding protein, such as Src Kinase, in mediating the internalization of K v1.2 channels into the early endosomal pathway. An unanswered question relates to whether or not dynein can mediate the regulation of endocytosis of Kv l .2 channels- upon phosphorylation of its C-terminus. 19 Recently, our laboratory has identified a role of the dynein motor complex in trafficking of early endosomal containing K v 1 .5 vesicles (Choi et al., 2005). Dynein disruption in HEK.293 cells, by overexpression of dynamitin, was shown to double K v 1 .5 current densities and surface expression through electrophysiological and proteinase K assays, respectively (Choi et al., 2005). Furthermore, treatment of the microtubule depolymerization agent to disrupt dynein-based motility, with nocodazole, was shown to enhance sustained currents in rat atrial myocytes preparations (Choi et al., 2005). These sustained currents were inhibited by 1 m M 4-AP and the K v 1 .5 specific antagonist D M M (100 irM). This verified that the disruption of the dynein motor system, in rat atrial myocytes, increased K v l ,5-associated membrane currents. In confocal images, where the dynein motor was disrupted by either dynamitin overexpression or nocodazole pretreatment in H E K 2 9 3 cells and rat atrial myocytes, respectively showed an increase in membrane K v 1.5 surface staining, at the expense of internal K v 1 .5 internal stores (Choi et al., 2005). In addition, we were able to demonstrate an interaction between K v 1 .5 and a component of the dynein motor complex, the dynein intermediate chain. These results suggest that not only dynein interacts with K v 1 . 5 , but that dynein can modulate the trafficking of K v l .5 channels within both mammalian cell lines and in rat atrial myocytes. Inhibitors of endocytosis, such as the dynamin inhibitory peptide, were able to mimic the effects of p50 overexpression by enhancing K v 1 .5 currents in HEK293 cells and in addition, confocal images of these cells showed colocalization of K v 1 .5 channels near the cell surface with the early endosomal marker, E E A 1 (Choi et al., 2005). Thus, disruption of the dynein motor complex may increase the number of functional K v l .5 channels at the cell membrane by interfering with the net removal of surface-resident channels. As well, 20 we observed a role for the N-terminus in mediating the interaction between dynein and K v 1 . 5 , as mutation of a single SH3 binding domain was insensitive to p50 overexpression in HEK293 cells (Choi et al., 2005). Therefore, we postulated a necessary role for an N -terminal SH3-binding domain in mediating endocytosis of K v 1 .5 channels at the plasma membrane. Overall, we concluded that dynein disruption impaired the uptake of newly endocytosed K v 1 .5 channels, thereby increasing the pool of K v 1 .5 channels resident at the cell membrane. The aim of this thesis is to further understand how cellular trafficking machinery, in particular the cytoplasmic dynein motor, may contribute and control K + channel surface expression in cardiac cells. We first hypothesize that surface expression of K v l .5 channels is modulated by the function of the dynein motor complex by mediating its internalization through an endocytic pathway. Second, we hypothesized that the surface expression of other cloned potassium channels in the heart also may be regulated by the dynein motor system, including Kj r2.1 channels and cardiac expressing K v 4 . 2 and K v 4.3 channels. In this report, we found that dynein regulates the steady-state surface expression of K v 1.5 channels in both mammalian cell lines and in rat atrial myocytes by possibly regulating the rate of internalization of these channels. Disruption of the dynein motor complex, via dynamitin overexpression, increases K v 1 .5 currents and protein levels at the cell surface. Furthermore, we also were able to demonstrate that other K v channels were regulated by the dynein motor system. We found that K v 4 . 2 and K v 4 .3 surface expression was downregulated with overexpression of dynamitin in H E K 2 9 3 cells and in addition, K v 4 . x like currents in rat atrial myocytes was reduced when the microtubule 21 depolymer iza t ion agents, nocodazole , was added. However , the I K I forming ion channel , K i , 2 . 1 , surface expression was unaltered w i t h both dynami t in overexpression and nocodazole treatment in Irk- cells . Moreover, , IKI currents were not altered upon nocodazole pretreatment wi th nocodazole . Taken together, this thesis suggests that dynein regulates the t raff icking o f various cardiac potassium ion channels differentially, and may also suggest distinct t raff icking pathways for potassium channels i n the heart. 22 Methods Cell Preparation and Transfection In most experiments, a stably transfected Human Embryonic Kidney cell line 293 (HEK293, American Tissue Culture Collection) was used as they expressed relatively high levels of surface human K v 1.5-T7 tagged, rat K v 4 .2 , and rat K v 4.3 protein. For electrophysiological studies on human Kj,2.1 channels, a mouse fibroblast Itk- cell line, stably expressing Kj r2.1, was used (Gift from Keiko Ishihara). The reason for using an Itk- cell line, rather then H E K 2 9 3 cells, to study Kj,-2.1 was due to the difficulty of producing a stable H E K 2 9 3 K„2.1 cell line observed in our laboratory. Cells were dissociated using Tryps in-ETDA and were plated onto coverslips containing minimum essential media ( M E M ) , 10% fetal bovine serum, 1% penicillin-streptomycin and 0.5 mg/ml geneticin and were incubated at 5% CO2 in air/37°C overnight. A l l tissue culture supplies were supplied by Invitrogen (Carlsband, C A , U S A ) . 24 hours later, cells were transfected with 2.0 ug of the desired plasmid construct and 3pl L I P O F E C T A M I N E 2000™ transfection reagent (Invitrogen) according to manufacturer's guidelines. Stable cell lines were constructed by transfecting a 5 ml flask of H E K 2 9 3 ce l l s /M- cells with 2.0 ug of the desired ion channel construct and L I P O F E C T A M I N E 2000™ transfection reagent suspended in M E M . Two days after transfection, the media was replaced with medium that contained Geneticin (0.5 ug/ml). A l l of the construct transfected contain a geneticin-resistance gene and thus cells which express the plasmid would survive and grow, whereas cells that lack the plasmid would die in geneticin-containing media. In the subsequent two weeks, the cell line was passaged and by the end of the 2"d week, cells were cultured using conventional cell culture techniques. For nocodazole (Sigma) experiments, cells were incubated with 35 u M nocodazole for 6 hr at 37°C before electrophysiological experiments were conducted. When using Dynamin Inhibitory Peptide in experiments, cells were incubated for 30 minutes with 50 u M myristoylated-DIP (Tocis Cookson Ltd., Bristol. U K ) or a scrambled control peptide. Plasmid Constructs For immunocytochemical detection, K v 1 .5 was N-terminally T7 tagged in pcDNA3 as described in Eldstrom et al. (2002). Briefly, K v l .5 was subcloned as a Hindlll-Notl fragment into pET28-a. The T7-tagged channel was then recovered by digesting the resultant plasmid with Ndel plus Nod and cloning the T7-tag- K v 1 .5 fragment into £coRV-A^ort-digested p c D N A 3 . The presence of in-frame fusions with the T7 tag was confirmed by D N A sequencing. Human p50 in pEGFP was a gift of Richard Vallee (Columbia University, N Y ) . Plasmid D N A was prepared for transfection using the Qiagen Plasmid M i d i Ki t (Qiagen Inc, Valencia, C A ) . Electrophysiological Experiments and Solutions For whole cell current recordings from transfected mammalian cells and myocytes, our standard patch pipette solutions contained (mM): KC1, 130; E G T A , 5; M g C b , 1; H E P E S , 10; N a i A T P , 4; OTP. 0.1; adjusted to pH 7.2 with K O H . The bath solution contained (in mM): 5, KC1; 130, N a C l ; 10, H E P E S ; 1, M g C b ; 1, C a C l 2 : and was adjusted to pH 7.4 using N a O H . However, when recording from rat atrial myocytes, 2 m M N i 2 + was added to eliminate calcium currents from L-type Calcium channels and the N a + / C a 2 + exchanger (Hinde et al., 1999). In myocytes electrical recordings, cells were held at -80 mV and given a brief 10 ms pulse to -40 mV to inactivate voltage-gated sodium channels. In our myocyte experiments, we chose to subtract peak current density from sustained current density to examine the effects of nocodazole on l t 0 currents. In some cases when studying K n 2 .1 channels in Itk- cells, we chose to use symmetrical YJ conditions as this gave us a broader range of voltages to test. In these experiments our pipette (intracellular) solution contained (mM): K C I , 5; E G T A , 5; H E P E S , 10; M g C ! 2 . 1; N a 2 A T P . 4; G T P , 0.1 and the final pH was adjusted of 7.2 using K O H and our bath solution (in mM): 5, K C I ; 130, NaCI; 10, H E P E S ; 1, M g C l 2 ; 1, C a C l 2 : and was adjusted to pH 7.4 using N a O H . A l l chemicals were from Sigma Aldrich Chemical (Mississauga. O N , Canada). Channel currents obtained were normalized to cell capacitance and expressed as channel current densities. Statistical significance was determined using a one-tailed Student's /-test, where P < 0.05 was considered statistically significant. Whole cell voltage clamp recordings and data analysis were done using an Axopatch 200B amplifier and pClamp 8 software (Axon Instruments, Foster City, C A ) . Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL) and polished by heating. We used pipettes which had resistances between 1-3 M Q . Compensation for cell capacitance and series resistance was performed manually in all whole cell recordings using mammalian cells. When recording from Kj,2.1 Itk- cells, we chose to break the cell-attached configuration at 0 m V to +10 mV, to reduce any ionic contamination when measuring capacitance and series resistance. But in all other cases, cells, were held at -80 m V and clamped to -70 m V for applying series resistance and capacitance compensation, where there is a low probability of 25 channel openings at these potentials. K;r2.1 expressing Itk- cells were depolarized to between -150 and +40 mV in 10 mV-steps from a holding potential of 0 mV, followed" by a -40 inV repolarization pulse to record Kjr2.1 currents. For electrical recordings of K v4.3 channels, cells were held at -100 mV and were depolarized/hyperpolarized between -110 mV to +80 mV at 10 mV increments and then returned to +40 mV for 400 ms where then cells were returned to the holding potential. A l l whole cell recordings were performed at room temperature (20-23 °C). Channel currents obtained were normalized to cell capacitance and expressed'as channel current densities. Data are presented as mean ± SEM. Statistical significance was determined using a one-tailed Student's Mes t . Myocyte Electrophysiology and Isolation Rat atrial myocytes isolated from adult male Wistar rats were prepared for immunocytochemical and electrophysiological experiments using the standard Langendorff dissociation technique (see below). In immunocytochemical experiments, isolated myocytes were plated on laminin-coated coverslips and suspended in M-199 medium (ImM DL-Carnitine; 0.1 mM Insulin; 25 mM HEPES; 10 mg/ml Fatty-acid free BSA: 100 lU/ml penicillin: and 100 mg/ml streptomycin) with 35 uM Nocodazole (in DMSO) or solvent (DMSO) alone. For electrophysiology experiments of rat atrial myocytes, cells were suspended in nocodazole/vehicle (DMSO) in M E M for 6 hours at 37°C, Cardiac atrial myocytes from rats were prepared using standard methods (Kagaya et al., 1995). Following anethesia (pentobarbital, 100 mg/kg), the heart was excised from 26 the chest and retrogradely perfused (at 37°C) through the aorta using the Langendorff method. After 10 minute perfusion with calcium-containing (2.2 mM) Tyrode's solution (121 m M N a C l , 5 m M K G . 2.8 m M NaAcetate, 1 m M M g C l 2 , 5 m M Glucose, 24 m M N a H C O j . 1 m M Na 2 HP04) , rat hearts were retrogradely perfused (10 min) with calcium free Tyrode's solution. Subsequently, digestion of the atria was accomplished by first, a 20 minute perfusion with collagenase-containing solution (120 ml normal Tyrode's solution, 120 mg collagenase, 300 mg taurine, and 125 u M C a C L ) and second, atria were removed form the Langendorff apparatus and digested in collagenase-containing solution for up to an additional 10 minutes. During this second stage, small amounts of supernatants were examined through a light microscope to monitor atrial isolation efficiency. When single atrial cells were observed, myocytes were isolated by low speed centrifugation. Myocytes were suspended in M E M containing media prior to treatment with nocodazole in an incubator (37 °C/5% C 0 2 ) for 6 hours. Myocytes chosen in our electrophysiological studies were tested based on their rod-shaped morphology and were used within 12 hours of isolation. Imaging For studies on the effects of p50 overexpression, HEK293 cells stably expressing T7-tagged K v 1 .5 or K v 4 . 2 were plated onto coverslips and later transfected with p50-GFP and cultured for a further 12 hr prior to fixation. The cells were fixed with 4% paraformaldehyde for 12 min at room temperature (RT). After three 5-min washes with l x phosphate-buffered saline (PBS; 137 m M N a C l , 2.7 m M K G , 4.3 m M N a 2 H P 0 4 , 1.4 m M K H 2 P O 4 ) , cells were incubated in a permeabilizing/blocking solution (PBS 27 containing 2% B S A and 0.2% Triton X-100) for 30 min at room temperature. A mouse monoclonal antibody to the T7 Tag (1:1000 dilution; Novagen) or K v 4 . 2 (1:500 dilution, Abeam) was diluted in blocking solution and incubated with cells for 2 hr at RT . Cells were then washed three times for 5 min in PBS on a rotator before incubation with secondary antibodies, Alexa 594-conjugated goat anti-mouse IgG antibody (1:1000; Molecular Probes) for 1 h on the rotator at RT. Coverslips were then washed three times with PBS prior to mounting with 10 ul of a 90% glycerol, 2.5% w/v D A B C O - P B S solution. Images were collected on a Nikon C l laser scanning confocal unit (Nikon DEclipse C l , Melvi l le , N Y ) using a 60X lens and processed using the operation software EZ-C1 for Nikon C l confocal microscope (Nikon). Images were prepared using the Adobe PhotoShop software package. For endosome imaging studies, H E K cells stably expressing K v 1 .5 were labeled with antibodies against the C-terminus of K v 1 . 5 (Fedida et al., 2003) and against E E A 1 (BD Biosciences, monoclonal antibody). For K V 1 . 5 - E E A 1 colocalization experiments, stable K v l .5-T7 lines were plated onto coverslips and treated with cycloheximide supplemented media (Cycloheximide, Sigma, 200 ug/ml) for 4 hr to arrest protein synthesis. After the first 2 hr of treatment at 37°C, cells were placed into a 20°C incubator for the last 2 hr treatment with cyclohexemide treatment. This temperature allows normal internalization but is not compatible with trafficking out of early endosomes or recycling to the plasma membrane (Dhani et al., 2003). The cells were rinsed and fixed with 4% paraformaldehyde for 12 min at room temperature. After three • 5-min washes with 1 x P B S , cells were incubated in permeabilizing/blocking solution 28 (PBS containing 2% B S A and 0.2% Triton X-100) for 30 min at room temperature. Rabbit polyclonal C-terminal K v 1 .5 antibody (1:500 dilution) and a mouse monoclonal E E A - 1 antibody (1:250 dilution, B D Biosciences) were diluted in permeabilizing/blocking solution and incubated with cells at 4°C overnight. Cells were then washed three times for 5 min in PBS on a rotator. Secondary antibodies, Alexa 594-conjugated goat anti-mouse IgG antibody and Alexa 488-conjugated goat anti-rabbit IgG antibody (1:1000; Molecular Probes) were then incubated with the cells for 1 h on the rotator at room temperature. The coverslips were once again washed three times with PBS prior to mounting with 10 pi of a 90% glycerol, 2.5% w/v D A B C O - P B S solution. Images were collected using a Deltavision Deconvolution Microscope using a 60X lens using Softworks software. Images were later viewed and prepared using Adobe PhotoShop software. For studies on the effects of nocodazole on rat atrial myocytes, the myocytes were treated with 35uM nocodazole (Sigma) for six hours then fixed and immunolabeled as previously described above. Myocytes were co-stained with anti-C-terminal K v 1 .5 (1:500 dilution) and monoclonal anti-f3-tubulin (1:200 dilution; Sigma) antibodies to verify the extent at which nocodazole was able to depolymerize microtubules. Image Analysis To quantitatively assess K v 1 .5 distribution in myocytes, line scanning analysis was performed using Image J software (NIH image). For each cell, eight 0.25 micron confocal slices were taken along the long axis of the cell, at 45-55% of the cell depth, 29 a p p r o x i m a t i n g t h e c e l l m i d d l e . T h r e e l i n e s w e r e d r a w n a c r o s s t h e w i d t h o f e a c h s l i c e c h o s e n a t p o s i t i o n s a p p r o x i m a t e l y VA, VI a n d V* o f t h e c e l l l e n g t h f r o m o n e e n d o f t h e c e l l . T h e l i n e w a s t h e n d i v i d e d i n t o e i g h t e q u a l s e g m e n t s . P a i r s o f s e g m e n t s ( i . e . , t h e o u t e r m o s t e i g h t h s , t h e e i g h t h s i m m e d i a t e l y i n s i d e a d j a c e n t t o t h o s e e i g h t h s , e t c ) w e r e c o m b i n e d t o f o r m ' q u a r t e r s ' a c r o s s t h e w i d t h o f t h e c e l l . T h e s e d a t a f r o m t h e l i n e s at t h e f o u r p o s i t i o n s a l o n g t h e l e n g t h o f t h e c e l l w e r e c o m b i n e d t o g e t a n a v e r a g e e s t i m a t e o f t h e fluorescence d i s t r i b u t i o n i n e a c h c e l l . T h e fluorescence i n t e n s i t y f o r e a c h q u a r t i l e w a s e x p r e s s e d a s a p e r c e n t a g e o f t h e t o t a l fluorescence i n t e n s i t y i n t h e l i n e s . A l s o , I m a g e J w a s u s e d f o r l i n e s c a n a n a l y s i s o f K v l .5 c o l o c a l i z a t i o n w i t h E E A 1 . L i n e s w e r e d r a w n t h r o u g h e n d o s o m e s a c r o s s s e v e r a l c e l l s i n c o n f o c a l i m a g e s a n d t h e i n t e n s i t i e s o f K v 1 . 5 a n d E E A 1 s i g n a l s at e a c h p i x e l w e r e p l o t t e d o n t h e s a m e g r a p h f o r c o m p a r i s o n . C o r r e l a t i o n c o e f f i c i e n t s , r , b e t w e e n K v 1 . 5 a n d E E A 1 s i g n a l s i n p i x e l s a c r o s s e a c h l i n e s c a n w e r e c a l c u l a t e d b y t h e f o r m u l a r = S ( x - x)(y - y) I S ( x - x ) 2 £ ( y ~ yf • T o a s s e s s t h e s i g n i f i c a n c e o f e a c h r v a l u e , t w a s d e t e r m i n e d a c c o r d i n g t o t = rl ( 1 - r~) /(TV - 2 ) a n d c o m p a r e d t o c r i t i c a l v a l u e s o f t. 30 Results PART 1: Kv1.5 Surface Expression Is Modulated by Retrograde Trafficking of Newly Endocytosed Channels by the Dynein Motor i) Kv1.5: Dynamitin/p50 Coexpression Significantly Increases Kvl.5 Current Levels and Surface Expression Prev ious ly , we have investigated the role o f the dynein motor on the surface expression o f K v 1 . 5 channels by using overexpression o f dynami t in as a tool to disrupt the dynein motor complex ( C h o i et a l . . 2005). In a H E K 2 9 3 ce l l l ine stably expressing K v l .5, overexpression o f dynami t in resulted i n approximately a 2-fold increase i n current amplitude and current density o f K v 1 . 5 in H E K 2 9 3 cells transfected w i t h dynami t in (F ig . IA) . Th is effect was not restricted to H E K 2 9 3 cel ls , but was also replicated i n Chinese Hamster Ova ry cells ( F i g . I B ) ; suggesting dynami t in increases K v 1 . 5 currents in other mammal i an ce l l lines (F ig . I B ) . These increases i n channel current density were not associated wi th changes in activation or inact ivat ion kinetics across the whole act ivat ion range o f K v 1 . 5 (F ig . I C ) , suggesting that the increase o f K v 1 . 5 current is due to an increase in the number o f functional channels inserted i n the plasma membrane. These results were later conf i rmed by Proteinase K assays. Proteinase K assays are a very sensitive test for w h i c h relative amounts o f surface expression o f membrane-destined proteins can be determined as the enzyme, w h i c h is impermeable to cel lu lar membranes, cleaves proteins w h i c h are exposed to the extracellular environment, such as in the case wi th ion channels. K v 1 . 5 channels that are not inserted into the membrane (i.e. the cytosol ic fraction) are uncleaved and run at a molecular weight o f 83 k D a on an S D S gel. 31 However, K v 1 .5 channels that are expressed on the cell surface, which are exposed to Proteinase K , run at a lower molecular weight, 47.5 kDa. Our results show an increased lower band intensity, approximately a 2-fold increase, of the digested form of K v 1 .5 in HEK293 cells overexpressing both K v 1 .5 and dynamitin compared to cells expressing K v l . 5 alone (Fig. IE ; (Choi et al., 2005)). The doubling of the intensity of the lower digested form of K v 1 .5 in p50-pGFP treated cells con-elates well with our electrophysiology results where we observed a doubling of K v 1 .5 current densities (Fig. IC). In confocal images taken from stably expressing T7-tagged K v 1 .5 H E K 2 9 3 cells, dynamitin overexpression resulted in a redistribution of internal K v 1 .5 channel stores such that dynamitin coexpression reduced the amount of visible K v l . 5 internal staining (Fig. ID). Our immunocytochemistry data shows that p50-pGFP overexpression resulted in a marked redistribution of K v 1 .5 staining. When K v 1 .5 was co-expressed with a control pGFP vector, prominent localization of K v l .5 staining in the cell interior proximal to the cell membrane was observed (Fig. ID). However, p50-pGFP overexpression resulted in an attenuation of K v 1 .5 staining in the cell interior and moreover, staining was concentrated at the cell exterior, proximal to the cell membrane. This is dramatically demonstrated by the lone cell that escaped transfection with p50-pGFP which contains significant internal staining of K v 1 .5 in Fig. ID (arrow). In agreement with Dhani et al. (2003), we were also able to identify a , direct/indirect interaction of K v 1.5 with one of the components of the dynein motor complex, the dynein intermediate chain, through coimmunoprecipitation (Choi et al., 2005). Taken together, our results support the notion that disruption of the dynein motor 32 D P G F P Kv1.5-T7 Merged -80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) Figure 1. p50/dynamitin coexpression significantly increases K v 1 .5 current levels and surface expression. A . Peak currents from IIEK293 cells stably expressing K v 1 . 5 transfected with empty vector (pGFP) or p50-pGFP. From -80 mV, cells were depolarized to between -70 and +80 m V in 10-mV steps, followed by repolarization to -40 mV. B , C H O cells transiently expressing both K v 1.5 and p50-GFP (solid line) and control cells transfected with K v 1 .5 pGFP alone (dashed line). Dotted line denotes the zero current level. C . Peak current amplitudes at +80 m V from controls (filled symbols) and p50-overexpressing cells (open symbols) were normalized to cell capacitance (n=12: **.P<0.01). D. Confocal images of HEK293 cells stably expressing T7-tagged K v 1 .5 (red) transfected with pGFP alone or p50-GFP (green). Scale bars=10 urn. E , H E K cells were cotransfected with T-7 tagged K v 1 .5 and either p50-GFP or pGFP and treated with externally applied Proteinase K (lanes 3 and 4) or buffer alone (lanes 1 and 2). The arrows indicate the Western blot position of surface K v 1 .5 (digested form) and intracellular K v 1 . 5 (undigested form). The immunoblot at the bottom indicates that equal amounts of protein were loaded in each lane. 33 complex leads to an upregulation of K v 1 .5 expression at the cell surface, possibly by influencing the distribution of the channel to membrane surfaces in H E K 2 9 3 cells. ii) Kyi.5 is Internalized Via a Dynamin-Dependent Endocytotic Pathway Recently, tyrosine phosphorylation of Shaker K + channel K v 1 . 2 has been demonstrated to suppress channel surface expression by accelerating channel endocytosis, a process that is mediated by the retrograde dynein motor complex (Nesti et al., 2004). Similarly, the chloride CIC-2 channel was shown to interact and is also regulated by the cytoplasmic dynein motor via the endocytotic pathway (Dhani et al., 2003). Given this information, we sought to test i f dynein disruption resulted in a defect of the retrograde movement of endocytotic vesicles containing K v l .5, since dynein is required for the transport of the majority of endosomes from the cell surface to the cell interior. To examine this further, we used confocal microscopy to identify i f K v l . 5 was localized in early endosomal compartments. We incubated cells with cycloheximide, an inhibitor of protein synthesis, to investigate whether newly endocytosed K v 1 .5 channels were internalized in the early endosomal pathway, similar to methods employed' by Dhani et al. (2003). To identify early endosomes, the early endosomal marker E E A 1 was used. Fig. 2A shows colocalization between K v 1 .5 in E E A 1 positive vesicles that lie proximal to the membrane. Through line-scan analysis, we examined the pixel correlation across nine groups of endosomes in the cell highlighted in Fig. 2 A and in endosome-rich regions of other cells. The pixel correlation between K v 1.5 and E E A 1 positive vesicles was remarkably strong (p < .0001, Fig. 2C, D). We also performed line scans analysis in 34 regions in which endosomal localization was absent but K v 1 .5 staining was present and observed no correlation between co-localization of the two proteins (Fig. 2E). ii) Kv1.5 is Internalized Via a Dynamin-Dependent Endocytotic Pathway One of the known roles of dynamin is to aid in the pinching off of clathrin-coated vesicles at sites of endocytosis. If interference with the dynein motor results in an accumulation of K v l .5-containg early endosomes, then direct disruption of dynamin should prevent the re-uptake of K v 1 .5 into the early endosomal secretory pathway. Dynein Inhibitory Peptide is a direct hrhibitor of this process and, i f dynein disruption is preventing endocytosis of K v 1 . 5 , should result in an increase amount of functional channels at the cell surface. This peptide inhibits dynamin function by binding to its amphiphysin binding site, a domain which is crucial for recruiting dynamin to immature endosomes, and therefore would inhibit dynamin-mediated endocytosis (Grabs et al., 1997; Kel ly , 1999) We tested this hypothesis by incubating Dynein Inhibitory Peptide (DIP), and a control scrambled peptide, to identify i f the current density of K v 1.5 increased with dynamin inhibition. DIP both increased current amplitude and current density of K v l .5 in HEK293 cells compared to cells incubated with a control scrambled peptide (Fig. 3; Peak current densities at +80 m V were 0.36 ± 0.05 nA/pF (n=15) in control cells and 0.76 ± 0.05 nA/pF (P< 0.01, n=l 1) in cells incubated overnight with 50 umol/L DIP (Choi et al., 2005). This data suggests that disruption of the dynein motor may increase K v l .5 surface expression by interfering with the net removal of channels from the cell surface and that internalization of K v 1 .5 channels from the plasma membrane is mediated by dynamin-dependent endocytosis. 35 Figure 2. K\ 1.5 is internalized via an endocytotic pathway. A , K v l .5 colocalizes with the early endosome marker, E E A 1 . Confocal images of cycloheximide treated H E K 2 9 3 cells stably expressing K v l .5 immunostained for K v l .5 (green) and E E A 1 (red), with yellow indicating colocalization. B , Dynamin inhibition reduces K v 1 .5 colocalization in E E A 1 positive early endosomes. Cells incubated with 50 mol/L dynamin inhibitory peptide (DIP; right) or scrambled control peptide (left) for 16 hours. A , line scan analyses of K\ 1 ,5-EEAl colocalization. In several cell confocal images, lines were drawn in Image.! through endosomes and the intensities of K v 1 .5 and E E A 1 signals at each pixel were plotted on the same graph for comparison. C and D are examples of plot profiles from cells that showed a high degree of colocalization between K v 1 .5 and E E A 1 . E illustrates a plot profile across several endosomes in a cell pretreated with Dynamin inhibitory peptide (DIP) before staining and examining for E E A 1 - K v 1 .5 colocalization. To ensure a stringent test for co-localization, pixel lengths ranged from 0.1064 microns to 0.134 microns in the scans across endosomes in which K V 1 . 5 - E E A 1 were expected to be present, and were 0.242 microns in the cells where K v l .5 and EEA1 were not obviously present together in endosomes. For all images, scale bar was set to 10 pm. 36 -G-J! -I 1 < 1 1 1 > 1 1 1 - 8 3 > 4 - 3 0 0 a « 6 0 M 1 0 0 vottageXifiV)-F i g u r e 3 . Internalization of K v 1 .5 is mediated by dynamin-dependent endocytosis internalized via an endocytotic pathway. A , Dynamin inhibition increases K v 1 . 5 currents. Cells incubated with 50 umol/L dynamin inhibitory peptide (DIP) or scrambled control peptide for 16 hours. Cells were depolarized from -70 to +80 m V in 10-mV steps and repolarized to -40 mV. Sample currents in control and a cell overexpressing DIP at +80 mV. C, Peak current K v l .5 amplitudes normalized to cell capacitance in control (filled symbols) and from DIP-overexpressing cells (open symbols; **/J<0.01). 37 iii) Nocodazole Pretreatment Increases Kv1.5, But Not Transient Outward if1" Currents in HEK293 Cells and in Rat Atrial Myocytes. K v l . 5 channels have been identified as the molecular correlate of I|<.ur, and are responsible for the repolarization of the action potential near the end of the plateau phase, forming the sustained current seen in human and rat atrial myocytes. Since the mechanism by which dynamitin overexpression disrupts the dynein motor system is undetermined, we attempted to mimic the actions of dynamitin by utilizing the drug nocodazole to interfere with dynein-based motility. Nocodazole prevents polymerization of microtubules and we expected that it would mimic the actions of dynamitin by trapping K v 1 .5 channels at the cell surface. We used a concentration of 3 5 u M nocodazole as this was the concentration that in our hands fully depolymerized (3-tubulin, a marker of microtubules (Fig. 4). Compared to vehicle treated myocytes (control. Fig. 4A) , 35 u M nocodazole was able to completely depolymerize the microtubule network in images where (3-tubulin was stained. A lower dose of nocodazole (17.5uM, Fig. 4) was ineffective in fully disrupting the microtubule cytoskeleton network, as aggregation of [3-tubulin was seen in perinuclear regions of rat atrial myocytes and in-the internal compartments within the cell. When performing electrical recordings from rat atrial cells, application of 35 u M nocodazole significantly increased the sustained current, and to a small extent, increased the inward potassium currents below -80 m V (Fig. 5A, B , C;(Choi et al., 2005)). However in these studies, the transient outward peak current densities were unaffected (Fig. 5D). From these results, we believed that dynein function, through an unknown 38 Figure 4. Concentration-dependent depolymerization of P-tubulin with nocodazole in rat atrial myocytes. Rat atrial myocytes wither treated with A , D M S O , B , 17.5 u M Nocodazole (in D M S O ) , and C, 35 u M Nocodazole (in D M S O ) for 6 hours at 37°C. Control myocytes treated with D M S O (A) displayed a reticular patter of P-tubulin (red) with staining that concentrated in the perinuclear region of rat atrial myocytes and at the cell periphery. After 17.5 u M Nocodazole treatment (B) P-tubulin staining appeared in small aggregates distributed within the cell, however most cells showed a clumped pattern of P-tubulin within the perinuclear region and there was a substantial loss of reticular P-tubulin staining extending from the nucleus to the cell surface. In contrast, treatment at a higher concentration of Nocodazole, 35 u M (C), had lost perinuclear clustering of P-tubulin and polymerization of P-tubulin had appeared to be completely diminished. Scale bar = 10 urn. 39 mechanism, can regulate the surface expression of K v l . 5 channels in HEK293 cells and in cardiac myocytes. In addition, these results suggested that nocodazole pretreatment did not influence the expression of I t 0 currents in rat atrial cardiac myocytes. Lastly, these results also suggested a possible upregulation of surface expression of channels that contribute to inward rectifying potassium current in the heart, I K I , as current densities in the inward direction were enhanced in nocodazole treated cells below the resting membrane potential (approximately'-70 to -80 mV;(Nattel, 2003)) of atrial myocytes (Fig. 5B). Imaging analysis of atrial myocytes treated with nocodazole under the same conditions revealed an interesting pattern, where myocytes treated with nocodazole carried the bulk of K v l .5 staining close to the cell surface compared to the interior of the cell (Fig. 6A, C). This was in contrast to control cells where a more equal distribution of K v l . 5 staining was observed within the cell (Fig. 6B, D). Line scan analysis of 10 randomly chosen control and nocodazole-treated myocytes showed that 26.9 ± 2.1% (mean ± % SEM) of K v l .5-positive pixels were within the quartile of the cell nearest to (and including) the plasmalemma in control cells and 40.5 ± 2.6% (Fig. 6C, D; unpaired / test, P < 0.001) of positive pixels were within these boundaries in nocodazole-treated myocytes. This correlates well with our imaging data taken from HEK293 cells (Fig. ID) where we observed a redistribution of K v 1.5 to the cell surface with p50-pGFP and our electrophysiological data from rat atrial myocytes (Fig. 5). These observations likely suggest that the drug nocodazole, which mimics the effect of dynein disruption by p50, results in an increase of K v l .5 channels at the cell surface. 40 < 5i 100 ms Nocodazole B rr i • Control (n=14) 80i A/p 60 a 40 > 20 c 0 +-* C -20 o -40--1 •oooooooeco Voltage (mV) so ^ 60 a s E I ° 3 -20 • Control (n«14) 0 Noccxtozol«(n=l4) -103-80 -60 -4C -20 0 20 40 60 80 Voltage (mV) 40 f » Q. & 20 •55 c « « 10 • Control (n.3) NocodaaXe (n«3) 3 O -10 0 g> o o o '-' 0 -1O0-80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) Figure 5. Nocodazole pretreatment increases i K s u s and IKI , but not I T 0 currents in rat atrial myocytes. A . Currents from rat atrial myocytes treated with 35 umol/L nocodazole or solvent ( D M S O ) alone. Cells were depolarized to +80 m V from -80 m V in 10-mV steps. B and C, Mean sustained current-voltage data ± nocodazole. **P<0.01, Student / test. D , Mean A-type peak current-voltage data ± nocodazole. To avoid contamination by sustained currents, the K v 1,5-specific blocker D M M (100 nM) was included in the bath solution. 41 Control 8S pM Hocodaxol* 1 2 3 4 1 2 3 4 Oiaunc* (quartart, Dislanc* (quarter*) Figure 6. Nocodazole pretreatment increases Kvl .5 surface expression in rat atrial myocytes. A and B. Myocytes incubated for 6 hours without (control) or with 35 uM nocodazole. fixed and immunostained for K v1.5 (green). Scale bar = 10 pm. C and D , Relative pixel intensity across cells divided into eighths and summed into quartile bins from edge to center, where 1 represents the total fluorescence intensity in the peripheral two-eighths of the cells and 4 represents the center-most quartile of the cells. **P<0.0\, 1-way A N O V A . 42 In addit ion to K v 1 . 5 , our results suggest that inwardly- rec t i fy ing channels may also be trafficked to the ce l l surface by microtubule-dependent molecular motors. H o w e v e r w h i c h molecular motors and w h i c h channels contr ibuting to I K I that are responsible for this effect remains undetermined. Thus, an addi t ional goal o f this thesis was to survey i f channels w h i c h form IKI currents i n the heart, such as K j r 2 . 1 , are trafficked to cel lular membranes by the dynein motor system. 43 PART 2: Disruption of the Dynein Motor Complex Does Not Alter the Surface Expression of K„2J Channels In the heart, K ; r 2 . x channels form the predominating inward-rectifying ( I K I ) current in the heart (Liu et al., 2001; Zaritsky et al., 2001; Preisig-Muller et al., 2002; Zobel et al., 2003). To date it is believed that the molecular basis of I K I in cardiac myocytes is composed of the heteromultimeric assembly of Kj r2.1 and Kj,-2.2 channels. In previous data from rat atrial myocytes, pretreatment of nocodazole at room temperature slightly increased an inward current below the resting membrane potential of atrial myocytes (-80 m V ; Fig. 5). Given that this temperature is not physiological, we have repeated these experiments at 37°C to examine the effects of microtubule disrupting drugs on the surface expression of various cardiac K + currents, including I K I , in rat atrial myocytes. Surprisingly, we found contradictory results, when compared to nocodazole pretreatment at room temperature (Fig. 5). At 37°C, nocodazole pretreatment did not alter inward current densities below the resting membrane potential (Fig. 7: between -70 and -80 m V in rat atrial cells (Nattel, 2003)). Current density of control rat atrial myocytes (at -140 m V , -16.14 pA/pF ± 3.44) was not significantly different from nocodazole treated cells (at -140 mV, -12.65 pA/pF ± 2.75) at 37°C (Fig. 7). Thus at more physiological temperatures, disruption of the microtubules, and therefore inhibition of kinesin and dynein motors which rely on microtubule tracks to transport their cargo, do not modify the surface expression of channels that contribute to IKI in rat atrial myocytes. Since K; r2.1 is the major inward rectifier K + channel in cardiomyocytes, we investigated the effects of dynein disruption, through p50 overexpression, on the membrane trafficking of this channel. In Itk- cells stably expressing K| r 2 .1 , transfection of p50-GFP did not alter 44 < e i 50 ms 40 i 50 ms Nocodazole LL a < a £ 10 c 2 0 § -10 u C o - 2 0 • Control (n=8) O Nocodazole (n=6) 8 o Q ' • 160 -140 -120 -100 -80 -60 -40 -20 0 20 40 60 80 100 Voltage mV Figure 7. Interference with microtubule-based trafficking downregulates A-type currents, but does not alter IKI currents at 37°C (A) Currents from rat atrial myocytes pre-treated with 35 u M nocodazole or solvent ( D M S O ) alone, at 37 ° C. Cells were initially held at -80 mV, and given a brief 10 ms pulse to -40 mV before a test pulse was applied from -150 m V to +90 m V in 10-mV steps. (B) Mean peak current density-voltage ± S E M data of control and Nocodazole treated myocytes Nocodazole from cells pre-incubated with nocodazole or solvent alone (n = 8 Control; n = 6 Nocodazole). • Represents statistically significant differences in current density at applied voltages between control and nocodazole pretreatment groups (p < 0.05; Student's t test). Between -150 m V to -80 mV, current densities represents peak currents-densities obtained. Between -70 m V to +90 m V , currents were analyzed as the difference between peak and sustained values (peak minus sustained). 45 K, r2.1 current densities compared to control cells expressing pGFP. Kj r2.1 mean peak current densities at -150 m V for control cells (n = 12) were -467.00 ± 42.73 pA/pF and for cells expressing p50-pGFP (n = 10). mean peak current densities were -451.29 ± 43.93 pA/pF (Fig. 8, p = 0.25, Student's / test). This insensitivity was surprising as our initial nocodazole-pretreated atrial myocytes data showed an increase of IKI - l i ke current density. Similar to our rat atrial myocytes data at 37°C, Itk- Ki r2.1 expressing cells that were pretreated with the microtubule-depolymerizing agent, nocodazole (35 uM) showed no statistical differences in Kj,2.1 current densities (Fig. 9). Mean current density at -150 m V for control ( D M S O treated; n = 5) cells were -446.62 ± 73.21 pA/pF and for nocodazole pretreated cells (n = 6), mean current densities were -440.36 ± 32.76 pA/pF (p = 0.37; Student's / test). B y visual inspection, inward rectification appeared to be unaltered in both groups, indicating that p50 and nocodazole does not alter the sensitivity of outward K + current block by divalent cations or polyamines. These results conflict with our initial results seen in rat atrial myocytes pretreated with nocodazole at room temperature. There may be several explanations for these contradictory results (see Discussion). Therefore, disruption of dynein motor function by either direct interference through p50 overexpression or introduction of microtubule depolymerization agent, nocodazole, did not alter the number of functional channels at the surface of Itk- cells. Taken together, our electrophysiological data from rat atrial myocytes and the mammalian cell line, Itk- cells, suggest that Kj,-2.1 channels and other channels that form I K I currents in the heart may not be modulated by the dynein motor, or even the kinesin motor system, at more physiological temperatures. 46 * L 50 ms Kir2.1 Control p50 +40 mV -150 mV 0 mV B o o c —i 3 - 0.1 3 (/) £ - 0 . 3 | - 0 . 4 •a "TJ -0.5 • pGFP(n=12) ^OOCrCrC O p50(n=10 fir -160 -120 -80 -60 -40 -20 0 20 40 Voltage (mV) Figure 8 . Kj, 2.1 currents are insensitive to disruption of the dynein motor by p50 overexpression in mouse Itk- cells. Stably expressing Kjr2.1 cells were transfected with either p50-GFP or an empty G F P vector alone (control). A , Individual current traces of Kjr2.1 expressing cells transfected with either p50 or pGFP. B , No significant difference in mean current-density voltage relationship of cells transiently transfected with p50 compared to controls (pGFP). A l l recordings were held at 0 mV in symmetrical K + at cells was pulsed at potentials ranging from -150 m V to +40 m V in 10 m V increments. 47 Kir2.1 B < = i 50 ms Control Nocodazole +40 mV -150 mV 0 mV uT 0.1 Q. 3 o.o c — -0.1 4 * » -0.2 c a c i-3 o -0.3 -0.4 -0.5 -0.6 © Control (n=5) O Nocodazole (n=6) -160 -120 -80 -40 Voltage (mV) 40 Figure 9. K , r 2 . l currents are insensitive to the microtubule depolymerizing drug, nocodazole, in mouse Itk- cells. A , Individual current traces of Kj,2.1 expressing cells treated with solvent ( D M S O ) or 35 p M Nocodazole for 6 hours. Mean current density-voltage relationship is plotted, B , where no significant difference of current densities were seen upon nocodazole pretreatment. A l l recordings were held at 0 m V in symmetrical K + at cells was pulsed at potentials ranging from -150 m V to +40 m V in 10 m V increments 48 PARTS: Disruption of Dynein Motor Function Reduces Expression of Transient Outward Potassium Channels ii) Disruption of the Microtubule Cytoskeleton Alters Potassium Currents in Rat Atrial Myocytes In rat atrial myocytes, we have previously shown that treatment at room temperature with the microtubule-depolymerizing agent nocodazole significantly enhances I K m - expression, while unaltering the current densities of I t 0 currents (Choi et al., 2005). When we replicated these experiments, we chose to pre-treat myocytes with the drug nocodazole at 37°C to look for effects of nocodazole treatment on other potassium currents under more physiological conditions. In the majority of cells (4/6 in nocodazole-treated group. 0/6 vehicle-treated group, data not shown), treatment of nocodazole (35 uM) resulted in the complete disappearance of transient-outward potassium currents compared to vehicle-treated controls atrial myocytes (Fig. 7). Subtractive peak-sustained current densities at +80 m V were 22.8 ± 3.5 pA/pF and 9.9 ± 2.7 pA/pF in control (n = 8) and nocodazole-treated cells (n = 6) pre-incubated at 37°C, respectively. Quantification of Ito was obtained by subtracting peak currents from sustained currents. When steady state currents densities were subtracted from the data, net peak density +80 mV was 22.8 ± 3.5 pA/pF and 12.6 ± 4.5 for vehicle and nocodazole pretreated myocytes, respectively. This is in disagreement with our previous report, obtained at room temperature, where we observed no alteration in I l 0 current levels between nocodazole and control rat atrial myocytes groups. This may suggest that at physiological temperatures (i.e. 37°C). I t 0 channels in the heart may be downregulated by dynein disruption and nocodazole treatment. 49 ii) Dynamitin Overexpression Downregulates Kv4.2 and Kv4.3 Channels in HEK293 Cells K v 4 .2 underlies l t 0 in rat atrial cardiac myocytes (Bou-Abboud and Nerbonne, 1999). We wanted to test whether the reduction'in I t 0 seen in nocodazole pretreated rat atrial myocytes was due to a defect in the trafficking of K v 4 .2 channels. In H E K 2 9 3 cells stably expressing K v 4 . 2 channels, we tested i f dynein disruption, via p50 overexpression, could alter K v 4 .2 current density levels. K v 4 .2 currents were reduced by almost 50 percent when p50 was overexpressed in these cells, compared to pGFP controls (Fig. 10). Kv-4.2 peak current density was reduced by p50 (n = 7) to 0.34 ± 0.02 nA/pF at +80 m V from 0.63 ± 0.08 nA/pF in controls (n = 12) (Fig. 10A). Kinetics of activation and inactivation kinetics appeared to be unaltered with dynamitin over-expressing, suggesting that dynein disruption reduces the number of functional channels inserted at the cell surface (not shown). Next, we wanted to confirm these results through confocal microscopy. In p50-overexpressing, K v4.2-expressing H E K 2 9 3 cells, a reduction of cell surface K v 4 .2 staining was seen compared to control cells, in which distinct membrane K v 4 . 2 staining was observed (Fig. 10B). However, p50-overexpressing cells contain substantial intracellular K v 4 .2 stores, which in our hands appeared in similar levels as control cells. Thus, this suggests that dynein disruption reduces K v 4 . 2 surface localization, * by possibly disrupting the forward trafficking of K v 4 .2 channels. We hypothesized that other members of the K v 4 . x channel subfamily might behave in a similar maimer to dynein disruption. K v 4 .3 is approximately 70% identical in sequence similarity to K v 4 .2 , thus we decided to test through electrophysiology i f K v 4.3 50 A B -80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) Figure 10. Dynamitin co-expression reduces K v 4 .2 currents and decrease K v 4 .2 staining at the cell surface in H E K 2 9 3 cells. (A) Examples of K v4.2currents and (B) the mean K v 4 .2 current density-voltage relationship measured in a H E K 2 9 3 cell line stably expressing K v 4 .2 with dynamitin (p50) or empty vector (pGFP). Dynamitin overexpression significant reduces current density of K v 4 . 2 (* p < 0.05, ** p <0.01, Student's / test). Cells were depolarized from -80 to +80 m V from a holding potential of -100 m V in 10-mV steps,. Peak current amplitudes were normalized to cell capacitance. 51 Figure 11. Dynamitin co-expression reduces K v 4.3 currents in H E K 2 9 3 cells. (A) Examples of K v 4.3 currents and (B) the mean current density-voltage relationship measured in a H E K 2 9 3 cell line stably expressing K v 4 .3 with dynamitin (p50) or empty vector (pGFP). p50 overexpression significantly attenuates the current density of K v 4.3 (•. p < 0.05, Student's t test). Cells were depolarized from -110 to +80 m V from a holding potential o f -100 m V in 10-mV steps, followed by repolarization to -40 mV. Peak current amplitudes were normalized to cell capacitance. 52 behaves in a similar manner to K v 4 .2 when dynamitin is overexpressed. Like K v 4 . 2 . stably expressing K v 4 .3 channels show a reduction of peak current levels by roughly a half. K v 4.3 peak current density was reduced by dynamitin overexpression (n = 5) to 634.91 ± 168.00 pA/pF at +80 m V from 1216.30 ± 171.65 pA/pF in control cells (n = 4; Fig. 11). Kinetics of activation and inactivation appeared to be unchanged by dynamitin overexpression, suggesting that dynein disruption reduced the number of functional channels inserted at the cell surface. Like K v 4 .2 , this may be caused by an impairment of the forward transport of Kv4!3 channels to the cell membrane. Thus, dynein disruption, by dynamitin overexpression may disrupt the forward trafficking of K v 4 . 2 and K v 4.3 channels in H E K 2 9 3 cells and in addition, disruption of microtubule dependent motors, via nocodazole, also may well disrupt the anterograde movement of l t o-forming channels in rat atrial myocytes. 53 Discussion Impairing Dynein Motor Function Alters the Surface Expression of KJ.5 This study was the first to determine a functional role for the dynein motor complex in mediating the trafficking of K v channels. In this report, interference of the dynein motor complex, via dynamitin overexpression in H E K 2 9 3 cells, increased K v 1 .5 surface expression approximately 2-fold as assayed by electrophysiological, biochemical, and immunofluorescent methods (Choi et al., 2005). Overexpression of dynamitin, to disrupt the dynein motor system, is a common way to study the role of the dynein motor system in the trafficking of various proteins, including ion channels. This method was employed by ourselves (Choi et al., 2005) and Dhani et al. (2005), who demonstrated an interaction of the dynein motor complex and the chloride conducting ion channel, C1C-2, and also showed a similar enhancement of C1C-2 channel surface expression upon dynamitin overexpression in COS7 cells. Not only does p50 overexpression increase the net surface expression of K v 1 .5 channels in H E K 2 9 3 cells, but it also leads to a redistribution of K v 1.5 internal stores (Fig. ID), such that K v 1 .5 is localized preferentially at cellular membranes when the dynein motor system is impaired. This led us to believe that disruption of dynein may increase surface K v l . 5 expression, possibly by increasing the pool of K v l . 5 containing endocytotic vesicles in submembrane compartments. This observation was replicated in nocodazole-treated rat atrial myocytes where a similar pattern of redistribution of K v 1 .5 expression was seen (Fig. 6). Nocodazole depolymerizes microtubules along which the dynein motor tracks. Our results with these myocytes were consistent with evidence previously obtained by us that nocodazole mimics the effects of p50 in p50-transfected K v 1 .5 expressing HEK293 cells, increasing 54 K v l . 5 current density (Choi et al., 2005). That the sustained current in these myocytes was underlain by K v l . 5 was confirmed using ion channel blockers. The sustained current was 4-AP sensitive and was inhibited by the specific K v 1 .5 blocker D M M (Choi et al., 2005). In addition, we have shown an interaction of K v 1 .5 with the dynein motor complex (Choi et al., 2005). Again, similar to Dhani et al. (2003), who also were able to show an in vivo and in vitro interaction between C1C-2 and dynein, using coimmunoprecipitation, we were able to identify an interaction between K v 1 .5 and the dynein intermediate chain, in H E K 2 9 3 cells and in rat cardiac myocytes (Choi et al., 2005). Whether or not this interaction of K v l .5 with dynein is direct remains uncertain and future studies wi l l be needed to clearly map the association of K v l .5 with the dynein motor system. Although a direct association is not necessarily required, it would imply a more direct role of the dynein motor complex in the trafficking of K v l . 5 channels. However, channels may indirectly interact with the retrograde motor by either being co-transported into endocytotic vesicles or by being affected indirectly by another protein that is directly transported by the complex. Several studies investigating the role of dyneins and kinesin on the trafficking of various ion channels suggest a bridging molecule may be required for the interaction of an ion channel with a molecular motor (Setou et al., 2000; Setou et al.. 2002). Therefore it may be more likely that K v 1 .5 interaction with the dynein motor involves a bridging molecule or complex. 55 Protein-Protein Interactions Likely Mediate the Interaction Between Molecular Motors and Ion Channel Substrates We have previously established that the proximal amino-terminal proline-rich domain is important in modulating dynein-mediated trafficking of K v 1 .5 channels (Choi et al.. 2005). We have also previously demonstrated that the intermediate chain of the dynein motor complex and K v 1 .5 are able to coimmunoprecipitate, indicating that K v 1 .5 directly or indirectly interacts with the dynein motor. This may imply that dynein may directly bind to K v 1 .5 channels, although this is unlikely. Other ion channel subunits, such as N R 2 B and GluR2 subunits, have been shown to require an intermediate protein complex to confer binding to their molecular motor kinesin (Setou et al., 2000; Setou et al., 2002). Specific transport of N R 2 B subunit of the N M D A receptor requires the binding of K i l l 7 to the P D Z binding domain of mLin lO, an adaptor molecule that is part of a multi-protein complex, along with N R 2 B (Setou et al., 2000). In addition, the kinesin isoform Kif5b interacts with the A M P A receptor subunit, GluR2, through GRIP-1 , a GluR2-interacting protein (Setou et al., 2002). Thus, it is reasonable that an adaptor protein, similar to mLin lO and GRIP-1 , are involved in mediating the interaction of dynein with K v l .5 and possibly K v 4 . 2 / K v 4.3 channels with kinesin. The dynein motor complex anchors its cargo through the p l 5 0 g l u e d complex and it would be interesting to whether K v l .5 interacts with the dynein motor complex by physically associating with the p l 5 0 g l u e d complex. 56 The IS-Terminal SH3 Binding Domain of Krl.S is Important for Endocytosis of Kvl. 5 Channels Although p50 overexpression prevents dynein from trafficking its cargo in the anterograde direction from the plasma membrane region, this does not imply that normal channel endocytosis and perhaps even recycling do not occur. In HeLa cells, dynamitin overexpression leaves endocytosis intact and the resultant endosomes do recycle to the cell surface (Valetti et al., 1999). Thus normal channel endocytosis and delivery to recycling endosomes may be unaffected by p50 overexpression. Kv-1.5 channels have a relatively short half life (~4hrs; (Takimoto et al., 1993)) and as such, impairments in rates of internalization or recycling may have relatively rapid effects in altering the surface expression of K v 1 .5 channels. Indeed, p50 overexpression results in a redistribution of early endosomes to the cell periphery in COS7 cells (Valetti et al., 1999). This may reflect an accumulation of endosomes that would normally be targeted for degradation or other processing but instead remain proximal to the cell surface as they cannot be further internalized. Thus conceivably, since forward transport of K v 1 .5 is still functional in p50 transfected H E K 2 9 3 cells, channels that would otherwise degrade may instead be recycled to the cell surface, resulting in higher K v l .5 functional expression upon p50 overexpression (Fig. 1). These endosomes may reincorporate into the cell surface by either simple diffusion alone or with the assistance of actin-dependent/microtubule-independent myosin motors (see below). We have demonstrated that K v 1 .5 colocalizes with the early-endosomal marker, E E A 1 , in cycloheximide-treated HEK293 cells (Fig. 2A, C, D) confirming that K v 1 .5 enters the 57 early endosomal pathway. Also, blockade of endocytosis, by the inhibition of dynamin via dynamin inhibitor peptide, enhanced K v 1.5 currents (Fig. 3) to an extent similar to that by p50 overexpression (Fig. IA) and reduced colocalization of E E A 1 - K v l . 5 in HEK293 cells (Fig. 2B, E), thus supporting our hypothesis that K v 1.5 is endocytosed by a dynamin-dependent and further internalized via a dynein-dependent pathway (Choi et al, 2005). Dynein-mediated enhancement of K v 1.5 current densities was abolished when the proline-rich domain of the K v l .5 amino-terminus was removed. The proline-rich domain in K v l . 5 harbors two putative SH3 binding domains; SH3 binding domains have been demonstrated to bind to tyrosine kinases such as Src Kinase (Holmes et al., 1996; Nitabach et al., 2002). Mutation of the first proximal amino-terminal proline-rich domain of K v l . 5 , and not the neighboring distal proline-rich domain, eliminated p50-induced upregulation of K v 1.5 currents in HEK293 cells (Choi et al., 2005). This data suggested to us that the first amino-terminal proline-rich domain was necessary in influencing the interaction between the dynein motor complex and K v 1.5, probably indirectly by way of its importance for endocytosis. This domain may be necessary in anchoring Src kinase (Nitabach et al., 2002), and suggests the possibility that the phosphorylation state of K v 1.5 may be an important factor in promoting dynein-mediated endocytosis of K v 1.5. In human ventricular tissue, K v 1.5 localizes to adhesion zones and has been shown to bind to Src Kinase through coimmunoprecipitation studies (Holmes et al., 1996). Interestingly, mutation of the first proline-rich domain expressed higher current densities than wild type K v l .5 channels (Choi et al., 2005). A potential explanation for this observation is that the 58 signal necessary for removal of K v 1 .5 channels at the cell membrane is absent and channels thus accumulate at the cell membrane leading to higher surface expression levels. Nesti et al. (2004) have shown in K v 1 .2 that dynamin-mediated suppression of the channel was dependent on the presence of a single tyrosine residue in its amino-terminus. It wi l l be interesting in the future to identify the important phosphorylation sites in K v l .5 that might mediate this process. However, studies have observed decreases of K v 1 .5 channel currents when Src kinase was coexpressed in oocytes (Nitabach et al., 2002) and heterologous cell lines (Holmes et a l , 1996). This alternatively may suggest that unidentified protein may bind near this region and increase K v 1 .5 surface expression by inhibiting phosphorylation of K v l .5. This may in turn mask the binding site of Src Kinase, and thereby prevent K v l . 5 internalization into the early endosomal pathway. To date, this question is unanswered and needs to be addressed in the future. Interestingly, other K v channels which lack this proline-rich domain at their amino-termini, such as K v l . 4 , Kv4.2 , and Kv4.3 channels, are downregulated when dynamitin is coexpressed in HEK293 cells (unpublished results, Fig. 10, 11). In addition, when we substituted the K v 1 .4 N-terminus with the N-terminus of K v 1 .5 and coexpressed the chimeric channel with dynamitin, we observed increases in channel current densities upon p50 overexpression (unpublished results). This supports our hypothesis that the proline-rich domain of K v 1 .5 is necessary for the promotion of dynein-mediated endocytosis of K v 1 . 5 . Insights for Anterograde Trafficking of Kvl.5 While anterograde and retrograde trafficking of kinesin and dyneins, respectively, is impaired in nocodazole-treated atrial myocytes, surface expression of K v 1 .5 is 59 increased in our study (Fig. 5, 6). Furthermore, dynein disruption increases K v 1 .5 surface expression with p50 overexpression (Fig. 1). These increases of K v 1 .5 surface expression with dynein disruption and microtubule depolymerization disruption have important implications in our current understanding of the mechanisms that govern forward trafficking of K v l .5. Through an unknown mechanism, K v l .5 is able to traffic to the cell surface when dynein and kinesin motor function is disrupted. Since forward trafficking of K v l . 5 is intact our study, kinesins are unlikely to be the sole mediators of K v 1 .5 forward transport. Studies have shown that inhibition of dyneins can influence the motility of kinesin motor, and vice versa (Hamm-Alvarez et a l , 1993; Valetti et al., 1999; Deacon et al., 2003); i.e., dynein inhibition may inhibit kinesin function in turn. The dynactin complex associated protein, p l 5 0 g l u e d , which links the vesicular cargo to its molecular motor, has previously been identified to influence kinesin-based motility of at least 1 kinesin isoform (Deacon et al., 2003). Thus, there remains the possibility that forward traffic of K v l .5 in nocodazole pretreated cells is transported to the cell membrane by a non-microtubule dependent route, Another distinct possibility is that K v 1 .5 is trafficked in the anterograde direction by the aid of the actin-dependent myosin motor, Myosin V , which has been demonstrated to assist in long-range kinesin-driven anterograde transport of vesicular cargo in neurons (DePina and Langford, 1999). Furthermore, simple diffusion of K v l .5 may be sufficient to traffic K v l .5 to the cell surface when the microtubule cytoskeleton is absent. In this case, K v 1 .5 may diffuse within the cell membrane by binding to a plasma membrane-associated high-affinity K v 1 .5 anchoring protein. However, the mechanism by which K v 1 .5 channels are trafficked in the anterograde direction by molecular motors is unclear at present and more work needs to 60 be completed to understand the precise mechanisms regulating anterograde transport of K v l . 5 in cardiac cells. Disruption of Microtubule-Based Motors Increases IKI, But Does Not Alter Iw Currents in Rat Atrial Myocytes at 20 °C Microtubule-depolymerization drugs, such as nocodazole disrupt microtubules by binding to S-tubulin and prevent the formation of interchain disulfide linkages and therefore in turn, block the motility of microtubule-based motors dynein and kinesins in cells. We have identified that I K S U S current densities are enhanced in nocodazole pretreated rat atrial myocytes at 20°C (Fig. 5) and the increases in I K S U S , upon nocodazole pretreatment are inhibited by application of a K v1.5-specific blocker D M M (Choi et al., 2005). Thus, it is highly likely that K v l .5 surface expression is increased in these myocytes. However, we were surprised to notice concurrent increases in inward currents at potentials negative to the resting membrane potential of myocytes pretreated with nocodazole at 20°C. This suggested to us that 1) an inward-rectifying potassium current were enhanced in these cells and/or 2) a chloride conductance was increased in nocodazole treated cells. Although studies were not earned out to identify which types of currents were upregulated at potentials negative to the resting membrane potential (approximately -70 to -80 mV in rat atrial myocytes (Nattel. 2003)), we sought to determine i f Kj r 2.1, a major component of I K I currents in the heart, would show similar effects with nocodazole pretreatment and p50 overexpression in heterologous cell systems. In Itk- cells, we have identified through electrophysiology that both p50 overexpression and nocodazole pretreatment, do not alter the expression levels of K.;r2.1 61 in these cells (Fig. 8, 9). A distinct possibility is that another Kj r2.x channel is responsible for upregulation of inward currents below the R M P of myocytes. Possibly, Kj,-2.2, which heterotetramerizes with K.jr2.1 channels in cardiac cells, may be responsible for the upregulation of these currents at 20°C. However, this hypothesis has not been tested and the possibility of a chloride conductance increasing upon these conditions remains. However, when the experiments were repeated at more physiological temperatures (i.e. 37°C), we observed vastly different results. We chose this temperature, as it is a better reflector of the physiological conditions of the heart. When pretreatment of nocodazole was completed at 37°C, we observed no differences in inward currents below the R M P of rat atrial myocytes suggesting that temperature was an important factor determining this response (Fig. 7). Since nocodazole pretreatment in Itk- cells was completed at 37°C, this correlates well with our report that K.u2.1 channel current densities are unaltered with the microtubule depolymerization drug, nocodazole (Fig. 9). One kinetic study concluded that the equilibrium binding K ( i values of nocodazole and (3-tubulin were identical at both 25°C and 30°C, suggesting that altering the pretreatment temperature of nocodazole has no overall effect of the ability of the daig to promote microtubule depolymerization (Head et al., 1985). Therefore, we cannot conclude that altering the incubation temperature of nocodazole in myocytes alters the ability of the drug to depolymerize microtubules. Moreover, the function of many proteins operates differently depending on temperature, and therefore this may help explain the differences we observed when recording from nocodazole pretreated atrial cells at core body and room temperature. 62 Most long-range trafficked proteins, including ion channels, are trafficked by dynein and kinesin motors. Since Kj r2.1 channel current density is unaltered in p50-transfected and nocodazole treated Itk- cells, this may suggest that disruption of the dynein motor system, via p50 overexpression, and disruption of the microtubule tracks of which dynein and kinesins rely on, via nocodazole pretreatment, may equally effect both forward and retrograde trafficking of Kj r2.1 expressing cells in heterologous cell systems. In nocodazole treated Itk- cells, dynein and kinesin function is impaired and thus forward and reverse transport of K, r2.1 by microtubule-dependent motors is absent. Our results support this explanation as we observed no changes in Kj,2.1 (Fig. 9) and I K i current density (Fig. 7) with nocodazole pretreatment. In addition, we observed no alterations in the current density of Kj ,2 , l channels in p50-transfected Itk- cells (Fig. 8). Again, dynein function has been shown to be coordinated with kinesin motility (Hamm-Alvarez et al., 1993; Valetti et a l , 1999; Deacon et al., 2003), and as such disruption of the dynein motor system in this study may impair kinesin function as well . In addition, dynein disruption is known to play a role in the forward E R to Golgi trafficking of proteins (Hamm-Alvarez et al., 1993; Valetti et al., 1999; Presley et al., 2002). This would suggest that some of the forward trafficking of K; r2.1 is impaired, along with retrograde transport in p50 overexpressed Itk- cells. However, only electrophysiological methods were used to explore the effects of dynein and microtubule disruption on IKI and K , r 2 . 1 currents and more biochemical assays need to be conducted to fully support the hypothesis that dynein disruption and microtubule disruption impair, in equal magnitudes, both the anterograde and retrograde trafficking of Kj,2.1. 63 Microtubule Depolymerization Downregulates It0 in Rat Atrial Myocytes We have previously shown that pretreatment of nocodazole at room temperature does not affect I t 0 currents in rat atrial myocytes (Figure 4;(Choi et al., 2005)). However, when similar experiments were conducted at 37°C, we noticed a reduction or, in the majority of cases (4 out o f 6 cells), an abolishment of I t 0 in nocodazole pretreated rat atrial myocytes (Figure 6). This was an interesting set of results when compared to our earlier study, which showed that 1,0 currents were unaffected by nocodazole treatment in rat atrial myocytes at 20°C (Fig. 5; (Choi et al., 2005)) suggesting that a temperature-controlled factor may determine these two different results seen at different temperatures. Another possible explanation is that like K v 1 .5 (Choi et al., 2005), channels that form I t 0 , i.e., K. v4.x channels, are present as a storage pool in submembrane compartments or vesicles. But unlike K v 1 . 5 , this storage pool of Kv4 .x channels is smaller such that disruption of dynein, via nocodazole, does not significantly alter the channel density of K v 4 . x channels. But rather, a likely explanation is that the anterograde (i.e. kinesin-based motility) secretory trafficking of K v 4 . x channels is impaired, leading to a reduction of K v 4 . x channels at the cell membrane. Also, degradation of K v 4 . x channels, through non-microtubule dependent routes, may be accelerated at 37°C versus at 20°C. This may explain why at 37°C, I l 0 is downregulated. and in the majority of cells, abolished upon nocodazole pretreatment when compared to incubation of the drug at room temperature (Fig. 5, 7). These are interesting theories that wi l l require more vigorous testing in the future. 64 Disruption of Forward Trafficking ofKv4.2 and Kv4.3 Channels Upon Dynein Inhibition Since l t 0 is largely underlain by K v 4 .2 in rat atrial myocytes (Bou-Abboud and Nerbonne, 1999), we predicted that the reduction in A-type currents seen in nocodazole-treated rat atrial myocytes was due to a reduction in K v 4 .2 surface expression. We were very interested by the opposing results observed with the response of K v 4 . 2 (Fig. 10) and K v 4.3 (Fig. 11) to dynamitin overexpression in HEK293 cells compared to K v 1 .5 channels, which surface expression of which increases with dynamitin overexpression (Fig. 1). Dynein disruption results in a rough halving of K v 4 .2 and K v 4 .3 channel current densities and reduces the intensity of K v 4 .2 at the cell membrane in immunofluorescent studies (Fig. 10, 11). This correlates well with our myocyte data as we observed a reduction of I t 0 current densities in rat atrial cells. The fact that functional expression of K v 4 . 2 , K v 4 . 3 , and K v 1 . 4 (Fig. 10, 11; unpublished results) channels responds to p50 overexpression in a manner opposite to that of K v 2.1 (unpublished results), K v 3.1 (unpublished results) and K v 1 .5 (Choi et al., 2005) is extremely interesting. Since dynein and kinesin motor activities are coordinated, perhaps disruption of one motor may impair the motility of the opposite microtubule-dependent motor (Hamm-Alvarez et al., 1993: Valetti et al., 1999; Deacon et al., 2003). Unlike K v 1 . 5 , K v 4 .2 and K v 4 .3 channels may have a greater dependency on kinesin based motors in regulating their surface expression. This is a likely explanation, as recently Chu et al. (2006) report that K v 4 . 2 trafficking to the cell surface of rat cortical neurons is dependent on a single kinesin motor, K i f l 7. However, K i f l 7 is a neuronal specific 65 kinesin motor, thus indicating that another kinesin motor expressed in HEK293 cells and in rat atrial myocytes may be involved in driving the forward transport of K v 4 . 2 and K v 4 . 3 channels to the cell surface. Another possibility is that the forward E R to Golgi trafficking of K v 4 . 2 and K v 4 .3 containing cargo requires dynein. With respect to K v 1 .5 and C1C-2 (Dhani et al., 2003) trafficking, forward E R to Golgi trafficking may not be mediated in a dynein dependent manner. Although most forward transport of secretory proteins along the microtubule cytoskeleton from the E R to Golgi requires the direct interaction between dynactin, a major component of the dynein motor complex, and COP11 coat proteins (Vaughan, 2005; Watson et al., 2005), previous studies have shown some support for a COPII-independent anterograde E R - G o l g i trafficking pathway (Fatal et al., 2002). in the future, it w i l l be interesting to understand which domains within K v 4 .2 and K v 4 .3 channels influence the behavior of dynein-mediated suppression of these currents in mammalian cells and in cardiac myocytes. Certainly, it wi l l be of interest to determine whether dynein disruption can produce similar effects when K C h i P ' s , a K v 4 . x interacting protein known to influence channel trafficking and alter its biophysical properties (Birnbaum et al., 2004), are coexpressed along with dynamitin. K C h i P ' s are known to interact with the N-terminus of K v 4 . x channels (An et al., 2000), and as such this experiment may serve to identify domains within K v 4 . x channels that interact with microtubule-dependent molecular motors. Physiological Significance of Distinct Trafficking Routes Employed by KY Channels in the Heart 66 It is interesting that dynein disruption and interference with microtubule assembly can result in extremely opposite effects on the surface expression of K v 1 .5 and members of the Shal-related K v 4 . x subfamily. This may suggest that these two channels are trafficked differently from one another. We have recently observed that the Shaker-related rapidly inactivating channel, K v 1 .4 , is downregulated by dynamitin overexpression in HEK293 cells (unpublished), whereas delayed- rectifying potassium channels, K v 3.1 (unpublished) and K v 2.1 are upregulated (unpublished). It is interesting that rapidly inactivating K v channels (K v 4.2, K v 4 . 3 , K v 1.4) are downregulated, while delayed rectifying K v channels (K v 1.5 , K v 3 . 1 , K v 2.1) are upregulated when the dynein motor complex is disrupted. If the mechanism by which dynein regulates K v 3.1 and K v 2.1 surface expression is similar to K v 1 .5 , it may suggest that cells may be able to compensate for increased cellular demand of these channels at a faster rate than channels that have last inactivating kinetics (K v 4.2, K. v4.3, and K v 1.4) . These channels contribute to the cardiac action potential at later phases than K v 4 . 2 , K v 4 . 3 , or K v 1 .4 channels, which play an important role in the earlier portion of the cardiac action potential profile. Cardiac cells may choose to alter the expression levels of delayed rectifying K v channels to reduce/increase action potential durations, thereby altering their excitability, rather than depending on ion channels that contribute to the early phase of action potential. Whatever the mechanism, this would have significant effects in altering cardiac excitability. Literature has shown that cardiac disease is accompanied by electrical remodeling and furthermore, structural remodeling, which is thought to include the rearrangements of the cytoskeleton. It wi l l be interesting to learn whether modification of the cellular cytoskeleton interferes with the trafficking of cardiac ion channels, possibly by 67 interfering with the ability of ion channels anchoring with their respective molecular motor(s). Conclusions In summary, this thesis has established a role for the dynein motor complex in altering the surface expression of voltage-gated K f channels expressed in the heart. We have demonstrated that dynein and microtubule depolymerizing agents can roughly double the surface expression of K v 1 .5 in mammalian heterologous cell expression systems and similarly in cardiac myocytes, possibly implicating a role for the dynein motor in mediating K v 1 .5 internalization through the early endosomal pathway. In contrast, we have shown that microtubule assembly inhibitors lead to downregulation of I T 0 currents in rat atrial myocytes and furthermore, dynein disruption reduces the surface expression of K v 4 .2 and K v 4 .3 channel currents by approximately one-half. Given the report that K v 4 . 2 anterograde trafficking is mediated by a single kinesin isoform in cortical neurons (Chu et al., 2006), we hypothesize that interference of dynein motor, via p50 overexpression and nocodazole treatment, may impair the ability of kinesin motors to drive the forward transport of K v 4 . x channels to the cell surface. Lastly, we have found no effect of microtubule depolymerization in altering the surface expression of IKI currents, and similarly when the dynein motor complex, via p50 overexpression and nocodazole, was disrupted in Kj,2.1 expression Itk- cells. Insensitivity of I K I and Kj,2.1 currents to dynein disruption and microtubule-depolymerization drugs may suggest an equal dependence of kinesin and dynein in mediating the trafficking of Kj r2.1 channels to cellular membranes. Overall, this thesis identifies variable functional dependencies of the 68 d y n e i n m o t o r i n m e d i a t i n g t h e t r a f f i c k i n g o f c a r d i a c p o t a s s i u m c h a n n e l s a n d m a y h a v e i m p o r t a n t i m p l i c a t i o n s i n o u r u n d e r s t a n d i n g t h e m e c h a n i s m o f h o w t r a f f i c k i n g o f c a r d i a c p o t a s s i u m c h a n n e l s i s a c h i e v e d . 69 Bibliography 1. Abdelhadi,R.H., Chung,M.K. , and Van Wagoner,D.R. (2004). New hope for the prevention of recurrent atrial fibrillation. Eur. Heart J 25. 1089-1090. 2. Al lan .V.J . , Thompson,H.M., and M c N i v e n , M . A . (2002). Motoring around the Golgi . Nat Cell B io l 4, E236-E242. 3. A n , W . 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