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Modulation of cardiac voltage-gated potassium channel functional expression by Kinesin I and small GTPases Cheng, Yvonne 2010

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      MODULATION OF CARDIAC VOLTAGE-GATED POTASSIUM CHANNEL FUNCTIONAL EXPRESSION BY KINESIN I AND SMALL GTPASES   by  Yvonne Cheng  B.Sc., The National University of Singapore, 2006     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE   in   The Faculty of Graduate Studies  (Pharmacology and Therapeutics)         THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)   June 2010    © Yvonne Cheng, 2010   ii ABSTRACT Functional expression of voltage-gated potassium (Kv) channels in the plasmalemma is essential for repolarization phase of the cardiac action potential. Therefore, changes in Kv channel plasmalemmal expression can impact cardiac action potential duration and thereby result in arrythmias. The expression of these Kv channels is modulated by a number of different mechanisms, and among these, the most important and potent one is the regulation of the number of channels in the plasma membrane through modulation of channel trafficking. The trafficking process of Kv channel in cardiac background is poorly understood. The purpose of the studies presented in this thesis was to identify the specific kinesin isoform that is required for cardiac Kv1.5 channel forward trafficking and to investigate the roles of small GTPases in the trafficking of Kv4.2 channels in adult rat ventricular myocytes. Overexpression of wild type Kif5b increased the Kv1.5-conducted current and this increase was dependent on Golgi function; a 6 h treatment with Brefeldin A reduced Kv1.5 currents to control levels in Kif5b-overexpressing cells. Expression of dominant negative isoform of Kif5b prior to induction of Kv1.5 in a tetracycline inducible system blocked surface expression of the channel in both HEK293 cells and H9c2 cardiomyoblasts. These data confirmed the requirement for Kif5b for forward trafficking of newly synthesized Kv1.5 channels. The involvement of several Rab GTPases as well as Sar1 in the trafficking of an endogenous cardiomyocyte potassium channel has also been established. Kv4.2 traffics out of the cardiac endoplasmic reticulum via a conventional pathway involving Sar1 and Rab1 and its trafficking to the sarcolemma is enhanced by overexpression of wild type Rab11. Internalization of the channels is dependent upon Rab5 function and block of Rab4 somehow also inhibits that   iii internalization. The internalized channels, if not recycle back into plasma membrane will be degraded via the proteasomal degradation pathway. Together, these studies enhance our knowledge of the players involved in the trafficking pathway of Kv channels in cardiomyocytes. In particular, the roles of kinesin and several small GTPases in regulating cardiac Kv channel plasmmalemmal expression through channel trafficking modulation.                     iv TABLE OF CONTENTS Abstract ......................................................................................................................................ii Table of contents........................................................................................................................iv List of figures............................................................................................................................vii List of abbreviations...................................................................................................................ix Acknowledgements....................................................................................................................xi Co-authorship statement............................................................................................................xii Chapter 1    General introduction.................................................................................................1      1.1       Overview....................................................................................................................1      1.2       Cardiac voltage-gated potassium channels ..................................................................2          1.2.1     Structure of voltage-gated potassium channels .......................................................2          1.2.2     Function of voltage-gated potassium channels in the heart .....................................4          1.2.3     Cardiac voltage-gated potassium channels and diseases .........................................5      1.3       Small GTPases in intracellular trafficking...................................................................7           1.3.1    General structure and activity of small GTPases.......................................................7           1.3.2     Biosynthesis and transport into the ER ................................................................10           1.3.3     ER-Golgi shuttle .................................................................................................11           1.3.4     Through the Golgi and on to plasma membrane ..................................................13           1.3.5     Internalization.....................................................................................................14           1.3.6     Slow and fast recycling of endocytosed channels ................................................15           1.3.7     Degradation of endocytosed channels..................................................................16      1.4       Cytoskeletal motors ..................................................................................................18           1.4.1     Structure of cytoskeletal motors ..........................................................................18           1.4.2     Roles of cytoskeletal motors in intracellular transport .........................................20      1.5       Research objectives ..................................................................................................22      1.6       References ................................................................................................................24 Chapter 2     Kif5b is an essential forward trafficking motor for the Kv1.5 cardiac potassium channel ................................................................................................................35      2.1        Introduction..................................................................................................................35      2.2        Materials and methods .............................................................................................36           2.2.1     Cell preparation and transfection.........................................................................36           2.2.2     Plasmid constructs ..............................................................................................36           2.2.3     Electrophysiological experiments and solutions ..................................................37           2.2.4     Imaging...............................................................................................................37           2.2.5     Tetracycline induction experiments.....................................................................39           2.2.6     Data statistics......................................................................................................39      2.3        Results.....................................................................................................................39           2.3.1     Kv1.5 functional expression is significantly increased by overexpression of Kinesin I (Kif5b)................................................................................................39   v           2.3.2     Kif5bWT and Kif5bDN increase in Kv1.5 current densities by different mechanisms .......................................................................................................41           2.3.3     Enhancement of Kv1.5 current density by Kif5bWT requires intact Golgi                        function...............................................................................................................43           2.3.4     Kif5b function is specifically required for Kv1.5 delivery to the plasma                        membrane ...........................................................................................................44      2.4        Discussion ...............................................................................................................46           2.4.1     Kif5b is essential for forward trafficking of Kv1.5 ..............................................46           2.4.2     Kv1.5-kinesin interaction ....................................................................................47           2.4.3     Microtubule transport as regulator of functional expression.................................47      2.5       References ................................................................................................................57 Chapter 3     Trafficking of endogenous Kv4.2 in adult ventricular myocytes ............................59      3.1        Introduction .............................................................................................................59      3.2        Materials and methods .............................................................................................62           3.2.1     Myocyte isolation and transfection......................................................................62           3.2.2     HEK 293 cell preparation and transfection ..........................................................63           3.2.3     Plasmid constructs ..............................................................................................63           3.2.4     Electrophysiology ...............................................................................................64           3.2.5     Imaging...............................................................................................................65           3.2.6     Kv4.2 colocalization assay ..................................................................................65           3.2.7     Tetracycline-induction experiments.....................................................................66           3.2.8     Data statistics......................................................................................................66      3.3        Results.....................................................................................................................66           3.3.1     Lipofectamine 2000-mediated transfection of ventricular myocytes ....................66           3.3.2     Sar1 involvement in Kv4.2 trafficking.................................................................67           3.3.3     Rab1DN significantly decreases Kv4 functional expression in cardiac                        myocytes.............................................................................................................68           3.3.4     Overexpression of Rab11 increases trafficking of newly synthesized Kv4.2 to the sarcolemma........................................................................................................69           3.3.5     Rab5 is involved in Kv4.2 internalization............................................................70           3.3.6     Rab4 involvement in Kv4.2 trafficking ...............................................................71           3.3.7     Kv4.2 degradation likely occurs in the proteasome..............................................73      3.4        Discussion ...............................................................................................................74      3.5        References ...............................................................................................................88 Chapter 4    Conclusion and recommendations for future work..................................................92      4.1        Overall summary and conclusions............................................................................92      4.2        Players involved in cardiac Kv channel forward trafficking .....................................95           4.2.1     Kinesin isoform Kif3, Kif5 or Kif17?..................................................................95           4.2.2     Sar1 and Rab1-dependent or not?........................................................................96           4.2.3     Rab11 mediates forward trafficking or slow recycling? .......................................98      4.3        Players involved in cardiac Kv channel retrograde trafficking..................................99           4.3.1     Kv channel internalization...................................................................................99           4.3.2     Rab4 and Rab5..................................................................................................100           4.3.3     Lysosomal or proteasomal degradation?............................................................101      4.4        Future directions ....................................................................................................102      4.5        Concluding remarks...............................................................................................103   vi      4.6        References .............................................................................................................107  Appendix 1    UBC animal care certificate...............................................................................110                    vii LIST OF FIGURES   Figure 1.1 Schematic representation of Kv channel structure.......................................................3   Figure 1.2 Outward, repolarizing currents carried by Kv channels that underlie the repolarizing phase of atrial (left panel) and ventricular (right panel) action potential .....................5   Figure 1.3 Rab GTPases cycle between a GDP-bound and a GTP-bound conformation...............9   Figure 1.4 Small GTPases implicated in the secretory pathway of membrane protein ................18   Figure 2.1 Transfections with Kif5bWT and Kif5bDN similarly affect Kv1.5 functional expression................................................................................................................47   Figure 2.2 Differential interactions of Kif5bWT and Kif5bDN with direct inhibitions of retrograde trafficking ...............................................................................................49   Figure 2.3 Brefeldin A treatment prevents increase in Kv1.5 currents associated with Kif5bWT overexpression.........................................................................................................51   Figure 2.4 Kif5bDN expression prevents functional expression of newly synthesized Kv1.5 .....52   Figure 2.5 Kif5bDN blocks surface expression of newly synthesized Kv1.5..............................54   Figure 3.1 Lipofectamine-mediated transfection of adult ventricular myocytes..........................75   Figure 3.2 Normal expression of Ito requires intact Sar1 function ..............................................76   Figure 3.3 Normal expression of Ito requires intact Rab1 function .............................................77   Figure 3.4 Expression of an inducible Kv4.2 in HEK293 cells is significantly reduced by coexpression of Rab1DN and Sar1DN .....................................................................78     viii Figure 3.5 Overexpression of Rab11WT substantially increases Ito in transfected ventricular myocytes .................................................................................................................79   Figure 3.6 Brefeldin A treatment prevents Rab11WT overexpression effect on Ito .....................80   Figure 3.7 Interference with Rab5 function increases Ito in transfected ventricular myocytes .....81   Figure 3.8 Rab4DN expression also increases Ito in transfected ventricular myocytes ................82   Figure 3.9 Internalized Kv4.2 colocalizes with Rab4 in ventricular myocytes............................83   Figure 3.10 Rab7 function appears irrelevant to maintenance of endogenous Ito ........................84   Figure 3.11 Incubation with proteasome inhibitor significantly increases the magnitude of Ito ...85   Figure 4.1 Functional expression of Kv1.5 and Kir2.1 is dependent on Rab1...........................103   Figure 4.2 Functional expression of Kv1.5 and Kir2.1 is dependent on Sar1............................104   ix LIST OF ABBREVIATIONS  APD   action potential duration ATP   adenosine-5’-triphosphate BFA                            brefeldin A BSA   bovine serum albumin CFTR   cystic fibrosis transmembrane conductance regulator C-terminus  carboxy terminus DABCO                      1,4-diazabicyclo2.2.2octane DIP   dynamin inhibitory peptide DN   dominant negative EEA1    early endosome antigen 1 protein EGFP   enhanced green fluorescent protein EGTA   ethylene glycol tetraacetic acid ENaC   epithelial sodium channel ER   endoplasmic reticulum ERES   endoplasmic reticulum exit sites FBS                             fetal bovine serum GAP   GTPase-activating protein GDP   guanosine-5’-diphosphate GEF   guanine nucleotide-exchange factor GFP   green fluorescent protein GRIP   glutamate receptor interacting protein GTP   guanosine-5’-triphosphate HA   hemagglutinin antigen hERG   human ether-a-go-go channel IK   delayed outwardly rectifying potassium currents IKr   rapidly activating delayed rectifying potassium currents IKs   slowly activating delayed rectifying potassium currents IKur   ultra rapidly activating delayed rectifying potassium currents Ito   transient outward potassium currents ITS                              Insulin-Transferrin-Selenium-A Supplement KATP   ATP-sensitive potassium channel KChIP   potassium channel interacting protein Kv   voltage-gated potassium channel LQTS   long QT syndrome MiRP   minK-related peptides N-terminus  amino terminus p50   dynamitin PBS   phosphate-buffered saline PKC   protein kinase C RILP   Rab7-interacting lysosomal protein RT   room temperature SEM   standard error of the mean SH3   src homology 3 domain SNAP25  synaptosomal-associated protein 25 SNARE  soluble N-ethylmaleimide-sensitive factor attachment protein receptors T1   tetramerization domain   x TdP   torsades de pointes arrhythmias TGN   trans-Golgi network VSVG   vesicular-stomatitis virus G-protein WT   wild type   xi ACKNOWLEDGEMENTS  First and foremost, I would like to express my sincere gratitude to my supervisor Dr. David Fedida for his excellent scientific guidance, constant support, and understanding throughout the course of this degree. I am also grateful to my supervisory committee members, Dr. James McLarnon and Dr. Stephanie Borgland, for all their insights and suggestions on my projects.  I would also like to extend my gratitude to my lab-mates and friends, who not only have committed valuable time and effort in helping me with the projects, but also have made the lab a warm and pleasant working place. I also owe a special thanks to Dr. David Steele and Dr. Zhuren Wang for sharing their invaluable knowledge and experience with me, as well as their words of encouragement and endless patience which have helped me tremendously. My particular thanks also go to Tiantian Wang for her understanding and support, and most of all, for being an extraordinary friend. Finally, I would like to thank the most important people in my life, my parents and my sister for their unconditional love, encouragement and continual support.                   xii CO-AUTHORSHIP STATEMENT  Chapter 2: Kif5b is an essential forward trafficking motor for the Kv1.5 cardiac potassium channel Alireza Dehghani Zadeh and David Steele developed the experimental design and protocols, I offered suggestion on the design of this study. Imaging data were collected by Alireza Dehghani Zadeh; electrophysiological experiments were performed by me, Hongjian Xu and Nathan Wong; the western blot experiment was performed by Charitha Goonasekara. All the electrophysiological data were analyzed by me, Hongjian Xu, Nathan Wong, and Zhuren Wang. David Steele contributed to the writing of the manuscript which was revised by David Fedida. I did not write but offered editing and feedback on this chapter.  Chapter 3: Trafficking of endogenous Kv4.2 in adult ventricular myocytes David Steele, Tiantian Wang and I developed the experimental design and protocols. Tiantian Wang and I performed all the electrophysiological experiments and analyzed all the electrophysiological data; Tiantian Wang and Charitha Goonesekara performed the immunocytochemistry assays. David Steele, Tiantian Wang and I wrote the manuscript which was revised by David Fedida.  1 CHAPTER 1 GENERAL INTRODUCTION 1.1 Overview Ion channels are pore-forming proteins that allow the selective flow of specific ions across the lipid bilayer to maintain and/or modify the electrochemical gradient across the membrane. Excitable cells such as neurons, muscle cells and endocrine cells have developed the ability to generate action potentials by exploiting exquisite sensitivities of the various channels to the voltage differences across the membrane as well as the selectivity of ion channels, resulting in rapid production and propagation of electrical signals to neighbouring cells. Functional ion channels are essential for important biological responses such as neurotransmitter release at synapses, cardiac muscle contraction and endocrine secretion.  In the heart, voltage-gated potassium channels (Kv channels) are responsible for the repolarization phase of the cardiac action potential. It is not hard to imagine how changes in Kv channel functional expression can impact cardiac action potential duration and thereby result in arrythmias. The activity of an ion channel is dependent on its functional membrane expression, which may be modulated by phosphorylation and other modifications, by association with accessory subunits or, perhaps most potently, by regulation of actual channel numbers in the plasma membrane through modulation of channel trafficking. Thus, ion channel surface expression is regulated by forward and retrograde trafficking of the channels, which includes insertion of newly synthesized channels, endocytosis and recycling of endocytosed channels, as well as degradation of channel proteins.  While the trafficking pathway of Kv channels in neurons has been studied extensively (Lai and Jan, 2006), little is known about that of cardiac Kv channels. To gain insights into the Kv channel trafficking pathways and the players involved in these processes, I have conducted two general investigations, the first investigating the molecular motor required for the trafficking of newly synthesized Kv channels from the endoplasmic reticulum or Golgi apparatus to the   2 plasma membrane and the second investigating the regulation of forward and retrograde trafficking of cardiac potassium channels by small GTPases.  1.2 Cardiac voltage-gated potassium channels  1.2.1 Structure of voltage-gated potassium channels   Kv channels belong to a large family of ion channel comprising 40 members that are further divided into 12 subfamilies. All channels in this family share the same basic structure, a tetramer of four α subunits with each contributing to the central pore that through which potassium ions flow and each containing a voltage sensor responsible for the voltage-dependent gating of that potassium-selective pore. A Kv channel α subunit contains six transmembrane domains, S1 to S6, as well as intracellular amino and carboxy temini that exist in the cytoplasmic aqueous phase (Packer et al. 2000; Wray 2000). Some Kv channel α subunits associate with auxiliary subunits, called the β subunits, associating in a α4β4 stoichiometry (Pongs et al. 1999). The association of β subunit modifies the kinetics and voltage dependence of gating of several Kv channels (Li et al. 2006).   The architecture of Kv channels allows them to perform three interrelated functions, namely voltage sensing, gating and ion permeation. The S1-S4 transmembrane helices confer the voltage sensing mechanism that detects changes in the voltage across the plasma membrane. In addition, these helices provide necessary structural elements for channel gating, which is a regulation of opening and closing of the pore. The pore itself is comprised of elements of the S5 helices, P-loops and S6 helices of the four α subunits. This pore enables fast and highly selective movement of potassium ions across the membrane.   The N- and C-terminal domains of Kv channels also play important roles in channel function. The N-terminal region of Kv channels generally consists of a disordered soluble   3 domain sometimes capped by a positively-charged ball important in the fast inactivation of some channels, a T1 tetramerization domain, and a linker between the T1 domain and the transmembrane body of the channel. The T1 domain serves several roles, including subunit assembly within families (Lu et al. 2001; Zerangue et al. 2000), as a platform for binding of the auxiliary β subunits (Gulbis et al. 2000) and in modulation of channel gating (Cushman et al. 2000; Minor et al. 2000). The C-terminal region of Kv channels have also been implicated in channel gating (Shamgar et al. 2006), channel assembly (Schmitt et al. 2000), Kvβ subunit binding (Cui et al. 2001) and trafficking (Etxeberria et al. 2007), although the precise roles of this domain in most of these processes remain poorly understood. Figure 1.1    Schematic representation of Kv channel structure. A Kv channel α subunit is composed of six membrane-spanning segments (S1 to S6), with the fourth membrane-spanning segment (S4, shown in red) containing positively-charged residues at approximately every third position. The residues between S5 and S6 (P-loop) form the ion selective pore. K+ channel α subunit co-assemble to form a tetrameric channel composed of four identical subunits (homotetramer).      4 1.2.2 Function of voltage-gated potassium channels in the heart   Kv channels are widely distributed across all cell types, although there is very substantial variation in expression of specific channel subtypes. Kv channels are the primary determinants of cardiac action potential repolarization and have substantial electrophysiological and functional diversity in the heart (Barry et al. 1996). Cardiac Kv channels display differences in pharmacological sensitivities and time- and voltage- dependent properties (Barry et al. 1996), the latter of which are essential for their differential functions. Two fundamental types of repolarizing currents have been discerned among the voltage-gated potassium channels, transient outward potassium currents (Ito) and delayed outwardly rectifying potassium currents (IK) (Barry et al. 1996).   The transient outward potassium current, Ito, exhibits rapid activation and rapid subsequent inactivation upon membrane depolarization to potentials positive to approximately - 30 mV during the early repolarization phase (Phase 1) of cardiac action potentials. The Kv channels that conduct Ito in rat and human are Kv4.2 and Kv4.3, respectively (Johns et al. 1997; Yeola et al. 1997).   The delayed outwardly rectifying potassium currents (IK) contain at least three components, IKur, IKr, and IKs, named after the distinctive activation time courses of the delayed rectifiers: ultrarapid, rapid, and slow, respectively. These currents underlie the rapid late repolarization phase (Phase 3) back to the diastolic potential of cardiac action potentials. Kv1.5 conducts the atrial-specific IKur in mouse, rat, dog, and human (Fedida et al. 1993; Snyders 1999). The Kv channels that conduct IKs in human are a complex of α subunit hERG and β subunit MiRP (Trudeau et al. 1995; Sanguinetti et al. 1995); while KvLQT1 channels when co- assembled with minK, another Kvβ subunit, form the channels that conduct IKr in human (Sanguinetti et al. 1996; Barhanin et al. 1996).   5          Figure 1.2    Outward, repolarizing currents carried by Kv channels that underlie the repolarizing phase of atrial (left panel) and ventricular (right panel) action potential. Ito transient outward current; IKur ultra rapidly activating delayed rectifying potassium current; IKr rapidly activating delayed rectifying potassium current; IKs slowly activating delayed rectifying potassium current. IKur is present in atria only. Phase 0, rapid depolarization; phase 1, rapid early repolarization phase; phase 2, slow repolarization phase; phase 3, rapid late repolarization phase; phase 4, resting membrane potential.    1.2.3 Cardiac voltage-gated potassium channels and diseases   Because of their roles in membrane repolarization and their impacts on action potential duration (APD), altered activity of any potassium current may contribute to arrhythmogenesis. A gain-of-function mutation in a Kv channel causes increased potassium currents, shortens APD and thereby facilitates potentially destabilizing re-entry into the depolarization phase of the next action potential. One study has shown that rapid atrial pacing increases Kv1.5 mRNA levels in   6 rats, which might contribute to the shortening of atrial refractoriness and the onset of atrial fibrillation (Yamashita et al. 2000). Kv1.5 conducts IKur which is specific to the atrium in humans and increasing Kv1.5 expression would increase IKur current density thereby shortening atrial refractoriness and favouring the re-entry of multiple wavelets specifically in the atria. Therefore, it is unsurprising that attention has been focused on IKur as a target for development of new antiarrhythmic drugs to prevent atrial fibrillation and flutter without a risk of ventricular proarrhythmia (Brendel et al. 2002).   On the other hand, a decrease in potassium currents due to a loss-of-function mutation in a Kv channel can prolong ventricular APD and result in Long QT Syndrome (LQTS). LQTS is a heart disorder characterized by prolongation of the time between the start of the Q wave and the end of the T wave in the heart’s electrical cycle. LQTS predisposes the heart to early after- depolarizations that may trigger torsades de pointes arrhythmias (TdP), which can deteriorate into ventricular fibrillation causing sudden cardiac death (Tomaselli et al. 1994). LQTS can be generated by multiple different mechanisms. It can be inherited as an autosomal dominant (Romano-Ward syndrome) or as a recessive (Jervell and Lange-Nielsen syndrome) disorder; it can also be acquired by certain medications that block cardiac potassium channels. Hereditary LQTS has been shown to be caused by mutations in any of several genes: KvLQT1, HERG, KCNE1, KCNE2 or SCN5A (Splawski et al. 2000; Kass and Moss, 2003).   As discussed above, the KvLQT1 and KCNE1 genes encode subunits that form the IKr channel while HERG and KCNE2 genes encode subunits that form the IKs channel. Most of the mutations in these channel subunits result in loss-of-function of the channels and cause LQTS, rare gain-of-function mutations in HERG and KvLQT1 shorten cardiac APD and cause arrhythmias known as short QT syndrome. Most loss-of-function mutations are missense mutations that result in improper protein folding, and/or lack of co-assembly between subunits   7 resulting in early degradation, disruption of trafficking or defects in plasmalemmal integration, altered voltage dependency, or impaired ion slectivity of the channel (Splawski et al. 2000; Kass and Moss, 2003; Zhou et al. 1998; Rajamani et al. 2002).   Long QT syndrome can result also from changes in other currents, especially in diseased hearts. It has been demonstrated that a decrease in Ito current prolongs APD and may contribute to cardiovascular diseases such as heart failure, cardiac hypertrophy and myocardial ischemia and infarction (Beuckelmann et al. 1993; Wettwer E et al. 1994; Pinto et al. 1999; Tomaselli et al. 1999; Oudit et al. 2001). Decreased expression of Kv4.2 and Kv4.3 mRNA was observed in the hypertrophied hearts of rats with renovascular hypertension (Takimoto et al. 1997). In fact, prolongation of the APD is a consistent electrophysiological abnormality in hypertrophied and failing hearts, a consequence of the electrical remodeling that is common in cardiovascular disease states (Marionneau et al. 2008).  1.3 Small GTPases in intracellular trafficking  1.3.1 General structure and activity of small GTPases   Small GTPases are monomeric GTPases with molecular weights ranging from 20 kDa to 40 kDa. They cycle between active GTP-bound and non-active GDP-bound forms. This process is aided by different small GTPase-associated proteins, such as guanine nucleotide-exchange factors (GEFs), which facilitate GDP release, and GTPase-activating proteins (GAPs), which activate the catalysis of GTP hydrolysis (Pereira-Leal and Seabra, 2000; Pfeffer 2007) (Figure 1.3).   Small GTPases are structurally classified into at least five families: (i) Ras, (ii) Rho, (iii) Rab, (iv) Sar1/Arf, and (v) Ran (Wennerberg et al. 2005) and each family has characteristic cellular functions. Ras members regulate gene expression; Rho members regulate cytoskeletal   8 reorganization and gene expression; Rab and Sar1/Arf GTPases regulate intracellular vesicle trafficking; Ran GTPases regulate nucleocytoplasmic transport during G1, S, and G2 phases of the cell cycle and microtubule organization during the M phase (Bourne et al. 1990; Hall 1990; Takai et al. 2001).   In humans, more than 60 members of the Rab family have been identified, most of which have been found to localize to distinct intracellular membranes where they perform specific cellular trafficking functions (Schwartz et al. 2007, Pereira-Leal and Seabra, 2001; Zerial and McBride, 2001). Sar1 GTPase localizes to endoplasmic reticulum (ER) membrane exit sites, binds directly to the Sec23 component of the COPII coat, and is required for vesicle formation from ER membrane in vitro and in vivo (Matsuoka et al. 1998; Bi et al. 2002). The vesicular trafficking functions of Rab and Sar1 are mediated by interactions with specific effector molecules (Segev 2001; Pfeffer 2001). For instance, Rab5 proteins regulate endosomal vesicle transport through recruitment of Early Endosome Antigen 1 protein (EEA1) which mediates endosome docking and, together with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), leads to membrane fusion (Christoforidis et al. 1999); Rab7 modulates lysosomal transport through Rab7-interacting lysosomal protein (RILP) (Jordens et al. 2001). Rab and Sar1 proteins further serve as instigators of membrane trafficking processes through their interactions with coat protein complex components, molecular motors, and SNAREs. SNAREs are thought to deform membranes, disturbing the hydrophobic-hydrophilic boundary and directly causing fusion (Jahn and Scheller, 2006). Different Rab proteins are involved in the regulation of the four major steps in intracellular vesicle transport: (i) vesicle budding from the donor membrane, (ii) targeting of the vesicle to the acceptor membrane, (iii) docking of the vesicle, and (iv) fusion of the vesicle with the acceptor membrane. Most Rab proteins regulate the targeting, docking or fusion processes and only some of them regulate the   9 budding process, which is primarily regulated by Sar1 proteins (Takai et al. 2001). The roles of Rab and Sar1 proteins in regulation of the expression of epithelial ion channels such as cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channels (ENaC) have been explored extensively (Saxena and Kaur, 2006; Saxena et al. 2006) but that of Kv channels, in cardiomyocytes in particular, is still not well understood.   Figure 1.3    Rab GTPases cycle between a GDP-bound and a GTP-bound conformation. Conversion of the GDP-bound Rab into the GTP-bound form occurs through the exchange of GDP for GTP, which is catalysed by a guanine nucleotide exchange factor (GEF) and causes a conformational change. The GTP-bound ‘active’ conformation is recognized by multiple effector proteins and is converted back to the GDP-bound ‘inactive’ form through hydrolysis of GTP, which is stimulated by a GTPase-activating protein (GAP) and releases an inorganic phosphate (Pi).   GEF RAB Rab GAP GTPGDP GDPGTP Effector Pi   10  1.3.2 Biosynthesis and transport into the ER   Like most eukaryotic membrane proteins, nascent Kv channel protein is co- translationally inserted into the endoplasmic reticulum (ER). The N-terminal tetramerization domain (T1 domain) of the nascent peptide co-assembles with other monomeric Kv channel α subunits to acquire its tertiary structure before exiting the ribosome (Lu et al. 2001; Robinson and Deutsch, 2005; Kosolapov and Deutsch, 2003; Kosolapov et al 2004). The T1 domain is highly conserved among Kv channels, with the exception of hERG and KvLQT channels, which instead contain a tetramerization domain functional analog in their C-terminal region; deletion of a short domain near the C-terminus of these two channels impairs tetramerization (Kupershmidt et al. 1998; Schmitt et al. 2000; Kupershmidt et al. 2002). In the former channels, the nascent peptide/ribosome complex is targeted to the ER membrane following T1-tetramerization and synthesis continues.   Protein exit from the ER is under a high degree of quality control. Only properly folded channels that appropriately exhibit forward trafficking signals and mask ER retention signals are exported from the ER. Chaperone proteins, such as Hsp 70 and Hsp 90, detect improperly folded ion channel complexes and shield forward trafficking signals, thereby preventing their transport to the ER exit sites (Ficker et al. 2003). The ER-resident protein, Calnexin functions to facilitate protein folding and assist in the assembly of immature proteins. It stabilizes Kv1.2 in the ER and improves its transport to the cell surface (Manganas and Trimmer, 2004). Thus far, no roles for Rab and Sar1 proteins have been found in the transport of membrane proteins to the ER exit sites.      11  1.3.3 ER-Golgi shuttle   As mentioned above, properly folded channel proteins have ER retention signals hidden in their tertiary structure and forward trafficking signals displayed. Forward trafficking signals are motifs that promote the export from the ER. In potassium channels, these signals are diverse, even in the same family of Kv channels. For example Kv1.4 harbours a VXXSL signal which potently facilitates its forward trafficking, Kv1.5 harbours a less potent VXXSN while hERG lacks both and may use instead its nucleotide-binding domains as forward trafficking signals (Zhu et al. 2003; Li et al. 2000; Akhavan et al. 2005). It has been suggested that forward trafficking signals interact directly or indirectly with COPII (Ma and Jan, 2002).   Anterograde transport of correctly folded channels beyond the ER is a GTPase-dependent process, mediated by Rab and Sar1 proteins, involving the production of COPII-coated vesicles that bud from ER exit points (the transitional ER) (Barlowe et al. 1994; Lee et al. 2004). COPII vesicles are formed at these specific ER exit sites (ERESs) (Orci et al. 1991). The formation of COPII is initiated by the activation of the small GTPase Sar1 through its guanine nucleotide- exchange factor (GEF) Sec12. This activation, exchanging GTP for the resident GDP, exposes the N-terminal amphipathic helix of Sar1 which then inserts into the ER membrane (Bi et al. 2002). Through direct interaction with Sec23, Sar1 recruits the heterodimer Sec23-Sec24 to form the stable pre-budding complex (Yoshihisa et al. 1993). Sec24 exhibits multiple independent cargo binding sites, and it is therefore thought that majority of cargo is captured through interaction with Sec24 (Miller et al 2003). After the incorporation of cargo and the formation of pre-budding complex, the outer layer of the coat is recruited to the ER membrane. Heterotetramers consisting of two Sec13 and Sec31 subunits then self-assemble into the cage- like structures with a cuboctahedral geometry (Stagg et al. 2006) that forms the outer layer of the coat. Crystal structure reveals greater flexibility of the COPII coat, compared with other coats   12 such as clathrin, suggesting an ability to accommodate cargo of different shapes (Stagg et al 2006). After budding, hydrolysis of Sar1-bound GTP causes the COPII vesicle to uncoat (Oka and Nakano, 1994), allowing the interaction of tethers and SNAREs on both the vesicle and the target membrane such as the Golgi membrane. Until now, among potassium channels, only the ER-to-Golgi trafficking of Kv4.2 in a heterologous cell system has been studied. Interestingly, in this system ER-to-Golgi trafficking of the Kv4.2 channel/ KChIP β subunit complex is Sar1- independent and vesicles harboring KChIP lack COPII (Hasdemir et al. 2005).   Retrograde transport in the ER-Golgi shuttle is performed by COPI, which is recruited to the newly formed transport vesicle in the Golgi, from where it retrieves recycling, ER-escaped and misfolded proteins back to the ER. The formation of COPI-coated vesicle is similar to that of COPII-coated vesicle, although different small GTPases and Sec proteins are involved. The major constituents of the coat structure are coatomer and its effector small GTPase Arf1 (Waters et al. 1991; Serafini et al. 1991). Coatomer is composed of seven conserved protein subunits and is recruited en bloc to membranes in an Arf1 and GTP-dependent manner (Hara-Kuge et al. 1994). Coatomer then binds directly to the dilysine motifs at the carboxy terminus of type I transmembrane proteins (Cosson and Letourneur, 1994). It has been demonstrated that the interaction between COPI and these dilysine motifs on cargo proteins is required for their retrograde transport from the Golgi to the ER (Letourneur et al. 1994).   Specific Rab proteins are also involved in ER and Golgi shuttling. These are Rab1, Rab2, and Rab6 (Figure 1.4). Localized to both the endoplasmic reticulum and the Golgi, these GTPases regulate the vesicle transport between the ER and the Golgi (Martinez et al. 1994; Plutner et al. 1991; Tisdale et al. 1992). Rab1 directs COPII vesicles for delivery to cis-Golgi membrane by recruiting p115 onto budding COPII vesicles, where p115 interacts directly with a select set of SNARE proteins which then facilitate vesicle-membrane fusion (Bernard et al.   13 2000). Rab6 regulates microtubule-dependent retrograde transport from the Golgi to the ER through its effector, Rabkinesin-6 (White et al. 1999), a member of the kinesin family (Echard et al. 1998). Rab2 has been implicated in retrograde transport from post-ER, pre-Golgi intermediates to the ER (Tisdale et al. 1992; Tisdale and Jackson, 1998; Tisdale 1999) and the budding of Rab2-mediated vesicle has been demonstrated to require atypical protein kinase C, PKC iota/lambda kinase activity (Tisdale et al. 2003).   1.3.4 Through the Golgi and on to plasma membrane   Once in the Golgi, the channel is further processed and packaged for delivery to the plasma membrane. Surface expression of some Kv channels is dependent on glycosylation in the Golgi. For instance, block of glycosylation of Kv1.4 decreased its protein stability and induced its intracellular retention, thereby decreasing its surface expression level (Watanabe et al. 2004). Glycosylation has also been demonstrated to increase the cell surface expression of other Kv1 family channels in mammalian cells, similarly by increasing the stability and proper folding of the channel protein (Khanna et al. 2001). Small GTPases are also involved in Golgi-to-plasma membrane trafficking. Rab8 mediates constitutive biosynthetic trafficking of many proteins from the trans-Golgi network (TGN) to the plasma membrane (Lukas et al. 1993). Similarly, Rab11 is present in the Golgi and participates in the trafficking of many proteins from the Golgi to the plasma membrane (Chen et al. 1998; Kessler et al. 2000; Zeigerer et al. 2002) (Figure 1.4).   The mechanisms by which cardiac Kv channels are targeted to the sarcolemma are poorly understood. Undoubtedly, complex processes are involved, the players including various motor proteins, the actin and microtubule cytoskeleton systems, scaffolding proteins and accessory subunits, likely analogous to the targeting of neuronal Kv to specific regions in neurons (Lai and   14 Jan, 2006). The role of cytoskeletal systems in channel trafficking is discussed further in section 1.4.   The final step in forward trafficking is the insertion of the channel into the sarcolemma. This step is believed to be mediated by SNARE proteins that catalyze the fusion of exocytic vesicles with the sarcolemma (Hong, 2005). SNAP25 and Syntaxin 1A have been shown to be involved in plasma membrane integration of Kv1.1 and Kv2.1 (Ji et al. 2002; Leung et al. 2003).   1.3.5 Internalization   The surface expression of Kv channels is a result of tightly regulated anterograde and retrograde trafficking processes. Retrograde trafficking includes the endocytosis and, in a sense, the forward trafficking associated with recycling of internalized channels to the plasmalemma. Thus, cardiac Kv channel density at the cell surface is regulated by a dynamic interplay of forward trafficking of newly synthesized channels combined with endocytosis and recycling of already sarcolemma-resident channels. Membrane protein internalization involves the rearrangement of actin filaments immediately adjacent to the plasma membrane, formation of nascent endocytic vesicle which is then cleaved by dynamin (McClure and Robinson, 1996), and activation of non-conventional myosins that carry the endocytosed vesicles (endosomes) along the actin filaments (Engqvist-Goldstein and Drubin, 2003), a process sometimes followed by transfer to microtuble-dependent carriers (Karcher et al. 2002). The endocytic trafficking of specific Kv channels has recently been addressed in tissue culture cells as well as in cardiomyocytes (Choi et al. 2005; Abi-Char et al. 2007). The endocytosis of Kv1.5 channels has been shown to involve dynamin-catalyzed scission of endocytic vesicles, as well as the dynein- dynactin complex and the microtubule cytoskeleton, which is required for long-range transport   15 of endocytosed channels from the cortical actin cytoskeleton to the cell interior (Choi et al. 2005).   Rab GTPases are also important players in the internalization process (Figure 1.4). One of them is Rab5, which is essential for clathrin-mediated endocytosis and early endosome formation. Rab5 is localized to early endosome and regulates the trafficking of CFTR from the plasma membrane to early endosomes (Gentzsch et al. 2004). Recently, it has been demonstrated that Kv1.5 channels and KCNQ1/KCNE1 channels are internalized via a Rab5-dependent pathway in H9c2 cardiac myoblasts and COS7 cells, respectively (Zadeh et al. 2008; Seebohm et al. 2007).   Phosphorylation of cytoplasmic domain(s) has been shown to be a key regulatory mechanism of the endocytosis of one Kv channel and which is likely operative in the endocytosis of other Kv channels. Rapid internalization of Kv1.2 was observed when a specific N-terminal tyrosine residue was phosphorylated. When this tyrosine residue was mutated to phenylalanine, Kv1.2 endocytosis was drastically reduced (Nesti et al 2004). On the other hand, the Kv1.5 N-terminus harbours two proline-rich SH3-binding domains which are consensus src kinase binding sites (Kay et al. 2000). One of these SH3-binding domains in N-terminus of Kv1.5 was found to be essential for Kv1.5 internalization, probably through tyrosine kinase activity, likely mediated through binding to the SH3 domain (Choi et al. 2005).   1.3.6 Slow and fast recycling of endocytosed channels   Endocytosed channels can have a variety of fates. They can be rapidly recycled back to the plasma membrane directly from the early endosome or, more slowly, via the recycling endosome; channels not recycled might persist in intracellular pools or they might undergo   16 proteasomal or lysosomal degradation. There are two main endosomal recycling pathways, a slow Rab11-dependent process and a rapid recycling process dependent on Rab4 (Figure 1.4).   Rab11 localizes to the trans-Golgi network (TGN), post-Golgi vesicles and recycling endosomes (Kessler et al. 2000). The role of Rab11 in mediating Golgi-to-plasma membrane trafficking has been mentioned above. Among its roles in ion channel trafficking, Rab11 has been shown to control trafficking of CFTR from early endosomes to the TGN and also in the recycling of endocytosed CFTR back to the cell surface (Gentzsch et al. 2004). KCNQ1 has also been shown to recycle in a Rab11-dependent manner (Seebohm et al. 2007). Recently, several groups have identified Rab11 as a mediator of Kv1.5 slow endocytic recycling through recycling endosome in atrial myocytes and cardiac myoblast cells (McEwen et al. 2007; Balse et al. 2009; Zadeh et al. 2008).   A more rapid recycling pathway is mediated by Rab4. Upon internalization, some proteins become associated with Rab4 in maturing early endosomes and recycle directly back to the plasma membrane, bypassing the slower Rab11-associated perinuclear recycling endosome system. The rapid recycling of transferrin receptors via the Rab4-mediated pathway is well established (Sheff et al. 2002). Kv1.5 has been shown to colocalize extensively with Rab4 within 10 minutes of internalization and, as colocalization with Rab11 occurs only to a lesser extent and was observed only after 24 hours of internalization, a Rab4-dependent pathway is very probably the primary pathway for Kv1.5 recycling (Zadeh et al. 2008).   1.3.7 Degradation of endocytosed channels   The two major intracellular pathways for protein degradation are the proteasomal and lysosomal pathways (Figure 1.4). The proteasomal degradation route usually requires polyubiquitination as the sorting signal. Although it is generally thought to be involved more in   17 the degradation of misfolded or excess proteins in the secretory pathway before ER exit, this pathway has also been shown to be responsible for degradation of certain surface membrane proteins (Bonifacino and Weissman, 1998). Proteasomal but not lysosomal inhibitors prolonged the half-life of Kv1.5 and IKur in cultured rat atrial cells (Kato et al. 2005), providing evidence that Kv1.5 is at least in significant measure degraded by the proteasome. Evidence does exist, however, for lysosomal degradation of Kv1.5 as well (Zadeh et al. 2008). The lysosomal degradation pathway is the major pathway by which membrane proteins are degraded. Early endosomes containing the membrane proteins, traffic via a Rab7-dependent process to the lysosomes, and then the proteins are digested by acidic hydrolases in the organelles.   Rab7 regulates trafficking from early to late endosomes and from late endosomes to lysosomes (Meresse et al. 1995; Bucci et al. 2000), probably through interaction with microtubule and dynein motors (Jordens et al. 2001). Zadeh et al. (2008) demonstrated that a dominant negative Rab7 increased Kv1.5 currents and overexpression of wild type Rab7 reduced Kv1.5 currents. Similarly, Kir6.2 conducted-currents have been shown to be increased by application of lysosomal inhibitors, indicating that Kir6.2 channels may be degraded via the lysosomal degradation pathway (Jansen et al. 2008). This degradation pathway has also been shown to be operative in hERG channel degradation (Guo et al. 2009); endocytosed hERG channels showed colocalization with markers for multivesicular bodies and lysosomes. Thus, no single Kv channel protein degradation pathway is operative; channels may be degraded by either or both pathways.   The choice of degradation pathways may be regulated. Targeting of endocytosed KATP channels to lysosomes is enhanced by PKC activation (Manna et al. 2010). Insulin secretion in pancreatic β cells is regulated by KATP, insulin is secreted when β cells is depolarized by closing of KATP channel. In addition, because stimulants of insulin secretion activate PKC, more   18 endocytosed KATP channels are targeted to lysosomes for degradation, therefore the findings by Manna et al. (2010) suggest that regulated endocytic trafficking, including degradation of channels may play a role in the regulation of insulin secretion.             Figure 1.4    Small GTPases implicated in the secretory pathway of membrane protein. ER- Golgi shuttle involves Sar1, Rab1, Rab2 and Rab6; Golgi-to-plasma membrane involves Rab8 and Rab11; Rab5 mediates internalization of membrane proteins; internalized membrane proteins recycle rapidly and slowly via Rab4- and Rab11-mediated pathways, respectively; membrane proteins are eventually degraded via Rab7-mediated lysosomal degradation and/or proteasomal degradation pathway.   1.4 Cytoskeletal motors  1.4.1 Structure of cytoskeletal motors    Rab proteins could not regulate trafficking were there not a molecular motor system to drive that trafficking. Three families of cytoskeletal motors perform that function: kinesins and Early Endosome Lysosome Recycling Endosome Internalization Lysosomal Degradation Fast Recycling Slow Recycling Cell Surface nucleus Endoplasmic Reticulum Golgi Apparatus proteasomal Degradation Rab 1 Sar 1 Rab 11 Rab 5 Rab 4 Rab7Rab 11 Rab 6 Rab 2 Rab 8   19 dynein, which run along the microtubule cytoskeleton and the actin-tracking motor myosin. These cytoskeletal motors are structurally and functionally different. Kinesins are a large family of motors that have a globular motor domain that shows high degrees of homology across family members and contains a microtubule-binding sequence and an ATP-binding sequence. Kinesins differ markedly in their cargo binding domains, however, with each kinesin isoform having a unique cargo-binding domain. It is the diversity of these cargo-binding domains that enables kinesins to transport numerous different cargos. Monomers of the classical kinesin, Kinesin I (also known as Kif5) have an N-terminal globular motor domain, and neck, stalk and tail domains, as well as two kinesin light chains at the C-teminus. These monomers combine to form a homodimer that is the functional Kif5 motor. Other kinesins have varied molecular shapes; some kinesins are monomeric, others form homodimers or are heterotrimeric. All use ATP hydrolysis to drive the conformational changes that generate motile force.   In contrast to kinesin, there are only three dynein forms, one of which, Dynein I, is predominant in cytoplasmic trafficking (Corthesy-Theulaz et al. 1992). Cytoplasmic dynein is composed of two heavy chains, which contain the ATPase activity and are responsible for generating movement along the microtubule, three intermediate chains which anchor dynein to its cargo, and four light intermediate chains which are less well understood. In addition to intermediate and light intermediate chains, cytoplasmic dynein is associated with the protein complex dynactin. It is the dynactin complex that forms the bridge between the dynein motor and the cargo it carries. Overexpression of a dynactin component, dynamitin (also known as p50), causes the dynactin complex to disassociate, practically preventing cargo movement by the dynein motor.   Superficially similar to some kinesins, myosins comprise a large family of motors. Composed of a head, neck and tail domain, myosins bind to the filamentous actin cytoskeleton   20 via the head domain and hydrolyze ATP to generate force to track along the actin filament. The neck domain acts as lever arm for transducing force generated by the motor domain. It also contains binding site for the myosin light chains that regulate motor functions. Cargo interactions are conducted by the tail domain, which can also regulate motor activity.   1.4.2 Roles of cytoskeletal motors in intracellular transport   Cytoskeletal motors convert the chemical energy contained in ATP into the mechanical energy of movement, thereby facilitating organelle and vesicular transport in cells. Long-range vesicular transport generally involves the microtubule cytoskeleton and the kinesin and dynein motors (Karcher et al. 2002). Microtubules serve as rails along which membranous organelles and macromolecular complexes can be transported (Hirokawa, 1998). Microtubules are long, hollow cylinders of diameter 25 nm that are formed as polymers of α- and β-tubulins. Microtubules have intrinsic polarity, with a fast-growing “plus end” towards the cell periphery and an opposite, slow-growing “minus end” at juxtanuclear centrosome (Desai and Mitchison, 1997). Hence, the plus-end-directed family of motors, i.e., the kinesins, almost always mediate anterograde (forward) transport of proteins towards the cell surface whereas the minus-end- directed motor, dynein, carries out retrograde transport of protein towards the cell interior.  Dynein has been implicated in the retrograde trafficking of cardiac Kv1.5, Kv2.1, Kv3.1, and hERG channels, as well as Kir2.1 (Choi et al. 2005; Loewen et al. 2009). Choi et al. (2005) disrupted dynein function by overexpression of dynamitin and observed a two-fold increase in Kv1.5 currents. In addition, treatment of rat atrial myocytes with the microtubule depolymerization agent, nocodazole, similarly enhanced Kv1.5 currents (Choi et al. 2005).  The involvement of kinesin in forward trafficking in the heart is less well understood. Kinesins’ roles in neuronal trafficking are better understood. Through the use of dominant   21 negative isoforms of several kinesins, Chu et al. (2006) determined that a specific isoform, Kif17, could regulate the dendritic targeting of Kv4.2 in dissociated cortical neurons; immunocytochemistry similarly showed that only the Kif17 isoform colocalized with Kv4.2, indicating that Kv4.2 is transported to dendrites by this kinesin isoform Kif17 (Chu et al. 2006). On the other hand, a dominant negative variant of another kinesin isoform, Kif5b, has been shown to specifically block axonal localization of endogenous Kv1 channels in cortical neurons (River et al. 2007), and the T1 domain of Kv1 channels was identified to be the site required for Kif5b interaction with the channel (Rivera et al. 2007).   The bidirectional transport by dynein and kinesin on the same microtubule cytoskeleton presents an obvious problem. Traffic must be somehow regulated to prevent chaos. One possible mechanism may involve the selective dissociation of dynein and/or kinesin in response to phosphorylation of the motors. Sato-Yoshitake et al. (1992) and Okada et al. (1995) have presented models by which this system may serve to regulate bidirectional transport. Alternatively, these authors argue, phosphorylation/dephosphorylaton may activate and inactivate these motors (Sato-Yoshitake et al. 1992; Okada et al. 1995). Another possibility is illustrated in the interesting observation that the direction of vesicle movement is regulated by the presence or absence of a tightly bound kinesin motor; that is, vesicles move retrogradely only when a retrograde motor is bound to the vesicles in the absence of an anterograde motor (Muresan et al. 1996).   Traffic along actin filaments is superficially similar to that on microtubules but is generally operative over much shorter distances. Myosins V and VI move along actin filament to drive short-range transport that typically occurs beneath the plasma membrane. Disruption of the actin cytoskeleton can have profound effects on potassium channel functional expression. In   22 both heterologous cells and cardiomyocytes, the expression of Kv1.5 (Maruoka et al. 2000; Mason et al. 2002) and Kv4.2 (Wang et al. 2004) is dramatically increased by such disruption.   While the studies in neurons provide important insights into how cytoskeletal motors affect intracellular transport, it is important to note that cardiomyocytes are structurally different from neurons and their trafficking system may employ different cytoskeletal motors to accomplish the analogous functions. It has been the object of the research described in this thesis to determine the mechanisms actually employed in cardiomyocytes in the trafficking of Kv channels.  1.5 Research objectives   The experimental work described in this thesis focused on the regulation of cardiac Kv channel functional expression at the cell surface with an aim to further our understanding of how cellular trafficking machinery such as cytoskeletal motors and small GTPases contribute to this regulation.   The first aim was to identify the specific kinesin isoform that is required for Kv1.5 channel forward trafficking in both heterologous cells and cardiac myoblasts. A specific kinesin isoform, Kif5b, was chosen to be investigated due to its ubiquitous tissue expression. Electrophysiology was performed to measure the IKur conducted by Kv1.5 channels, which gives an indication of functional expression of the channel; surface expression of Kv1.5 was observed directly using immuocytochemistry. The requirement for Kif5b for forward trafficking of newly synthesized Kv1.5 channels was demonstrated.   The second aim of the thesis was to investigate the roles of small GTPases in the trafficking of Kv4.2 channels in adult rat ventricular myocytes. Wild type and dominant negative isoforms of small GTPases were expressed in these myocytes. The effects of these isoforms on   23 Ito, the current conducted by Kv4.2, were measured in patch clamp experiments. 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Biochem J 375, 761-768.       35 CHAPTER 2 KIF5B IS AN ESSENTIAL FORWARD TRAFFICKING MOTOR FOR THE Kv1.5 CARDIAC POTASSIUM CHANNEL1  2.1 Introduction Voltage-gated K+ channels (Kv channels) are required for repolarization and the termination of electrical excitation in all cardiomyocytes, and their function depends upon the presence of active channels at the plasmalemma. Surface expression has long been known to be regulated by changes in gene expression, phosphorylation and interactions with accessory subunits (reviewed in Steele et al. 2007), and several groups have investigated the roles of motifs within K+ channels that affect trafficking of the channels to the cell surface (Li et al. 2000; Manganas et al. 2001; Zhu et al. 2001, 2003). More recently, we have shown the dynein motor to be important in the maintenance of normal expression levels of several Kv channels (Choi et al. 2005; Loewen et al. 2009). Also, several Rab GTPases (Seebohm et al. 2007; McEwen et al. 2007; Zadeh et al. 2008) and dynamin (Nesti et al. 2004; Choi et al. 2005) have all been implicated in Kv channel trafficking. Beyond the roles of the motifs mentioned above, however, little has been earned about the forward trafficking of newly synthesized channels. Surface expression requires movement from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane, and several studies have investigated channel determinants that affect this trafficking process (Li et al. 2000; Manganas et al. 2001; Zhu et al. 2001, 2003). In neurons, a kinesin isoform, Kif17, has been implicated in the trafficking of Kv4.2 (Chu et al. 2006) and there is evidence that Kif5b is involved in axonal but not dendritic trafficking of some Kv1 channels (Rivera et al. 2007). The involvement of kinesins in the trafficking of cardiac ion channels has not, however, been established to date. Here we have used electrophysiology,  1 A version of this chapter has been published. Zadeh AD, Cheng Y, Xu H, Wong N, Wang Z, Goonasekara C, Steele DF, Fedida D (2009). Kif5b is an essential forward trafficking motor for the Kv1.5 cardiac potassium channel. J Physiol 587, 4565-4574.   36 molecular biology and imaging techniques to show that the major isoform of kinesin I (Kif5b) is required for the normal trafficking of Kv1.5 to the cell surface in both heterologous cells and in a cardiomyoblast cell line. Overexpression of a Kif5b construct caused a significant increase in Kv1.5 surface expression as did expression of a dominant negative Kif5b construct, the latter probably because of negative interactions with the dynein motor. The delivery of newly synthesized Kv1.5, on the other hand, was almost completely blocked by the dominant negative. Thus, Kif5b is required for the normal delivery of Kv1.5 to the plasma membrane.  2.2 Materials and methods   2.2.1 Cell preparation and transfection  HEK293 cells and H9c2 cardiac myoblasts were cultured and transfected as described previously (Fedida et al. 2003). Transfections were by a liposome-mediated method. One day before transfection, cells were plated on a coverslip in 35mm dishes at 40–50% confluence. After 1 day’s growth, transfections were performed using 1.5 µg of each relevant plasmid and Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions.   2.2.2 Plamid constructs  Human p50 in pEGFP was a gift of Richard Vallee (Columbia University, NY, USA). p50 was amplified by PCR and inserted in the C-terminus of mCherry in pcDNA3 as described previously (Zadeh et al. 2008). Human Kif5b was a gift of Ronald Vale (University of San Francisco, CA, USA). Kif5b–EGFP was similarly constructed by inserting PCR-amplified Kif5b at the C-terminus of EGFP in pcDNA3. A dominant negative version of Kif5b–EGFP (Kif5bDN–EGFP) was constructed by removing the motor domain, 366 amino acids from the N   37 terminus of Kif5b and inserting PCR-amplified Kif5bDN at the C-terminus of EGFP in pcDNA3. pcDNA3–T7–Kv1.5–HA (haemagglutinin antigen)(S1–S2) was described previously (Zadeh et al. 2008). In generating a tetracycline-inducible Kv1.5, EGFP fused to the N-terminus of Kv1.5 was inserted into pcDNA4-TO. Plasmid DNA was prepared for transfection using the Qiagen PlasmidMidi Kit (Qiagen Inc., Valencia, CA, USA).   2.2.3 Electrophysiological experiments and solutions  Solutions and methodology for the recording of ionic currents were as previously reported from our laboratory (Fedida et al. 2003). The standard bath solution contained (in mM): NaCl, 135; KCl, 5; MgCl2, 1; sodium acetate, 2.8; HEPES, 10; CaCl2, 1; adjusted to pH 7.4 using NaOH. The standard pipette filling solution contained (in mM): KCl, 130; EGTA, 5; MgCl2, 1; HEPES, 10; Na2ATP, 4; GTP, 0.1; adjusted to pH 7.2 with HCl. All chemicals were from Sigma (Mississauga, ON, Canada). Whole-cell current recording and data analysis were done using an Axopatch 200B amplifier and pClamp 9 software (Axon Instruments, Foster City, CA, USA). Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL, USA) and polished by heating. Pipette resistances were between 1 and 3MΩ. Compensation for capacitance and series resistance was performed manually in all whole-cell recordings. All recordings were performed at room temperature (RT, 20–23°C).   2.2.4 Imaging  Cells were prepared for imaging according to previously published methods (Zadeh et al. 2008). Briefly, the cells were rinsed and fixed with 4% paraformaldehyde for 12 min at room temperature (RT). After three 5 min washes with 1× phosphate-buffered saline (PBS; 137 mM   38 NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4), cells were incubated with monoclonal mouse-anti-HA antibody (Clone 12CA5, Roche) diluted (1:1000) in PBS supplemented with 0.2% BSA to label the externally tagged Kv1.5 for 1 h at RT. The cells were washed three times for 5 min in PBS on a rotator before incubation with secondary antibody, Alexa 594-conjugated goat anti-mouse IgG antibody (1:1000; Molecular Probes) for 1 h on the rotator at RT. Cells were then washed three times with PBS prior to mounting with 10 µl of a 90% glycerol, 2.5% w/v DABCO (1,4-diazabicyclo2.2.2octane)–PBS solution. In experiments testing the effects of pre-expression of Kif5bDN on Kv1.5 surface expression, H9c2 myoblasts and HEK293 cells were transfected with Kif5bDN–EGFP then, 24 h later, with T7–Kv1.5–HA. Twenty-four hours after the second transfection, the cells were fixed and stained with rabbit-α-HA (Zymed Laboratories) without permeabilization to label the surface Kv1.5. The cells were then permeabilized and stained with mouse-α-T7 (Novagen) to label total Kv1.5. α-Mouse-Alexa 647 and α-rabbit-Alexa 594 were used to detect the total or surface Kv1.5, respectively. Images were collected on an Olympus Fluoview 1000 laser scanning confocal microscope equipped with a 60× (NA 1.4) oil immersion objective. EGFP was excited using the 488 nm line of an Ar laser set at 5% transmission and emission collected using the variable bandpass filter set at 500–530 nm. Alexa 594 was excited using a 543 nm He–Ne laser set at 25% transmission and emission collected using the variable bandpass filter set at 555–625 nm. Alexa 647 was excited with a 633 nm He–Ne laser set at 5% transmission and emission collected using the variable longpass filter set at >650 nm. A 60×, 1.35 NA oil-immersion objective was used for imaging. An optical zoom of 2× was often used. Images were acquired at 512×512 pixel resolution. Identical acquisition settings were used for producing the images. The images were analysed using ImageJ software (NIH).    39  2.2.5 Tetracycline induction experiments  pcDNA4/TO–Kv1.5–EGFP was co-transfected with tetracycline repressor, pcDNA6/TR, at a ratio of 1:6 into HEK293 cells or H9c2 myoblasts in control cells. Cells used to test the effects of Kif5bDN and p50 were co-transfected also with Kif5bDN–mCherry or p50–mCherry, respectively. Twenty-four hours later, tetracycline was added at a final concentration of 2 µg/ml to one culture to induce Kv1.5 expression on the evening prior to electrophysiological analysis. Electrophysiology was performed on the next day. Western analysis for Kv1.5–EGFP expression was performed using 15 µg of protein loaded, and probed with anti-GFP.   2.2.6 Data statistics  Results are expressed as mean±S.E.M. Statistical analyses were conducted using Student’s t test (paired and unpaired) or by one-way ANOVA, as appropriate.  2.3 Results   2.3.1 Kv1.5 functional expression is significantly increased by overexpression of Kinesin I (Kif5b)  There are many kinesin isoforms. Some are expressed only in specific cell types; others are present in all cell types (reviewed in Hirokawa & Noda 2008). As the kinesin I isoform Kif5b is expressed across essentially all cell types, we chose to investigate whether this form of kinesin was involved in the trafficking of Kv1.5. Unlike Kif5a and Kif5c, the expression of which appears to be restricted to neurons, Kif5b is ubiquitously expressed (Hirokawa & Noda 2008). To carry out this work, we constructed an N-terminally EGFP-tagged wild type Kif5b clone (Kif5bWT) in pcDNA3 and a similarly tagged dominant negative Kif5b construct (Kif5bDN) in which the N-terminal 366 amino acid residues, comprising the kinesin motor domain, were   40 deleted. H9c2 cardiomyoblast cells and HEK293 cells each stably expressing a Kv1.5 construct (T7–Kv1.5–HA), tagged N-terminally with T7 and externally (in the S1–S2 linker) with an HA tag, were transfected with these Kif5b–EGFP and Kif5bDN–EGFP as a first test of whether Kif5b is important to Kv1.5 trafficking. Figure 2.1A and B, and C and D show, respectively, representative images of HEK293 cells and H9c2 cardiomyoblasts stably expressing T7–Kv1.5–HA, transfected with the Kif5bWT and Kif5bDN constructs. Both the Kif5bWT (Fig. 2.1A and C, left panels) and DN constructs (Fig. 2.1B and D, left panels) were expressed at high levels in both cell types. Surface Kv1.5 distribution, detected with anti-HA was quite uniform in HEK cells, whether transfected with the Kif5b wild type or dominant negative (Fig. 2.1A and B, right panels). The distribution of the channel at the surface of H9c2 myoblasts was also robust, but more punctate than in the HEK cells (Fig. 2.1C and D, right panels). No obvious difference in Kv1.5 surface staining could be detected between the cells expressing the wild type and dominant negative Kif5b isoforms. Electrophysiological analysis indicated that both the wild type and dominant negative Kif5b proteins increased Kv1.5 functional expression relative to control, EGFP-transfected cells. Overexpression of Kif5bWT significantly increased Kv1.5 current densities in both the HEK293 cells (Fig. 2.1E and F) and in the H9c2 myoblasts (Fig. 2.1G and H). Current density was increased from 389±50.0 pA/pF in control, EGFP-tranfected HEK293 cells to 614±74.3 pA/pF in the Kif5bWT overexpressing cells at +80 mV and from 317±50.3 to 580±90.9 pA/pF, respectively, in the H9c2 myoblasts (Fig. 2.1F and H). Kif5bDN similarly increased current densities in these cell lines. In the myoblasts, the current density in Kif5bDN-transfected cells was 600±105 pA/pF (Fig. 2.1H) and in the HEK293 cells, the density was 742±86.8 pA/pF (Fig. 2.1F). Activation and inactivation kinetics were unchanged by any of the treatments (data not   41 shown). Thus, overexpression of both wild type and dominant negative Kif5b increases Kv1.5 surface expression in these cells that stably express Kv1.5.   2.3.2 Kif5bWT and Kif5bDN increase in Kv1.5 current densities by different mechanisms One explanation for the fact that both wild type and dominant negative forms of Kif5b increase Kv1.5 surface expression is that overexpression of the wild type kinesin is promoting the anterograde trafficking of newly synthesized channel to the cell surface whereas the dominant negative is interfering generally with the microtubule-dependent trafficking system. It is well established that blocking retrograde microtubule-dependent trafficking by p50 overexpression, which blocks dynein function, causes an increase in surface expression of several ion channels (Dhani et al. 2003; Loewen et al. 2009), including Kv1.5 (Choi et al. 2005), very probably by interfering indirectly with channel endocytosis (Choi et al. 2005). It is also established that block of retrograde trafficking can cause a reduction in anterograde trafficking and vice versa (Hamm-Alvarez et al. 1993; Valetti et al. 1999; Martin et al. 1999). Thus, it is expected that a dominant negative Kif5b would cause an increase in Kv1.5 functional expression. As a first test of whether a negative effect on retrograde transport indeed underlies the increase in currents seen when the Kif5bDN is expressed, we co-transfected the Kv1.5- expressing HEK293 line with the Kif5b dominant negative, plus p50. p50 expression alone increased Kv1.5 currents by approximately 50% above control levels (Fig. 2.2A), as we have reported previously (Choi et al. 2005). Co-expression of the Kif5bDN with p50 had no further effect. Current densities in p50-overexpressing cells were 661±91.9 pA/pF at +80 mV and 662±192 pA/pF in cells co-expressing also Kif5bDN. In striking contrast, the effects of co-   42 overexpression of p50 and Kif5bWT on Kv1.5 currents were additive. p50 and Kif5bWT co- overexpression increased Kv1.5 current density to 1062±170 pA/pF at +80 mV, significantly higher than the 661 pA/pF in cells overexpressing p50 alone. To confirm that Kif5bDN acts through a mechanism similar to that of p50, and that the mechanism of action of Kif5bWT is different, we tested for additivity of the wild type and dominant negative with two additional manipulations known to be non-additive with p50 overexpression, an SH3-binding domain deletion in the N-terminus of Kv1.5 and a direct block of endocytosis using dynamin inhibitory peptide. In previously published work, we showed that the p50-dependent increase in Kv1.5 expression depends on the presence of a specific SH- binding domain in the channel (Choi et al. 2005). Removal of this domain (generating Kv1.5_SH3(1)), increases Kv1.5 functional expression to a level that cannot be further increased by p50 overexpression. We tested whether the effect of the Kif5bDN is similarly attenuated in cells expressing Kv1.5_SH3(1). As shown in Fig. 2.2B, this was indeed the case. Co-expression of Kif5bDN failed to increase the functional expression levels of this mutant channel. Current densities at +80 mV were 981±174 pA/pF in EGFP-transfected HEK293-Kv1.5_SH3(1) cells and 1094±179 pA/pF in the cells transfected with Kif5bDN. Again indicating that Kif5bWT acts by a different mechanism, overexpression of this motor protein increased Kv1.5_SH3(1)- dependent currents nearly 2.5-fold. Current densities were enormous in these cells, reaching 2433±495 pA/pF. The results of the experiments with dynamin inhibitory peptide (DIP), which blocks endocytosis by interfering with nascent vesicle scission from the plasma membrane, were similar. Kif5bWT overexpression increased Kv1.5 current density over and above that produced by a 16 h treatment with 50 µM DIP whereas the Kif5bDN did not. DIP alone increased Kv1.5 current densities from control levels of 352±35.5 pA/pF at +80 mV in scrambled peptide-treated   43 cells to 567±71.9 pA/pF in the DIP-treated cells (compare Fig. 2.2C and D). When added to Kif5bWT-overexpressing cells, DIP further increased current densities to a similar extent but there was no additivity of DIP and Kif5bDN expression. Current densities in Kif5bWT- overexpressing cells were 908±104 pA/pF at +80 mV in the DIP-treated Kif5bWT-expressing cells (Fig. 2.2C), some 50% higher than the 583±51.5 pA/pF measured in Kif5bWT-transfected cells treated with the control peptide (Fig. 2.2D). In the Kif5bDN-expressing cells, current densities in the Kif5bDN-expressing cells±DIP were not significantly different. Densities at +80 mV were 648±103 pA/pF in the DIP-treated cells and 651±132 pA/pF in the Kif5bDN- expressing cells treated with the scrambled control peptide. Thus, Kif5bDN very probably acts by generally impeding microtubule-dependent trafficking and, like p50, indirectly interfering with channel endocytosis. Kif5bWT, on the other hand, clearly operates by a different mechanism.   2.3.3 Enhancement of Kv1.5 current density by Kif5bWT requires intact Golgi function   To further test whether wild type Kif5b is indeed promoting delivery of new channels to the plasma membrane, experiments with Brefeldin A were conducted. Brefeldin A blocks ER-to- Golgi transport (Klausner et al. 1992) and thus should deprive kinesin of cargo to deliver to the plasma membrane. Immunocytochemistry confirmed that 5 µg/ml of the drug caused dissolution of the Golgi apparatus in these cells (data not shown). Whereas treatment with the drug for 6 h only marginally attenuated the increase in currents associated with Kif5bDN expression (Fig. 2.3A and B), it completely blocked the increase in Kv1.5 expression associated with Kif5bWT expression (Fig. 2.3A and C). Current densities at +80 mV were 341±94.0 pA/pF in EGFP- transfected cells treated with Brefeldin A only (shown in Fig. 2.3C) and 703±98.8 and 560±161 pA/pF in cells transfected with KifbDN untreated and treated with Brefeldin A, respectively.   44 Current densities in cells overexpressing Kif5bWT were reduced from 1139±194 pA/pF in untreated cells to 570±152 pA/pF when treated with Brefeldin A.   Together, the experimental results described above strongly indicate that overexpression of Kif5bWT promotes the forward trafficking of new Kv1.5 channel to the plasma membrane whereas the dominant negative probably acts by reducing microtubule-dependent trafficking in both directions and, thereby, indirectly interfering with endocytosis of plasma membrane resident channels.  2.3.4 Kif5b function is specifically required for Kv1.5 delivery to the plasma membrane  That Kif5bWT overexpression increases delivery of Kv1.5 to the cell surface does not prove that the kinesin isoform is a requirement for delivery to occur at all. In order to test whether Kif5bWT is required for this delivery, we employed a system in which Kv1.5 expression could be delayed relative to that of transfected motor. HEK293 cells and H9c2 myoblasts were co-transfected with tetracycline-inducible EGFP-tagged Kv1.5 (Kv1.5–EGFP) and the tetracycline repressor (see Methods). As shown in Fig. 2.4A, Kv1.5 expression, as assayed by Western blot, is very low under control conditions but levels of the protein increased 24 h after induction with 2 µg/ml tetracycline. For electrophysiology, these cells were simultaneously co-transfected with the inducible Kv1.5–EGFP system±either p50 tagged with mCherry fluorescent protein (p50–mCherry) or similarly tagged Kif5bDN (Kif5bDN–mCherry). On the next day, Kv1.5 expression was induced with tetracycline and, 12 to 24 h later, patch clamping was performed. As shown in the upper panels of Fig. 2.4B and D, tetracycline treatment increased Kv1.5 current densities to very high levels in control cells in both HEK293 cells and H9c2 cardiomyoblasts. In all cases, induction of Kv1.5–EGFP expression was clearly   45 visible under the light microscope as bright green fluorescence, and p50 or Kif5bDN was visible as red fluorescence.  As expected, induction of Kv1.5–EGFP expression in p50-transfected HEK cells and H9c2 myoblasts resulted in moderately lower, but still very high, Kv1.5 current densities than were obtained in control cells transfected with the inducible Kv1.5 system alone (Fig. 2.4C and E). This is consistent with previous reports that block of dynein function also causes a reduction in kinesin function (Hamm-Alvarez et al. 1993; Valetti et al. 1999; Martin et al. 1999). Tetracycline-induced current densities in the control cells were 1530±458 and 1020±172 pA/pF in the HEK293 cells and H9c2 myoblasts, respectively. Induced current densities in p50- expressing HEK293 cells were reduced to 1222±237 pA/pF and, in H9c2 myoblasts to 512±68.9 pA/pF. By 2 days post-induction, current densities in p50-co-expressing cells substantially exceeded those of the control cells (Fig. 2.4F). This is consistent with p50 overexpression strongly interfering with Kv1.5 internalization and more moderately impeding forward trafficking of the channel. In striking contrast to the p50 results, the Kif5bDN nearly eliminated surface expression of the induced Kv1.5–EGFP. In HEK293 cells, densities were 262±111 pA/pF and in the H9c2 myoblasts, current densities were 210±67.4 pA/pF in these Kif5bDN- expressing cells. Separate immunocytochemical experiments, in which H9c2 myoblasts and HEK293 cells were transfected with Kif5bDN–GFP 24 h prior to transfection with Kv1.5–HA yielded similar results. All cells expressed high levels of internal Kv1.5 (Fig. 2.5A–D, right panels). H9c2 myoblasts and HEK293 cells untransfected with Kif5bDN showed robust surface expression of Kv1.5–HA (Fig. 2.5A and C, middle panels) whereas Kif5bDN-transfected cells expressed almost no surface Kv1.5 (Fig. 2.5B and D, middle panels). This was despite the fact that internal   46 expression of the channel was robust (Fig. 2.5B and D, right panels). Kif5b function is clearly essential to the normal trafficking of Kv1.5 to the cell surface.  2.4 Discussion  2.4.1 Kif5b is essential for forward trafficking of Kv1.5   Using both immunocytochemistry and quantitative electrophysiological techniques, we have shown that the ubiquitous kinesin I isoform Kif5b is essential for the normal trafficking of a cardiac potassium channel to the plasma membrane in both HEK293 cells and H9c2 cardiac myoblasts. This is the first identification of a role for a kinesin isoform in the trafficking of any such cardiac ion channel. Block of Kif5b function by expression of a dominant negative isoform prevents the delivery of newly synthesized channels to the plasma membrane, as indicated by current density measurements and immunocytochemistry. Overexpression of Kif5b dramatically increased Kv1.5 functional expression. Interestingly, expression of the dominant negative increased Kv1.5 current density in cells pre-expressing the channel. This is almost certainly due to an indirect reduction in the endocytosis of the channel, as it is well established that interference with kinesin function interferes also with dynein function (Hamm-Alvarez et al. 1993; Valetti et al. 1999). Further, we have previously established that interference with dynein function by p50 overexpression increases Kv1.5 surface expression by both imaging and current density measurements. This effect requires the presence of a specific SH3-binding domain in the channel and is not additive with block of endocytosis by a dynamin inhibitory peptide (Choi et al. 2005), all properties shared by the Kif5bDN. Our demonstration that the effect of the Kif5bDN to increase channel surface density is also non-additive with that of p50 overexpression firmly establishes that the Kif5bDN acts in the same way as does the p50, i.e. indirectly inhibiting endocytosis of the channel. That the Kif5b wild type is additive with the   47 effects of p50, DIP and the SH3-binding domain deletion in increasing Kv1.5 functional expression firmly establishes that this kinesin isoform carries the channel to the cell surface.   2.4.2 Kv1.5-kinesin interaction   The motifs in Kv1.5 required for interaction with Kif5b have yet to be identified. Attempts to co-immunoprecipitate Kif5b with Kv1.5 proved unsuccessful (data not shown), indicating that the channel probably does not interact directly with the motor. This is not surprising. The ion channels GluR2 and NR2B interact with kinesins via PDZ-containing proteins that interact with those channels’ C-terminal PDZ-binding motifs (Setou et al. 2000, 2002). GluR2, for example, is linked to Kif5b via the GRIP PDZ protein (Setou et al. 2002). Like GluR2 and NR2B, Kv1.5 contains a PDZ-binding motif at its extreme C-terminal end that could be involved in indirect kinesin binding. Deletion of this motif, however, does not appreciably reduce surface expression of this channel (Eldstrom et al. 2003; Mathur et al. 2006). Another possibility is that a non-canonical PDZ-binding domain present in the Kv1.5 N-terminus (Eldstrom et al. 2002) is involved in linking the channel to the kinesin. Alternatively, pore (Zhu et al. 2001) and/or C-terminal motifs (Li et al. 2000) in the channel may be responsible for linkage to the kinesin motor. Clearly, further work is required to resolve this issue.   2.4.3 Microtubule transport as regulator of functional expression The involvement of both the dynein (Choi et al. 2005) and kinesin motors in the trafficking of Kv1.5 to and from the cell surface implies that modulation of microtubule- dependent trafficking is probably an important mechanism by which cardiomyocytes regulate the functional expression of its resident ion channels. Several additional potassium channels are known to be affected by modulation of dynein motor function (Loewen et al. 2009) and this   48 further supports the hypothesis that the microtubule transport system is fundamental to the regulation of channel surface localization. It has been reported that the abundance of microtubules is increased in the cardiomyocytes of patients with congestive heart failure (Aquila et al. 2004). It will be of great interest to determine if changes in trafficking along these microtubules are associated with the channel remodelling inherent in this condition and other chronic cardiac diseases.                                     49                                                   50 Figure 2.1 Transfections with Kif5bWT and Kif5bDN similarly affect Kv1.5 functional expression A–D, representative images of cells stably expressing Kv1.5 transfected with the Kif5bWT–EGFP and Kif5bDN–EGFP constructs. A, HEK293 cell stably expressing Kv1.5–HA transfected with Kif5bWT–EGFP showing Kif5bWT expression (left) and Kv1.5–HA surface expression (right). B, HEK293 cells stably expressing Kv1.5–HA transfected with Kif5bDN- EGFP showing Kif5bDN expression (left) and surface expression of Kv1.5–HA (right). C, H9c2 myoblasts stably expressing Kv1.5–HA transfected with Kif5bWT–EGFP showing Kif5bWT expression (left) and Kv1.5–HA surface expression (right). D, H9c2 myoblasts stably expressing Kv1.5–HA transfected with Kif5bDN–EGFP showing Kif5bDN expression (left) and Kv1.5–HA surface expression (right). Scale bar, 10 µm. E–H, effects of Kif5bWT and Kif5bDN on Kv1.5 current density in myoblasts and HEK293 cells. Cells were depolarized from −60 to +80 mV in 10 mV, 200 ms steps and repolarized to −80 mV between pulses. Data are represented as mean ± S.E.M. Current densities from control cells (EGFP), Kif5bWT–EGFP and Kif5bDN–EGFP transfected cells are plotted against voltage. E and G, sample traces of control, Kif5bWT and Kif5bDN-transfected HEK293 cells and H9c2 myoblasts, respectively. Both cell lines stably express Kv1.5. F and H, current density versus voltage plot for data recorded 36–48 h post transfection for HEK293 cells and H9c2 myoblasts, respectively. *P < 0.05, comparing Kif5bWT–EGFP and Kif5bDN–EGFP to EGFP.             51                  52 Figure 2.2 Differential interactions of Kif5bWT and Kif5bDN with direct inhibitions of retrograde trafficking HEK293 cells stably expressing Kv1.5 were depolarized from −60 to +80 mV in 10 mV, 200 ms steps and repolarized to −80 mV between pulses. Data are represented as mean ± S.E.M. A, current densities, 36–48 h post-transfection, measured from cells transfected with EGFP (control), p50–mCherry, Kif5bWT–EGFP + p50–mCherry and Kif5bDN–EGFP + p50–mCherry plotted against voltage. *P < 0.05, comparing Kif5bDN–EGFP + p50–mCherry to Kif5b–EGFP +p50 and p50–mCherry alone. B, current densities, 36–48 h post transfection, from control Kv1.5_SH3(1) + EGFP expressing cells, Kv1.5_SH3(1) + Kif5bWT EGFP and Kv1.5_SH3(1) + Kif5bDN–EGFP-transfected cells plotted against voltage. *P < 0.05 comparing Kv1.5_SH3(1) + Kif5bWT–EGFP to Kv1.5_SH3(1) + Kif5bDN–EGFP and to Kv1.5_SH3(1) + EGFP. C and D, current densities from control cells (EGFP), Kif5bWT– EGFP transfected cells + DIP (C) or control peptide (D) and Kif5bDN–EGFP transfected cells + DIP (C) or control peptide (D) plotted against voltage. C, current density versus voltage plot for data from DIP-treated cells recorded 36–48 h post-transfection. D, current density versus voltage plot for data from control peptide-treated cells recorded 36–48 h post-transfection. *P < 0.05, comparing, in C, Kif5bWT–EGFP + DIP to Kif5bDN–EGFP + DIP and to DIP alone and, in D, Kif5bWT–EGFP + control peptide and Kif5bDN–EGFP + control peptide to control peptide alone.    53   Figure 2.3 Brefeldin A treatment prevents increase in Kv1.5 currents associated with Kif5bWT overexpression HEK293 cells stably expressing Kv1.5 were depolarized from −60 to +80 mV in 10 mV, 200 ms steps and repolarized to −80 mV between pulses. Data are represented as mean ± S.E.M. A, sample traces of Brefeldin A-treated EGFP-, Kif5bWT- and Kif5bDN-transfected cells, and untreated Kif5bDN- and Kif5bWT-transfected cells. B, current density versus voltage plot for data recorded from Kif5bDN-transfected cells ± Brefeldin A treatment 36–48 h post-transfection. C, current density versus voltage plot for data recorded from Kif5bWT-transfected cells ± Brefeldin A treatment and EGFP-transfected Brefeldin A- treated cells 36–48 h post-transfection. *P < 0.05, comparing Kif5bWT–EGFP to Kif5bWT– EGFP + Brefeldin A.    A B   54             55  Figure 2.4 Kif5bDN expression prevents functional expression of newly synthesized Kv1.5 HEK293 cells and H9c2 myoblasts were transfected with Kv1.5–EGFP in pcDNA4/TO and pcDNA6/TR and either Kif5bDN–mCherry or p50–mCherry. Kv1.5–EGFP expression was induced 24 h later and electrophysiological experiments were performed the following day. A, Western blot showing induction of Kv1.5 by 2 µg/ml tetracycline in HEK293 cells 24 h after transfection with the pcDNA4 and pcDNA6 constructs. B and D, sample traces of control, p50 and Kif5bDN-transfected HEK293 cells and H9c2 myoblasts co-transfected with the tetracycline-inducible Kv1.5 system, respectively, 12–24 h post-tetracycline-induction of Kv1.5 expression. C and E, current density versus voltage plot for data recorded 12–24 h post-induction from HEK293 and H9c2 cells, respectively. F, current densities in HEK293 cells co-expressing p50 and tetracycline inducible Kv1.5–EGFP relative to control cells expressing tetracycline- inducible Kv1.5 EGFP only at 12–24 h (Day 1) and 36–48 h (Day 2) post-tetracycline induction of Kv1.5–EGFP expression. Values are normalized to control values for each day ± S.E.M. Day 1: control, n = 11; p50, n = 10; Day 2: control, n= 10; p50, n = 10. *P < 0.05, **P < 0.01.     56    Figure 2.5 Kif5bDN blocks surface expression of newly synthesized Kv1.5 H9c2 myoblasts and HEK293 cells were transfected with Kif5bDN–EGFP then, 24 h later, with T7–Kv1.5–HA. A and C, examples of control H9c2 (A) and HEK293 (C) cells transfected with T7–Kv1.5–HA but untransfected with Kif5bDN. Kif5bDN–EGFP is absent (left panel) and T7–Kv1.5–HA surface expression is robust (right panel). B and D, examples of H9c2 myoblasts (B) and HEK293 cells (D) transfected first with Kif5bDN–EGFP then, 24 h later, with T7–Kv1.5–HA. Scale bar, 10 µm.  57 2.5 References Aquila LA, McCarthy PM, Smedira NG, Young JB & Moravec CS (2004). Cytoskeletal structure and recovery in single human cardiac myocytes. J Heart Lung Transplant 23, 954– 963.  Choi WS, Khurana A, Mathur R, Viswanathan V, Steele DF & Fedida D (2005). Kv1.5 surface expression is modulated by retrograde trafficking of newly endocytosed channels by the dynein motor. Circ Res 97, 363–371.  Chu PJ, Rivera JF & Arnold DB (2006). A role for Kif17 in transport of Kv4.2. J Biol Chem281, 365–373.  Dhani SU, Mohammad-Panah R, Ahmed N, Ackerley C, Ramjeesingh M & Bear CE (2003). Evidence for a functional interaction between the ClC-2 chloride channel and the retrograde motor dynein complex. J Biol Chem 278, 16262–16270.  Eldstrom J, Choi WS, Steele DF & Fedida D (2003). 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Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites. Nature 417, 83–87.  Steele DF, Eldstrom J & Fedida D (2007). Mechanisms of cardiac potassium channel trafficking. J Physiol 582, 17–26.  Valetti C,Wetzel DM, Schrader M, Hasbani MJ, Gill SR, Kreis TE & Schroer TA (1999). Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170. Mol Biol Cell 10, 4107–4120.  Zadeh AD, Xu HJ, Loewen ME, Noble GP, Steele DF & Fedida D (2008). Internalized Kv1.5 traffics via Rab-dependent pathways. J Physiol 586, 4793–4813.  Zhu J,Watanabe I, Gomez B & Thornhill WB (2001). Determinants involved in Kv1 potassium channel folding in the endoplasmic reticulum, glycosylation in the Golgi, and cell surface expression. J Biol Chem 276, 39419–39427.  Zhu J,Watanabe I, Gomez B & Thornhill WB (2003). Trafficking of Kv1.4 potassium channels: interdependence of a pore region determinant and a cytoplasmic C-terminal VXXSL determinant in regulating cell-surface trafficking. Biochem J 375, 761–767.  59 CHAPTER 3 TRAFFICKING OF ENDOGENOUS KV4.2 IN ADULT VENTRICULAR MYOCYTES2  3.1 Introduction Voltage-gated K+ channels (Kv channels) are essential to the repolarization phase of the action potential in cardiac cells. Because together, they determine the duration of the action potential and of the refractory period, minor differences in Kv channel functional expression can have dramatic effects on cardiac electrophysiology. Channel functional expression may be modulated by phosphorylation and other modifications, by association with accessory subunits, and in large measure via control of channel trafficking into and out of the plasma membrane. This trafficking is regulated by a dynamic interplay between anterograde and retrograde trafficking pathways. Surface expression of Kv1.5 in a myoblast cell line, for example, requires a specific kinesin isoform (Kif5b), which is essential for the forward trafficking of the channel (Zadeh et al. 2008) and, like several other potassium channels (Loewen et al. 2009), the numbers remaining at the plasma membrane are modulated via a dynein-dependent process (Choi  et al. 2005). To date, the internalization and trafficking of already plasma membrane-resident potassium channels has been best studied. Several Rab GTPases have been implicated in this dynein-dependent internalization and recycling process of Kv1.5 (McEwen et al. 2007; Zadeh et al. 2008), and that of another potassium channel, KCNQ1 (Seebohm et al. 2007). These small GTPases regulate diverse processes in eukaryotic cells, and are very important in regulating intracellular membrane trafficking (Pucadyil et al. 2009; Stenmark 2009; Takai et al. 2001). These Rab proteins are intimately involved in the regulation of vesicle trafficking in all eukaryotic cells (Zerial and McBride 2001), including budding, delivery, tethering and fusion  2 A version of this chapter will be submitted for publication. Wang T, Cheng Y, Goonesekara C, Dou Y, Steele DF and Fedida D (2010). Trafficking of endogenous Kv4.2 in adult ventricular myocytes.   60 (reviewed in (Grosshans et al. 2006)), and they play significant roles in channel internalization and recycling. Rab5, localized to the early endosomes, acts as a primary effector of the rapid endocytosis of KCNQ1/KCNE1 and Kv1.5 (Seebohm et al. 2007; Zadeh et al. 2008). Rab4 associates with at least a subset of early endosomes and is involved mainly in fast recycling of internalized membrane proteins back to the plasmalemma. A GDP-locked dominant negative Rab4 mutant significantly increases Kv1.5 surface expression, probably by indirectly blocking Rab5-dependent endocytosis (Zadeh et al. 2008). After internalization, Kv channels may also be delivered into Rab11-associated recycling endosomes, and slowly recycled back to cell surface (Akyol et al. 2007; Balse et al. 2009; Seebohm et al. 2007). Rab7 WT overexpression reduces Kv1.5 surface expression in H9c2 myoblasts, very probably by enhancing the lysosomal degradation of the channel (Zadeh et al. 2008). Less well studied has been the forward trafficking of potassium channels. While roles for retention signals and chaperones have been demonstrated in the trafficking of several potassium channels out of the endoplasmic reticulum (ER) (reviewed in (Steele et al. 2007)), the actual paths followed upon ER exit are less well understood. It is clear, nevertheless, that there is considerable variation in the pathways employed by different channels. While some channels clearly traffic to the Golgi apparatus via COPII-coated vesicles (Taneja et al. 2009; Wang et al. 2004), others have been shown to traffic independently of these vesicles, at least in some cell types (Flowerdew and Burgoyne, 2009; Hasdemir et al. 2005). Trafficking through COPII vesicles is dependent on the Sar1 GTPase, which regulates the formation of the COPII coat- associated protein complex (Barlowe 2002; Barlowe et al. 1994). Rab1 recruits tethering factors into the cis-SNARE complex and facilitates fusion between ER budded vesicles and Golgi compartments (Allan et al. 2000). However, it has been reported that secretory protein H-Ras is trafficked independently of Sar1 (Zheng et al. 2007) and cystic fibrosis transmembrane   61 conductance regulator (CFTR) trafficking does not require Rab1 (Yoo et al. 2002), suggesting alternate ER-to-golgi trafficking. The ER-to-Golgi trafficking of newly synthesized Kv4.2 channels in Neuro2A cells has been shown to involve KChIP1 and to be independent of Sar1 but nevertheless dependent on Rab1 function (Flowerdew and Burgoyne, 2009; Hasdemir et al. 2005). Unlike in neurons, cardiac Kv4.2 forward trafficking is dependent on KChIP2; the involvement of Sar1 and Rab1 in the trafficking of this complex in cardiac cells has to date not been established. Here we have used electrophysiological and imaging techniques to identify the small GTPases that regulate the trafficking of endogenous Kv4.2, well established to underlie Ito in rat ventricular myocytes (Schultz et al. 2005; Yeola and Snyders, 1997). We report that, as in neurons, ER-to-Golgi trafficking of the channel requires Rab1 but, unlike in neurons, this trafficking is dependent also upon Sar1 function. Once at the sarcolemma, channel internalization involves Rab5 activity; expression of a Rab5DN greatly increases endogenous channel expression. Similar to previously reported findings on Kv1.5 expression, interference with Rab4 function by a Rab4DN also increased the Kv4.2-dependent current, possibly by indirectly blocking Rab5-mediated endocytosis. Rab11WT overexpression caused an increase of Kv4.2 surface expression which was additive with the effects of dynamin inhibitory peptide block of endocytosis. In addition, Rab11WT-associated increase of Kv4.2 functional expression was blocked by Brefeldin A, suggesting involvement of Rab11 in the trafficking of newly synthesized Kv4.2 to the sarcolemma. Finally, interference with Rab7 function had no detectable effect on endogenous Kv4.2 expression, suggesting that either the channel is not subject to lysosomal degradation or that shunting to that compartment is rare or slow.     62 3.2 Materials and methods  3.2.1 Myocyte isolation and transfection Rat ventricular myocytes were isolated from the hearts of male Wistar rats weighing 250- 300 g using a conventional horizontal heart Langendorf apparatus. Rats were dosed with 500 units of heparin via an intraperitoneal injection and then euthanized according to protocols approved by the Committee on Animal Care of the University of British Columbia in accordance with the regulations of the Canadian Council on Animal Care. Rapidly beating hearts were excised and placed in cold rinse buffer containing 121 mmol/L NaCl, 5 mmol/L KCl, 24 mmol/L NaHCO3, 5.5 mmol/L glucose, 2.8 mmol/L sodium acetate, 1 mmol/L MgCl2, 1 mmol/L Na2HPO4, 1.5 mmol/L CaCl2, pH 7.4.  After cannulating the aorta, hearts were retrogradely perfused with warm (37˚C) rinse buffer gassed with 5.2 % CO2, 94.8 % O2 until the perfusate was clear of blood. Perfusion was continued with a low-Ca2+ (5 µmol/L CaCl2) rinse buffer for 5-10 min before being switched to the collagenase solution containing in addition 20 mmol/L taurine, 12.5 U/ml type II collagenase (Worthington, Lakewood, NJ, USA), 0.1 U/ml type XIV protease (Sigma-Aldrich), 40 µmol/L CaCl2, for 4-5 min. The ventricles were then cut from the heart and minced roughly into 20 mL of incubation solution containing 240 U/ml type II collagenase, 2.0 U/mL type XIV protease, 10 mg/mL essentially fatty acid free BSA (Sigma- Aldrich), 100 µmol/L CaCl2. The tissue was gently agitated at 37˚C and the supernatant monitored periodically until a maximal yield (60-80%) of rod-shaped Ca2+-tolerant myocytes was obtained, at which point the enzyme-containing solution was removed and the incubation quenched by adding 40 mL of enzyme-free incubation solution. Cells were plated on laminin- coated coverslips within 1 hour of isolation. After 30 minutes, the overlaying Storage Buffer was replaced with 1 ml M199 (Sigma-Aldrich), pH7.4, supplemented with 2 mM EGTA, 0.6 µg/mL insulin, 5 mM creatine, 2 mM DL-carnitine, 2 mM glutamine, 5 mM taurine, 50 U/mL penicillin   63 and 50 µg/mL streptomycin. Media with 1% FBS, 50 U/mL penicillin and 50 µg/mL streptomycin, 1/1000 GIBCO™ Insulin-Transferrin-Selenium-A Supplement (ITS, 100X, invitrogen) can also be used. The cells were incubated at 37˚C overnight in a 1% CO2 incubator. On the second day, prior to transfection, the media was replaced with 1 ml M199 containing 5% FBS, 1/1000 GIBCO™ Insulin-Transferrin-Selenium-A Supplement (ITS). Transfections were carried out using 6 µl of lipofectamine 2000 (Invitrogen) in 300 µl of Opti-MEM mixed with 3 µg of relevant plasmid and added to the cells after replacing the media with 1 ml M199 containing 5% FBS, 1/1000 ITS. After 4 h, the medium was replaced with 1 ml M199 supplemented with 5% FBS, 1/1000 ITS, 50 U/mL penicillin and 50 µg/mL streptomycin. The cells were then incubated overnight at 37˚C in 5% CO2 incubator.   3.2.2 HEK 293 cell preparation and transfection HEK293 cells were cultured and transfected as described previously (Fedida et al. 2003). One day before transfection, cells were plated on a coverslip in 35 mm dishes at 40-50% confluence. After one day’s growth, transfections were performed using 1.5 µg of each relevant plasmid and lipofectamine 2000 transfection reagent according to the manufacturer's instructions.   3.2.3 Plasmid constructs Plasmid constructs were generally as described previously (Zadeh et al. 2008). The wild type (WT) and dominant negative (DN) Sar1 and Rab1 clones were kind gifts of Terry Hebert (McGill University, Montreal, CA). Sar1WT, Sar1DN, Rab1WT and Rab1DN were amplified by PCR and fused N-terminally to mCherry in pcDNA3 as described previously (Zadeh et al. 2008). In generating a tetracycline-inducible Kv4.2, mCherry fused to the N-terminus of Kv4.2 was   64 inserted into pcDNA4/TO. Plasmid DNA was prepared for transfection using the Qiagen Plasmid Midi Kit (Qiagen Inc, Valencia, CA).   3.2.4 Electrophysiology Whole cell voltage-clamp experiments were performed at room temperature using an Axopatch 200B amplifier and pClamp software (Axon Instruments, Foster City, CA). Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL) and polished by heating. Patch pipettes had a resistance of 1 to 3 MΩ. The standard bath solution contained (in mmol/L) 135 NaCl, 5 KCl, 1 MgCl2, 2.8 sodium acetate, 10 HEPES, and 1 CaCl2, adjusted to pH 7.4 using NaOH. The standard pipette filling solution contained (in mmol/L) 130 KCl, 5 EGTA, 1 MgCl2, 10 HEPES, 4 Na2ATP, and 0.1 GTP, adjusted to pH 7.2 with KOH. Compensation for capacitance and series resistance was performed manually in all whole cell recordings. Currents were elicited by 500 ms pulse stepping from a holding potential of -80 mV to test potentials of -80 to +90 mV in 10 mV increments followed by 100 ms pulse to +60 mV. All recordings were performed at room temperature (20-23 °C).  For experiments with dynamin inhibitory peptide (DIP; Tocris Bioscience), 5 mM DIP stock solution was prepared in H2O and stored at 4˚C. Cells were incubated with 50 µM DIP for 6 h prior to electrophysiological recordings. For experiments with Brefeldin A (BFA), 10 mg/ml BFA stock solution was prepared in ethanol and stored at -20˚C. Cells were incubated with 0.5 µg/ml BFA for 6 h prior to electrophysiological recordings. For experiments with the proteasomal inhibitor MG132, 50 mM MG132 stock solution was prepared in DMSO and stored at -20˚C. Cells were incubated with 5 µM MG132 for 6 h prior to electrophysiological recordings.    65  3.2.5 Imaging Myocytes were rinsed and fixed with 2% paraformaldehyde at room temperature (RT). After 10 minutes, fixative was removed and the cells were washed with Glycine Buffer and phosphate-buffered saline (PBS; 137 mmol/L NaCl, 2.7 mmol/L KCl, 4.3 mmol/L Na2HPO4, 1.4 mmol/L KH2PO4), for 10 minutes each. In experiments in which fluorescent proteins were employed and antibodies were not needed, cells were mounted in DABCO/Glycerol (90% glycerol, 2.5% w/v DABCO–PBS solution) at this stage. In other experiments, the cells were then incubated with PBS containing 2% BSA +/- 0.2% Triton X-100 for 10 min followed by three 10 min washes with PBS buffer. After the permeabilization step, cells were incubated with appropriate primary and secondary antibodies one hour each before mounting.  Images were collected on an Olympus Fluoview 1000 laser scanning confocal microscope. EGFP and Alexa 488 were excited using the 488 nm line of an argon laser set at 5% transmission and emission collected using the variable bandpass filter set at 510 nm. Alexa 594 and mCherry were excited using a 543 nm He-Ne laser set at 25% transmission and emission collected using the variable longpass filter set at 618 nm. A 60x, 1.35 NA oil immersion objective was used for imaging. A digital zoom of 2x was often used. Images were acquired at 512x512 resolution. The images were analyzed using ImageJ software (NIH).   3.2.6 Kv4.2 colocalization assay In Rab4 colocalization assays, myocytes were co-transfected with Kv4.2-HA and EGFP- tagged-Rab4 and incubated at 37°C for 24 h. Surface Kv4.2 was labelled with mouse anti-HA (Roche) on ice for 1 h, washed 3 times with cold culture medium, then incubated at 37°C for various times to allow internalization of the labelled Kv4.2-HA. The myocytes were then fixed with 2% paraformaldehyde and permeabilized in PBS containing 0.1% Triton X-100, and non-   66 specific binding was blocked by incubation in 2% BSA in PBS for 30 min at room temperature. Following this, the cells were labelled with Alexa 594 conjugated goat anti-mouse antibody (Molecular Probes) for 1 h to detect Kv4.2-HA. Samples were imaged and analysed as described above.   3.2.7 Tetracycline-induction experiments   pcDNA4/TO-Kv4.2-EGFP was co-transfected with tetracycline repressor, pcDNA6/TR, at a ratio of 1:6 into HEK293 cells. Control cells were also co-transfected with mCherry while cells used to test the effects of Sar1DN and Rab1DN were co-transfected with Sar1DN-mCherry or Rab1DN-mCherry, respectively. Twenty-four hours later, tetracycline was added at a final concentration of 2 µg/ml to culture to induce Kv4.2 expression on the evening prior to electrophysiological analysis.   3.2.8 Data statistics Results are expressed as mean±S.E.M. Statistical analyses were conducted using Student’s t test (paired and unpaired), or by one-way ANOVA, as appropriate.  3.3 Results  3.3.1 Lipofectamine 2000-mediated transfection of ventricular myocytes   Freshly isolated adult rat ventricular myocytes were transfected using a lipofectamine- mediated transfection protocol modified from that reported in Dou, et al., 2010 (see Materials and methods). This modified procedure, which employs an M199-based media unsupplemented with BSA, produces transfected myocytes, at an efficiency of several percent that retain intact morphology and endogenous current profiles without the need for a Biolistic gene gun. Figure   67 3.1A shows wide field fluorescent and bright field views of two myocytes transfected with mCherry in this manner. Red fluorescence is bright in these transfected myocytes and completely absent in the untranstected examples seen alongside. Close up fluorescent, wide field and overlaid views of the myocyte visible in the lower left of the wide field view are shown in Figure 3.1B. Rod-shaped morphology is retained and mCherry fluorescence is readily detectable. Electrophysiological analysis demonstrated that current profiles in mCherry-transfected myocytes matched those of untransfected myocytes cultured under the same conditions (Figure 3.1C and 1D), both of which were essentially identical to those of freshly isolated myocytes.   3.3.2 Sar1 involvement in Kv4.2 trafficking Sar1 is a GTPase that controls the assembly of COPII complexes on ER exit membranes. It is required for transport vesicle formation in the conventional ER-to-Golgi protein transport pathway. Kv4.2 trafficking has been reported to be independent of this GTPase in neurons and a heterologus expression system (Hasdemir et al. 2005). To test whether Sar1 influences Kv4.2 functional expression in ventricular myocytes, electrophysiological experiments were performed on transfected myocytes. Ventricular myocytes were co-transfected with Sar1WT or a dominant negative isoform (Sar1T39N; Sar1DN), and a mCherry construct to allow the ready identification of transfected myocytes. Peak transient outward current (Ito) was decreased to 19.9±2.3 pA/pF at +90 mV in myocytes transfected with Sar1DN plus mCherry from the 37.2±5.6 pA/pF measured in myocytes transfected with mCherry alone. Thus, unlike in HeLa cells, Kv4.2 trafficking in cardiac myocytes is at least partially dependent on Sar1 function.      68  3.3.3 Rab1DN significantly decreases Kv4 functional expression in cardiac myocytes   Another small GTPase known to be important in the trafficking of most membrane proteins from the ER to the Golgi is Rab1. Rab GTPases define the various intracellular trafficking vesicular compartments, regulating the formation of these compartments and recruiting the various effectors required for their function (Pfeffer 2001). Rab1 is required for vesicular traffic from the ER to the cis-Golgi, and for transport between the cis and medial compartments of the Golgi stack (Plutner et al. 1991). The GDP-locked Rab1 S25N (Rab1DN) has been reported to efficiently block trafficking of the Kir3.1/Kir3.4 complex to the plasma membrane (Robitaille et al. 2009). We tested whether this Rab1 dominant negative would similarly affect the trafficking of Kv4.2 in ventricular myocytes. As illustrated in Figure 3.3B, Rab1DN transfection decreased Ito to 21.3±2.0 pA/pF at +90 mV, significantly lower than 33.0±5.6 pA/pF measured in myocytes transfected with mCherry alone. Indicating that endogenous Rab1 levels were not rate limiting in the process, current density in myocytes overexpressing Rab1WT averaged 31.4±5.2 pA/pF at +90 mV, a value indistinguishable from control.   To confirm the essential nature of Sar1 and Rab1 for normal Kv4.2 trafficking in ventricular myocytes, we attempted to employ an inducible expression system in the myocytes so that expression of an introduced tagged channel could be delayed relative to that of the dominant negative GTPases. We were unable to successfully transfect the myocytes with the combination of three plasmid DNAs necessary to accomplish the experiment, however. In light of these failures, we chose to perform the experiment in HEK293 cells, instead. HEK293 cells have proven to traffic another Kv channel, Kv1.5, similarly to its trafficking in a myoblast line (Zadeh et al. 2008). Thus, HEK293 cells were simultaneously transfected with inducible Kv4.2- mCherry ± either Sar1DN or Rab1DN tagged with EGFP. On the next day, Kv4.2 expression   69 was induced with tetracycline and, 12 to 24 hours later, patch clamping was performed. As shown in Figure 3.4, tetracycline induced Kv4.2 expression was very high, at 121.9±14.3 pA/pF at +60 mV in the absence of either of the dominant negatives. Rab1DN pre-expression reduced this current by more than two-thirds, confirming its importance in the normal forward trafficking of the channel. The Sar1DN also reduced expression of the channel, although to a lesser extent. Whether this lower effectiveness of the Sar1DN is due to ineffectiveness of the mutant in completely blocking Sar1 function or whether a parallel, Sar1-independent trafficking pathway for Kv4.2 exists in these cells will be of great interest to determine in future experiments.  3.3.4 Overexpression of Rab11 increases trafficking of newly synthesized Kv4.2 to the sarcolemma Rab11 is also involved in the forward trafficking of many proteins. It localizes to the trans-Golgi network (TGN), to post-Golgi vesicles, as well as to pericentriolar recycling endosomes (Chen et al. 1998; Choi et al. 2005; Ullrich et al. 1996). It mediates both the trafficking of many newly synthesized proteins to the same location and recycling of many endocytosed proteins to the plasma membrane (Balse et al. 2009; Gentzsch et al. 2004; McEwen et al. 2007; Seebohm et al. 2007; Zadeh et al. 2008). As shown in Figure 3.5, expression of a Rab11 dominant negative in rat ventricular myocytes had little effect on Ito. However, in contrast to all other Rabs tested, overexpression of wild type Rab11 substantially increased this current in the cardiac myocytes. Overexpression of Rab11WT caused a significant increase of peak current density in these cells, to 37.9±4.8 pA/pF at +90mV, much higher than the 21.9±2.2 pA/pF present in control, mCherry-transfected myocytes (Figure 3.5B). Indicating that overexpression of Rab11 increases delivery of internal channel into the plasma membrane, the increase in Kv4.2 functional expression mediated by Rab11 overexpression was additive with treatment with   70 dynamin inhibitory peptide (DIP), a compound that blocks endocytosis from the plasma membrane. Peak current density in DIP-treated myocytes overexpressing Rab11WT was 55.6±8.0 pA/pF at +90 mV, much higher than either the 36.0±3.4 pA/pF seen in DIP-treated control myocytes or the 37.9±4.8 pA/pF exhibited by Rab11WT-overexpressing myocytes untreated with DIP (Figure 3.5C and D).  As Rab11 is involved in both recycling of endocytosed channels (Balse et al. 2009; McEwen et al. 2007; Seebohm et al. 2007; Zadeh et al. 2008) and in the trafficking of newly synthesized proteins to the cell surface (Urbe et al. 1993), overexpression of this GTPase might increase Kv4.2 functional expression either by promoting the insertion of channel into the sarcolemmma from an internal pool in recycling endosomes or by promoting the forward trafficking of newly synthesized channel to that membrane (or both). To distinguish between these possibilities, we employed an inhibitor of ER-to-Golgi transport, Brefeldin A (BFA).  As illustrated in Figure 3.6, BFA treatment of Rab11WT-ransfected myocytes 6 h prior to electrophysiological analysis completely blocked the increase in Ito. Whereas Rab11WT overexpression increased Ito current density at +90 mV to 50.9±3.6 pA/pF from the contol 31.3±0.7 pA/pF, in the presence of BFA, no significant difference in current densities between Rab11WT-transfected cells (19.5±10.7 pA/pF) and control cells (20.9±2.3 pA/pF) was evident (Figure 3.6B). Thus, overexpression of wild type Rab11 increases Ito in ventricular myocytes by increasing the forward trafficking of newly synthesized channel to the sarcolemma in these cells.   3.3.5 Rab5 is involved in Kv4.2 internalization Once delivered to the plasma membrane, a channel must eventually be reinternalized. To study the mechanisms by which Kv4.2 is internalized and recycled to the plasma membrane, we investigated the involvement of several Rab GTPases previously shown to affect the trafficking   71 of other potassium channels. Rab5 is required for clathrin-mediated endocytosis and early endosome formation (Bucci et al. 1992). It has been shown to be involved in Kv1.5 endocytosis; a Rab5 dominant negative (Rab5DN) mutant, Rab5S34N, increases Kv1.5 surface expression by interfering with endocytosis of the channel (Zadeh et al. 2008). We tested electrophysiologically whether Rab5 was involved, also, in the trafficking of Kv4.2. As shown in Figure 3.7, transfection with Rab5DN significantly increased Ito current densities in transfected ventricular myocytes. Peak current density at +90 mV was 55.3±6.9 pA/pF in Rab5DN expressing cells and 21.9±2.2 pA/pF in mCherry transfected control cells (Figure 3.7B). Overexpression of Rab5WT had no effect on Ito in these myocytes, indicating that endogenous levels of this GTPase are not rate limiting for Kv4.2 endocytosis. To confirm that Rab5DN affects Ito by interfering with the endocytosis of the underlying Kv4.2 channel, we applied Dynamin Inhibitory Peptide (DIP) to the transfected myocytes 6 h prior to performing electrophysiological experiments. An inhibitor of dynamin GTPase activity, DIP blocks endocytosis by competitively inhibiting the interaction of dynamin with amphiphysin (Shupliakov et al. 1997; Wigge et al. 1997). When applied to ventricular myocytes, DIP treatment significantly increased peak current density at +90 mV from 27.1±4.6 pA/pF of control cells to 49.8±4.9 pA/pF, but was not additive with Rab5DN (peak current density at +90 mV = 50.0±5.7 mV) (Figure 3.7C and D). Thus, it is highly likely that both Rab5DN and DIP are interfering with Kv4.2 trafficking by inhibiting endocytosis of the channel.   3.3.6 Rab4 involvement in Kv4.2 trafficking Rab4 associates with early endosomes shortly after their formation and regulates the rapid recycling of internalized surface proteins. Previous observations in cell culture systems indicate that Rab4 is involved in the rapid recycling of internalized Kv1.5 channels (Zadeh et al.   72 2008). We therefore investigated whether Rab4 is involved in endogenous Kv4.2 trafficking. As shown in Figure 3.8, when transfected into ventricular myocytes, Rab4DN expression led to a significant increase in peak current density. Peak current density in Rab4WT-overexpressing myocytes was 25.1±3.2 mV at +90 mV, no different from the 21.7±4.1 pA/pF measured in control myocytes transfected with mCherry alone (Figure 3.8B). Expression of a Rab4DN in the myocytes, however, resulted in a significant increase in peak Ito, i.e., to 37.4±4.4 pA/pF at +90 mV. This result is similar to our previously published finding that Kv1.5 surface expression is increased by this dominant negative (Zadeh et al. 2008). In that case, we demonstrated that the Rab4DN interfered with channel internalization, quite possibly by preventing maturation of early endosomes. To test whether the Rab4DN was interfering with endogenous Kv4.2 internalization, we tested whether its effects were additive with direct block of endocytosis by dynamin inhibitory peptide. As illustrated in Figure 3.8C and D, the effects of the two treatments indeed were not additive. Peak current densities in mCherry-transfected and mCherry plus Rab4DN myocytes treated with DIP were statistically identical at 49.5±4.0 and 46.1±2.6 pA/pF, respectively, at +90 mV, higher than the control 28.1±3.9 pA/pF in untreated mCherry transfected myocytes. Thus, it is highly likely that Rab4DN is, like DIP, interfering with the endocytosis of endogenous Kv4.2.  The interference by the Rab4DN with the endocytosis of Kv4.2 does not demonstrate that Rab4 is normally involved in the trafficking of the channel. As Rab4 function is generally in the trafficking of membrane proteins back to the plasmalemma, the dominant negative isoform might be simply mucking up endocytosis in general and artifactually interfering with Kv4.2 expression. To determine whether the channel does indeed traffic through a Rab4-positive compartment, we looked for colocalization of the channel with wild type Rab4. To accomplish this, freshly isolated ventricular myocytes were transfected with an extracellularly HA-tagged   73 Kv4.2 construct (a kind gift of Dr. Robert Bähring, University of Hamburg, Germany) and Rab4WT-GFP. The next day, the myocytes were incubated with anti-HA at 37˚C for variable times before fixation and staining for the anti-HA with Alexa594. As illustrated in Figure 3.9, there was no colocalization of Kv4.2 and Rab4 at 0 minutes (Figure 3.9, top panels) but, by 45 minutes, Rab4 colocalization with internalized Kv4.2 was readily apparent (Figure 3.9, middle panels). By six hours internalization time, this colocalization was quite extensive (Figure 3.9, bottom panels). Thus, internalized Kv4.2 does indeed traffic through the Rab4-positive compartment, very probably recycling rapidly to the cell surface.   3.3.7 Kv4.2 degradation likely occurs in the proteasome Recycled, or not, eventually channels must be degraded. We have previously shown that a portion of internalized Kv1.5 in a heterologous cells and a cardiomyoblast cell line is shunted to the lysosome for degradation via Rab7-positive endosomes (Zadeh et al. 2008). Overexpression of Rab7WT decreased Kv1.5 functional expression in the myoblast cell line whereas similar expression of a Rab7DN in HEK293 cell line increased functional expression of the channel. Rab7 reportedly localizes to early endosomes as they mature into late endosomes and has been shown to be critical for trafficking through the lysosome (Bucci et al. 2000; Meresse et al. 1995). To determine whether Rab7 plays a significant role in the expression of endogenous Kv4.2 in ventricular myocytes, we tested the effects of overexpression of Rab7WT and DN isoforms in this cell type. As shown in Figure 3.10, neither Rab7WT nor Rab7DN had any effect on Ito. Current densities in Rab7WT and DN-transfected myocytes were 53.5±7.3 and 40.2±9.4 pA/pF at +90 mV, indistinguishable from the 40.9±3.9 pA/pF measured in control myocytes transfected with mCherry alone. Indicating that endogenous Kv4.2 in these myocytes may instead be degraded by   74 the proteasome, the proteasome inhibitor MG132 increased peak current densities by roughly 40% from 25±3.4 to 34.3±3.3 pA/pF (Figure 3.11).  3.4 Discussion   This work has established for the first time the involvement of several Rab GTPases as well as Sar1 in the trafficking of an endogenous cardiomyocyte potassium channel. The trafficking of Kv4.2 out of the ER, on to the Golgi and the sarcolemma, and, thereafter, its internalization from and its recycling to the sarcolemma have been mapped. Kv4.2 traffics out of the cardiac endoplasmic reticulum via a conventional pathway involving Sar1 and Rab1 and its trafficking to the sarcolemma is enhanced by overexpression of wild type Rab11. Once at the sarcolemma, the channel behaves in much the same way as Kv1.5 was previously demonstrated to traffic in a myoblast cell line (Zadeh et al. 2008). Like Kv1.5, internalization is dependent upon Rab5 function and block of Rab4 somehow also inhibits that internalization. Unlike the results previously reported with Kv1.5, however, overexpression of Rab11 wild type greatly increases surface expression of endogenous Kv4.2 as assayed electrophysiologically and the channel does not appear to traffic to the lysosome for degradation. Proteasomal degradation of the channel appears more likely to be operative.   For the most part, the trafficking of endogenous Kv4.2 in ventricular myocytes appears to be conventional. That Kv4.2 traffics through a conventional ER-to-Golgi pathway in cardiomyocytes is significant. This is very different than the situation in a HeLa cells transfected with the channel and KChIP1 (Flowerdew and Burgoyne, 2009; Hasdemir et al. 2005).  In the HeLa cells, Kv4.2/KChIP1 traffic independently of Sar1, traveling from the ER to the Golgi instead via vesicles uncoated with COPII and, in a majority of cells, reaching the plasmalemma even in the presence of a Sar1 dominant negative that completely blocked the vesicular-   75 stomatitis virus G-protein (VSVG) traffic to that membrane (Flowerdew and Burgoyne, 2009). Hasdemir et al. (2005), hypothesize that Kv4.2 traffics from the ER to the Golgi via ‘KChIP vesicles’ that, in the case of KChIP1 at least, are demonstrably independent of Sar1 function. KChIP1 is predominant in brain (Pourrier et al. 2003) but not in the heart (Schultz et al. 2005). Thus, a prime possibility is that KChIP2-dependent trafficking of Kv4.2 is very different than that driven by KChIP1. KChIP2 (3 splice variants) is predominant in cardioymyotes (Schultz et al. 2005). Perhaps KChIP2 traffics very differently than KChIP1. Indeed, Flowerdew et al. (2009) found that KChIP2-mediated Kv4.2 trafficking did not involve the same SNARE protein intermediaries (VAMP-7 and Vti1A) as that mediated by KChIP1.   Also of interest is the increase in endogenous Kv4.2 expression when Rab11 wild type is overexpressed in the ventricular myocytes. Kv1.5 expression in H9c2 myoblasts was unaffected by overexpression of wild type Rab11 and affected by the dominant negative only after prolonged co-expression (Zadeh et al. 2008). The dominant negative did, however, block the increase in Kv1.5 associated with cholesterol depletion in atrial myocytes (Balse et al. 2009). In these ventricular myocytes, the dominant negative, like its effect on Kv1.5 in the myoblasts, is negligible. The increase in Ito associated with overexpression of wild type Rab11, however, contrasts dramatically with the findings with Kv1.5. There are numerous pathways through which a channel can potentially traffic from the Golgi to the plasmalemma. In addition to traffic through Rab11-associated compartments, membrane proteins can traffic instead through several additional pathways (reviewed in (Stenmark 2009)). Thus, it is not surprising that different channels are differentially affected by manipulation of one of the pathways. The failure of the dominant negative Rab11 mutant to significantly reduce Kv4.2 currents in the myocytes, however, makes interpretation of the phenomenon more difficult. Perhaps Kv4.2 is quite stable at the cell surface, or internalizes/recycles rapidly and independently of Rab11 function. In this   76 scenario, a loss of forward trafficking due to block of Rab11 function would have little effect on net surface expression. The fact that internalized Kv4.2 colocalizes well with Rab4-positive endosomes, known to generally drive rapid recycling, is consistent with this hypothesis. In addition to its role in Kv1.5 recycling, Rab4 is known to be involved with the rapid recycling of other membrane proteins such as the transferrin receptor (Sheff et al. 2002) and the amiloride- sensitive sodium channel, ENaC (Saxena et al. 2006).   Our data indicates that degradation of internalized Kv4.2 likely differs, at least in part, from that of Kv1.5 as well. Whereas Kv1.5 has been shown to traffic to the lysosome via Rab7- positive endosomes (Zadeh et al. 2008) and to be degraded also by the proteasome (Kato et al. 2005), endogenous Kv4.2 in ventricular myocytes very likely does not traffic to the lysosome. Functional expression is unaffected by overexpression of wild type or dominant negative Rab7 isoforms. That incubation with the proteasome inhibitor MG132 increases Ito indicates that Kv4.2 most likely is, like CFTR (Ward et al. 1995) and hERG channels (Gong et al. 2005), degraded by the proteasome.   In summary, we have implicated several Rab GTPases plus Sar1 in the trafficking of endogenous Kv4.2 in adult ventricular myocytes. It will be interesting to determine how the trafficking of Kv4.2 intersects with that of other potassium channels in these myocytes and how these trafficking pathways are modulated both in health and disease.    77                    Figure 3.1 Lipofectamine-mediated transfection of adult ventricular myocytes (A) Wide- field fluorescent (left) and bright field (right) image of adult rat ventricular myocytes transfected with mCherry. B. Close-up views of the transfected myocyte visible in the lower left of the panels in (A). C. Representative currents from untransfected and mCherry-transfected rat ventricular cardiomyocytes recorded 12-24 hours post-transfection or, in the case of untransfected myocytes, a similar culture period. Currents were elicited by 500 ms pulse stepping from a holding potential of -80 mV to test potentials of -80 to +90 mV in 10 mV increments followed by a 100 ms pulse to +60 mV. D. Current density versus voltage plot for data for mCherry-transfected and untransfected myocytes, respectively. Data are represented as mean ± SEM.              BA C 100 µm 20 µm CherryControl D -80 -60 -40 -20 0 20 40 60 80 100 Pe ak  c ur re nt  d en si ty  (p A /p F) 0 10 20 30 40 Cherry transfected (n=15) Untransfected (n=7) 2 nA 200 ms 2 nA 200 ms Pulse potential (mV)P ea k cu rr en t d en si ty  (p A /p F)   78                                Figure 3.2 Normal expression of Ito requires intact Sar1 function A. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post-transfection with mCherry, mCherry + Sar1WT or mCherry + Sar1DN. Current protocols were as described in Figure 3.1. B. Current density versus voltage plot for data for mCherry-, mCherry + Sar1WT-, and mCherry + Sar1DN (T39N) transfected myocytes, respectively. Data are represented as mean ± SEM. * p<0.05, comparing Sar1DN + mCherry to control, mCherry-transfected myocytes.            Cherry Sar1DNSar1WT A B 200 ms Pulse potential (mV) -80 -60 -40 -20 0 20 40 60 80 100 T39N (n=10) Control (n=19) Sar1WT (n=11). * * ****0 10 20 30 40 Pe ak C ur re nt  d en si ty  (p A /p F) 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms Pe ak C ur re nt  d en si ty  (p A /p F)   79                                Figure 3.3 Normal expression of Ito requires intact Rab1 function A. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post-transfection with mCherry, mCherry + Rab1WT or mCherry + Rab1DN. Current protocols were as described in Figure 3.1. B. Current density versus voltage plot for data for mCherry-, mCherry + Rab1WT-, and mCherry + Rab1DN (S25N) transfected myocytes, respectively. Data are represented as mean ± SEM. * p<0.05, comparing Rab1DN + mCherry to control, mCherry-transfected myocytes.            Pe ak C ur re nt  d en si ty  (p A/ pF ) -80 -60 -40 -20 0 20 40 60 80 100 0 10 20 30 40 50 * ***** * *** Control (n=10) Rab1WT (n=10) Rab1DN (n=12) Pulse potential (mV) Cherry Rab1 WT Rab1 DNA B 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms Pe ak C ur re nt  d en si ty  (p A/ pF )   80   A                               Figure 3.4 Expression of an inducible Kv4.2 in HEK293 cells is significantly reduced by coexpression of Rab1DN and Sar1DN HEK293 cells were transfected with Kv4.2-EGFP in pcDNA4/TO and pcDNA6/TR and one of mCherry, Sar1DN-mCherry and Rab1DN-mCherry. Kv4.2-EGFP expression was induced 24 hours later and electrophysiological experiments were performed the following day. A. Representative currents from rat ventricular cardiomyocytes recorded. B. Current density versus voltage were plotted from data recorded 12-24 hours post- induction. * p<0.05, relative to Kv4.2 control.    20 40 60 80 100 120 140 -80 -70 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60 70 Pulse potential (mV) Pe ak  C ur re nt  D en si ty  (p A /p F) * * * * * * * *** * * * * * Kv4.2 control (n=16) Kv4.2 + Sar1DN (n=13) Kv4.2 + Rab1DN (n=9) 0 Pe ak  C ur re nt  D en si ty  (p A /p F) Kv4.2 control Kv4.2 + Sar1DN Kv4.2 + Rab1DN B   81                                Figure 3.5 Overexpression of Rab11WT substantially increases Ito in transfected ventricular myocytes A. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post-transfection with mCherry, mCherry + Rab11DN or mCherry + Rab11WT. Current protocols were as described in Figure 3.1. B. Current density versus voltage plot for data for mCherry-, mCherry + Rab11WT-, and mCherry + Rab11DN transfected myocytes, respectively. Data are represented as mean ± SEM. * p<0.05, comparing Rab11WT + mCherry to control, mCherry-transfected myocytes. C. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post-transfection with mCherry or Rab11-WT + mCherry, incubated for 6 hours with dynamin inhibitory peptide (DIP) as indicated. Current protocols were as described in Figure 3.1. D. Current density versus voltage plot for myocytes thus treated. Data are represented as mean ± SEM. * p<0.05, relative to control, mCherry-transfected myocytes.     Pulse potential (mV) -80 -60 -40 -20 0 20 40 60 80 100 Pe ak  c ur re nt  d en si ty  (p A/ pF ) 0 10 20 30 40 50 Rab11DN (n=14) Rab11WT (n=6) Cherry control (n=6) Pulse potential (mV) -80 -60 -40 -20 0 20 40 60 80 100 Pe ak  c ur re nt  d en si ty  (p A/ pF ) 0 10 20 30 40 50 60 70 Rab11 WT + DIP (n=7) Cherry + DIP (n=9) Cherry control (n=5) * * * * * * * * * * * * * * * * * * * * * * * Cherry Rab11WT+DIPCherry+DIP B A C D *********** Cherry Rab11DN Rab11WT 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms Pe ak  c ur re nt  d en si ty  (p A/ pF ) Pe ak  c ur re nt  d en si ty  (p A/ pF )   82                Figure 3.6 Brefeldin A treatment prevents Rab11WT overexpression effect on Ito A. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post- transfection with mCherry, or mCherry + Rab11WT incubated for 6 hours with Brefeldin A as indicated. Current protocols were as described in Figure 3.1. B. Current density versus voltage plot for data from myocytes treated as described in A. Data are represented as mean ± SEM. * p<0.05, relative to control, mCherry-transfected myocytes.             A 2 nA 200 ms 2 nA 200 ms Rab11WT+BFA Cherry +BFA Rab11WT Cherry B Pulse potential (mV) -80 -60 -40 -20 0 20 40 60 80 100 Pe ak  c ur re nt  d en si ty  (p A/ pF ) 0 10 20 30 40 50 60 Rab11 + BFA (n=4) Cherry control + BFA (n=4) Rab11 (n=5) Cherry control (n=5) * * * ** * * * * * * * * * * ** 2 nA 200 ms 2 nA 200 ms Pe ak  c ur re nt  d en si ty  (p A/ pF ) Pe ak  c ur re nt  d en si ty  (p A/ pF )   83                       Figure 3.7 Interference with Rab5 function increases Ito in transfected ventricular myocytes A. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post-transfection with mCherry, mCherry + Rab5WT or mCherry + Rab5DN. Current protocols were as described in Figure 3.1. B. Current density versus voltage plot for data for mCherry-, mCherry + Rab5WT-, and mCherry + Rab5DN transfected myocytes, respectively. Data are represented as mean ± SEM. * p<0.05, comparing Rab5DN + mCherry to control, mCherry- transfected myocytes. C. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post-transfection with mCherry or Rab5-DN + mCherry, incubated for 6 hours with dynamin inhibitory peptide (DIP) as indicated. Current protocols were as described in Figure 3.1. D. Current density versus voltage plot for myocytes thus treated. Data are represented as mean ± SEM.    CA Pulse potential (mV) -80 -60 -40 -20 0 20 40 60 80 100P ea k cu rr en t d en si ty  (p A /p F) 0 10 20 30 40 50 60 70 Rab5DN (n=8) Rab5WT (n=9) Cherry control (n=10) Pulse potential (mV) -80 -60 -40 -20 0 20 40 60 80 100Pe ak  c ur re nt  d en si ty  (p A/ pF ) 0 10 20 30 40 50 60 Rab5DN + DIP (n=9) Cherry + DIP (n=9) Cherry control (n=15) Rab5WT * * * * * * * * * * * * Cherry +DIPRab5DN+DIPCherry B D Cherry Rab5DN 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms Pe ak  c ur re nt  d en si ty  (p A /p F) Pe ak  c ur re nt  d en si ty  (p A/ pF ) Pe ak  c ur re nt  d en si ty  (p A /p F) Pe ak  c ur re nt  d en si ty  (p A/ pF )   84                 Figure 3.8 Rab4DN expression also increases Ito in transfected ventricular myocytes A. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post- transfection with mCherry, mCherry + Rab4WT or mCherry + Rab4DN. Current protocols were as described in Figure 3.1. B. Current density versus voltage plot for data for mCherry-, mCherry + Rab4WT-, and mCherry + Rab4DN transfected myocytes, respectively. Data are represented as mean ± SEM. * p<0.05, comparing Rab4DN + mCherry to control, mCherry-transfected myocytes. C. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post-transfection with mCherry or Rab4-DN + mCherry, incubated for 6 hours with dynamin inhibitory peptide (DIP) as indicated. Current protocols were as described in Figure 3.1. D. Current density versus voltage plot for myocytes thus treated. Data are represented as mean ± SEM.    A Rab4DNCherry Rab4WT Pulse potential (mV) -80 -60 -40 -20 0 20 40 60 80 100Pe ak  c ur re nt  d en si ty  (p A /p F) 0 10 20 30 40 50 Rab4DN (n=6) Rab4WT (n=8) Cherry control (n=7) Pulse potential (mV) -80 -60 -40 -20 0 20 40 60 80 100 Pe ak  c ur re nt  d en si ty (p A/ pF ) 0 10 20 30 40 50 60 Rab4DN + DIP (n=14) Cherry + DIP (n=11) Cherry control (n=18) * * * * Cherry Rab4DN+DIP Cherry+DIP C B D 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms Pe ak  c ur re nt  d en si ty  (p A /p F) Pe ak  c ur re nt  d en si ty (p A/ pF ) Pe ak  c ur re nt  d en si ty  (p A /p F) Pe ak  c ur re nt  d en si ty (p A/ pF )   85                 Figure 3.9 Internalized Kv4.2 colocalizes with Rab4 in ventricular myocytes Ventricular myocytes were transfected with externally HA-tagged Kv4.2 plus EGFP-tagged Rab4, then, 24 hours later incubated with mouse anti-HA at 37oC for the times indicated prior to fixation then staining with Alexa 594 goat anti-mouse and imaged using an Olympus Fluoview 1000 confocal microscope (see Materials and Methods). Arrows highlight some of the vesicles in which Kv4.2HA and Rab4-EGFP colocalize. Scale bar, 20 µm.       Kv4.2HA (Alexa 594) Rab4GFP Colocalization 45 min 0 min 6 hours   86                Figure 3.10 Rab7 function appears irrelevant to maintenance of endogenous Ito A. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post- transfection with mCherry, mCherry + Rab7DN or mCherry + Rab7WT. Current protocols were as described in Figure 3.1. B. Current density versus voltage plot for data for mCherry-, mCherry + Rab7WT-, and mCherry + Rab7DN transfected myocytes, respectively. Data are represented as mean ± SEM.       Rab7DN Pulse potential (mV) -80 -60 -40 -20 0 20 40 60 80 100 Pe ak  c ur re nt  d en si ty  (p A /p F) 0 10 20 30 40 50 60 70 Rab7DN (n=9) Rab7WT (n=8) Cherry control (n=9) A B Cherry Rab7WT 2 nA 200 ms 2 nA 200 ms 2 nA 200 ms Pe ak  c ur re nt  d en si ty  (p A /p F)   87                           Figure 3.11 Incubation with proteasome inhibitor significantly increases the magnitude of Ito A. Representative currents from rat ventricular cardiomyocytes recorded approximately 24 hours post-isolation treated, where indicated, with MG132, for 6 hours immediately prior to recording. Current protocols were as described in Figure 3.1. B. Current density versus voltage plot for data for the myocytes thus treated. Data are represented as mean ± SEM. * p<0.05, comparing mean densities in MG132-treated myocytes to control,untreated myocytes.    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J Biol Chem 282, 25760-25768.     92 CHAPTER 4 CONCLUSION AND RECOMMENDATIONS FOR FUTURE WORK 4.1 Overall summary and conclusions   The rhythmic pumping action of the heart is dependent on action potential generation and propagation, followed by relaxation and a period of refractoriness until the next impulse is generated. Due to different time- and voltage-dependent activities of the various channels underlying it, the action potential is achieved through sequential activation and inactivation of Na+, Ca+, and K+ channels. When ion channel expression and activity are normal, unidirectional propagation of action potentials are produced and normal cardiac rhythms are generated. However, when the activity of ion channels is perturbed, action potential duration will be affected and can lead to the generation of life-threatening arrhythmias. Changes in activities or surface expression of Kv channels contribute to most of the inherited and acquired cardiac arrhythmias. Due to their abundant expression in the heart and importance in the repolarization phase of the action potential, it is of great interest to understand the regulatory mechanisms behind Kv channels’ functional surface expression.   The studies presented in this thesis first investigated the molecular mechanisms implicated in the delivery of cardiac Kv1.5 channel to the plasma membrane in heterologous cells and cardiac myoblasts, providing important insights into the requirement for a cytoskeletal motor, kinesin I (Kif5b) for cardiac Kv channel forward trafficking. In this study, wild type (WT) and dominant negative (DN) isoforms of Kif5b were transfected into the cells and the effects of these motor proteins on Kv1.5 current density and surface expression were determined via electrophysiological and immunocytochemical analyses. Surprisingly, both WT and DN Kif5b isoforms increased Kv1.5 channel surface expression and current density. We hypothesized that these increases in Kv1.5 channel functional expression were brought about by   93 very different mechanisms. To discern the mechanisms, a number of interventions were employed. It is known that interference with at least some kinesins indirectly interferes also with dynein function (Hamm-Alvarez et al. 1993; Valetti et al. 1999). So, we hypothesized that whereas wild type Kif5b was likely simply increasing forward trafficking of Kv1.5 to the plasma membrane, the dominant negative might be indirectly interfering instead with retrograde trafficking of the channels. Confirming our suspicions, we found that manipulations known to increase Kv1.5 functional expression by blocking retrograde transport – i.e., a) by disrupting the dynein-dynactin complex through overexpression of the dynactin component p50, b) blocking vesicle scission from the plasma membrane or c) deleting SH3 domain known to be required for internalization of Kv1.5 channel, – were all additive with the effects of overexpressing Kif5bWT but non-additive with the effects of Kf5bDN expression. These results strongly suggested that Kif5bWT was likely to be involved in transporting Kv1.5 channel to the cell surface while Kifb5DN-associated increase in Kv1.5 current was likely to be due to indirect interference with endocytosis of the channel. The enhancement of Kv1.5 current by Kif5bWT was blocked by a six hour treatment with Brefeldin A, a drug which blocks transport of newly synthesized protein from the ER to the Golgi. Brefeldin A had no such effect on the Kif5bDN-associated increase in Kv1.5 current. Finally, directly demonstrating that Kif5b function is required for the delivery of Kv1.5 to the cell surface, expression of Kif5bDN prior to induction of Kv1.5 expression in a tetracycline-inducible system almost completely blocked Kv1.5 current. Collectively, these results demonstrate that Kif5b is required for the forward trafficking of newly synthesized Kv1.5 channel to the plasma membrane. Thus, the motor is closely involved in the regulation of Kv1.5 surface expression.   94   After the first study was completed, our lab developed new techniques that, for the first time, allow the ready transfection of adult rat ventricular myocytes with standard mammalian expression vectors (Dou et al. 2010). Therefore in the second study, we decided to investigate cardiac Kv channel trafficking using ventricular myocytes. Because Kv1.5 channels have no known functional expression in rat ventricular myocytes, we shifted our attention to Kv4.2 channels which are expressed abundantly in these cells. In this second study, the ER-to-Golgi anterograde as well as the retrograde trafficking machineries underlying the expression of cardiac Kv4.2 were explored. The channel was found to traverse pathways involving several Rab GTPases and the Sar1 GTPases. Specifically, the effects of expressing WT and DN isoforms of Sar1, Rab1, Rab4, Rab5, Rab7, and Rab11 in myocytes were assessed using electrophysiology and imaging techniques. It was found that Sar1DN and Rab1DN prevented endogenous Kv4.2 channel from reaching the plasmalemma. Indicating that it, too, may be involved in the forward trafficking of the channel, Rab11WT overexpression caused an increase of Kv4.2 surface expression, an effect that was additive to DIP block of endocytosis. This Rab11WT-associated increase of Kv4.2 functional expression was blocked by Brefeldin A, again suggesting involvement of Rab11 in Kv4.2 forward trafficking. Rab5DN greatly increased Kv4.2 current, very probably by interfering with the channel internalization from plasma membrane. Rab4DN also increased Kv4.2 current, possibly by indirectly blocking Rab5-mediated endocytosis. Finally, Rab7, important in lysosomal targeting of internalized proteins, appeared to have no significant role in Kv4.2 trafficking.   Together, these two studies not only identified the players in the forward and retrograde trafficking pathways, but also improved our understanding of how Kv channel functional expression can be regulated by the trafficking machinery.   95 4.2 Players involved in cardiac Kv channel forward trafficking  4.2.1 Kinesin isoform Kif3, Kif5 or Kif17?   It is well known that trafficking and targeting of Kv channels in neurons are kinesin motor-dependent. Chu et al. (2006) introduced dominant negative constructs of several kinesins found in dendrites, and observed that only a dominant negative isoform of Kif17 dramatically inhibited the transport of endogenous and exogenous Kv4.2. They also showed that Kv4.2 colocalizes with Kif17 but not with other kinesin isoforms and these two proteins co- immunoprecipitate from brain lysate. Thus, Kif17 is very probably the motor that transports Kv4.2 from its sites of synthesis in the cell body to the dendrites. On the other hand, Kv1.2 axonal targeting has been shown to require another kinesin isoform, Kif3; siRNA knockdown of endogenous Kif3 in hippocampal neurons impairs Kv1.2 axonal targeting (Gu et al. 2006). Another group demonstrated that in cortical neurons, Kif5b is involved in the transportation and axonal targeting of endogenous Kv1.1, Kv1.2, and Kv1.4 channels, they showed that a dominant negative isoform of Kif5b, but not a Kif17DN, prevents axonal localization of both endogenous and introduced Kv1 channels (Rivera et al. 2007).   However, until the work described in this thesis was done, there was not a single study published on the possible role(s) of kinesin isoforms in cardiac Kv channel trafficking. Thus, it was an interesting question whether cardiac Kv channels are trafficked by Kif3, Kif5, Kif17 or other kinesin isoforms. Our study, presented in Chapter 2, found that knocking down endogenous Kif5b function almost completely abolished newly synthesized Kv1.5 current and surface expression; it was the first to demonstrate that Kif5b isoform is essential for Kv1.5 channel forward trafficking with a cardiac background. Although one type of kinesin motor may transport multiple types of vesicular cargoes, there are a large number of different kinesin   96 isoforms in the cell and several kinesins may be involved in mediating vesicular transport of cardiac Kv channels. Therefore, other cardiac Kv channels may require different kinesins for their plasmalemmal expression and the requirement for a specific kinesin isoforms for those Kv channels would definitely be worth looking into.   Recently, it was found that the cell surface level of Kv1.5 in HL-1 atrial myocyte cell line was decreased by expression of a dominant negative version of Kif17 (Cai et al. 2009). In addition, Cai et al. (2009) also observed, by using live cell imaging, that the Kif17 dominant negative isoform decreased Kv1.5 vesicle motility, both in the average distance travelled and average net velocity of the vesicles but none of these was observed with the expression of a dominant negative version of Kif5 isoform. The authors therefore suggested that Kif5 does not have a role in Kv1.5 trafficking in atrial myocytes. Despite their claim, however, it is not surprising that their findings are not in agreement with ours. Cai et al. employed a dominant negative Kif5c construct instead of a Kif5bDN. Although the two Kif5 isotypes are highly homologous, Kif5c expression is restricted in retina and brain and is not expressed in the heart whereas the Kif5b isoform is ubiquitously expressed including, robustly, in the heart (Cai et al. 2001). Thus, it is likely that Kif5b is indeed involved in cardiac myocyte trafficking of Kv1.5 and Kif17 likely plays a role as well.   4.2.2 Sar1 or Rab1-dependent or not?   The conventional ER-to-Golgi trafficking pathway has been elucidated in great detail largely by following the traffic of a temperature-sensitive mutant of the vesicular-stomatitis virus G-protein (VSVG). Transport of VSVG from the ER includes budding of COPII-coated vesicles as regulated by Sar1 (Kuge et al. 1994) followed by fusion of those vesicles with the cis-Golgi in   97 a Rab1-dependent process (Allan et al. 2000). However, there is growing evidence that this ‘conventional’ pathway is not the only pathway via which vesicles are transported from the ER to the Golgi. For example, Hasdemir et al (2005) have shown that the Kvβ subunit, KChIP1 traffics via a Sar1- and COPII-independent pathway in both heterologous (HeLa) cells and hippocampal neurons. KChIPs are constitutive β subunits of Kv4 channels; they promote the trafficking of Kv4 channels to the plasma membrane (Shibata et al. 2003). In the absence of KChIP expression, the bulk of the channels remain trapped in the ER (Hasdemir et al. 2005). The trafficking of the CFTR has also been demonstrated to be non-conventional. Traffic of these channels is Rab1-independent and at least a fraction of the trafficking may skip the Golgi altogether, involving instead the late endosomal system and recycling pathway (Yoo et al. 2002). Another example of transmembrane protein that traffics Rab1-independently is α2B-adrenergic receptor, which was shown to retain its functional surface expression and downstream signalling when endogenous Rab1 was knockdown by siRNA and dominant negative mutant (Wu et al. 2003).   As mentioned above, previous studies on small GTPase involvement in cardiac potassium channel trafficking were conducted in heterologous cell systems or myoblast cell lines. Our work is the first to define the pathways by which an endogenous Kv channel traffics in actual cardiac myocytes. Also, as mentioned above, we found (Chapter 3) that endogenous cardiac Kv4.2 trafficking pathway is both Sar1- and Rab1-dependent, a situation unlike the Sar1-independence previously reported in hippocampal neurons. Interestingly, though, we have found that other potassium channels in the heart do traffic independently of Sar1. We also investigated the effect of expressing Sar1 and Rab1 dominant negative isoforms on the functional expression of endogenous Kir2.1 and transiently transfected Kv1.5 in our myocyte system. The dominant   98 negative Rab1 mutant decreased current densities of Kv1.5 and Kir2.1 (Figure 4.1) while Sar1DN had no effect on the current densities of either channel (Figure 4.2). Clearly, the anterograde trafficking of potassium channels in the heart varies according to channel type. It will be very interesting to more precisely define the pathways and to ascertain which channel(s) traverse which pathways and how traffic through those pathways is regulated.   4.2.3 Rab11 mediates forward trafficking or slow recycling?   Rab11 has been well established to be involved in both recycling of some internalized membrane proteins to the plasma membrane and in forward trafficking from the Golgi apparatus to the same membrane (Zerial and McBride, 2001). Indicating that it is not normally a major player in Kv1.5 recycling, Zadeh et al. (2008), reported that internalized Kv1.5 colocalizes with Rab11 only after prolonged periods of internalization and that the combined effects of disruption of microtubules by nocodazole plus Rab11 dominant negative expression were additive. These results probably reflect the involvement of Rab11 in forward trafficking of newly synthesized Kv1.5 to the plasmalemma as well as the recycling of internalized channels, only the latter of which was touched on by imaging. However, Zadeh et al. (2008) did not determine the mechanisms behind the Rab11 wild type-associated increase in Kv1.5 current. In Chapter 3, we reported wild type of Rab11 causes an increase in endogenous Kv4.2 currents. We went on to investigate whether the increase was due to enhanced forward trafficking of newly synthesized channel or was somehow related to enhanced recycling. When the trafficking of newly synthesized Kv4.2 channels was blocked through the use of Brefeldin A, we found that the Rab11 wild type-associated increase was completely eliminated. This strongly suggests that   99 Rab11 is involved in forward trafficking and, if it is involved at all in the slow recycling of Kv4.2, that involvement is slight.  4.3 Players involved in cardiac Kv channel retrograde trafficking  4.3.1 Kv channel internalization   Phosphorylation by tyrosine kinases is an important mechanism for the regulation of at least several Kv channels. The first evidence of this was found in a study of Kv1.2 in 1993 (Huang et al. 1993). Within a few years, other members of the Kv1 family, such as Kv1.3 and Kv1.5 were also found to undergo tyrosine kinase-dependent regulation (Holmes et al. 1996; Fadool and Levitan, 1998). However, the mechanisms by which tyrosine kinases modulate any of the Kv channels was not well understood until Nesti et al. (2004) demonstrated that Kv1.2 current suppression and channel internalization were both eliminated when a specific Kv1.2 N- terminal tyrosine residue was mutated to phenylalanine. Incubation of cells expressing the wild type channel with a dynamin inhibitory peptide also blocked this tyrosine kinase-associated internalization, confirming the role of endocytosis in this down regulation of Kv1.2 functional expression.   Besides phosphorylation, modification by ubiquitin ligase can also regulate endocytosis of Kv channels. Overexpression of the ubiquitin ligase Nedd4-2 reduced Kv1.5 current by ubiquitinating and thereby reducing Kv1.5 plasma membrane expression (Boehmer et al. 2008). Plasmalemmal expression of Kv1.3 has also been shown to be down regulated by Nedd4-2 and upregulated by expression of serum and glucocorticoid inducible kinase SGK1, which inactivates the ubiquitin ligase Nedd4-2 (Henke G et al. 2004).  Clearly, there are signals behind at least some Kv channel internalization. But, random processes may also be operative. It is known that   100 post-internalization fate of the transferrin receptor, which at least superficially traffics similarly to Kv1.5 (Zadeh et al. 2008), is stochastic. Whether the receptor is recycled or shunted off to degradation is essentially random (Lakadamyali et al. 2006).   4.3.2 Rab4 and Rab5   It is well established that retrograde trafficking of Kv1.5, Kv4.2 and several other cardiac Kv channels is a dynein and microtubule-dependent process (Choi et al. 2005; Loewen et al. 2009). Dynamic retrograde trafficking of cardiac Kv via the microtubules also requires Rab proteins. The involvement of Rab proteins in Kv1.5 trafficking has been studied extensively in heterologous cells and atrial myocytes, where Rab4, Rab5 and Rab11 were implicated in cardiac Kv1.5 endocytosis and recycling (McEwen et al. 2007; Zadeh et al. 2008). Transient expression of dominant negative isoforms of Rab4 or Rab11 decreased the apparent surface fluorescence of tagged Kv1.5 (McEwen et al. 2007) and Zadeh et al. (2008) showed that retrograde trafficking of Kv1.5 involves Rab4, Rab5 and Rab11, and endocytosed Kv1.5 vesicles colocalized with all of these Rab proteins. Additionally, Zadeh et al. (2008) demonstrated that all dominant negative versions of Rab4, Rab5 and Rab11 decreased Kv1.5 current density when assayed electrophysiologically. Rab5 and Rab11 have also been implicated in the endocytosis and recycling of KCNQ1/KCNE1 potassium channels; functional expression of KCNQ1/KCNE1 channels was greatly reduced when functioning of either Rab5 or Rab11 was disrupted (Seebohm et al. 2007).   In Chapter 3, to elucidate the involvement of these Rab proteins in the Kv4.2 endocytic and recycling trafficking, we surveyed the roles of Rab proteins in modulating the retrograde trafficking and surface expression of the channel in ventricular myocytes. We found that   101 endogenous Kv4.2 current density was increased when either Rab4 or Rab5 dominant negative was transiently expressed in myocytes. Moreover, internalized HA-tagged Kv4.2 colocalized with fluorescence-tagged Rab4, indicating a direct role of Rab4 in the trafficking of the channels. The data presented in Chapter 3, together with results from McEwen et al. (2007) and Zadeh et al. (2008) suggest common roles for Rab4 in endocytosis and Rab5 in rapid recycling of Kv1.5 and Kv4.2. Therefore, it is possible that cardiac Kv channels use a common retrograde trafficking pathway that is mediated by Rab4 and Rab5.   4.3.3 Lysosomal or proteasomal degradation?   Membrane proteins can generally be degraded by two degradation pathways, the proteasomal and lysosomal pathways. Kato et al. (2005) showed that proteasome but not lysosomal inhibitors prolonged the half-life of Kv1.5 in cultured rat atrial cells. Additionally, the lysosomal inhibitor, MG132 increased the protein level of Kv1.5, as well as the level of its ubiquitinated form in a dose-dependent manner, suggesting Kv1.5 undergoes degradation in the proteasome. Another study demonstrated that internalized Kv1.5 colocalized with a lysosomal marker; and that expression of a Rab7DN increased Kv1.5 current as did incubation with a lysosomal inhibitor (ammonium chloride), implying that lysosomal degradation of Kv1.5 also occurs (Zadeh et al. 2008). Proteasomal degradation has also been shown to be operative in hERG channel degradation where internalized hERG channels and ubiquitin colocalized. Ubiquitination was promoted under low-K+ conditions (Guo et al. 2009). Interestingly, Guo et al. also showed colocalization of endocytosed hERG channels with lysosomal markers, suggesting multiple degradation pathways responsible for hERG degradation in low-K+ conditions.   102   In Chapter 3, the effects of transiently overexpressing Rab7 or Rab7DN on Kv4.2 currents were presented. It was observed that neither expression of wild type nor dominant negative Rab7 in the myocytes had any effect on Kv4.2 current densities. Therefore, we hypothesized that Kv4.2 degrades via a proteasomal pathway. The lysosomal inhibitor GM123 was used to test our hypothesis and we found that incubation of MG132 for six hours increased Kv4.2 current levels. These findings suggest that Kv4.2 undergoes proteasomal degradation.   4.4 Future directions   The requirement for Kif5b for Kv1.5 surface expression has been investigated but it is still unknown whether Kif5b is commonly required for forward trafficking of other cardiac Kv channels and the extent to which other kinesins may be involved. We attempted but failed to co- immunoprecipitate Kif5b with Kv1.5, indicating that there is probably no direct interaction between the channel and the motor. This is not surprising since simple inclusion in vesicles being trafficked by the motor would be sufficient to affect the trafficking. Other kinesins might bind directly to specific motifs on channel protein or indirectly via interaction with proteins containing binding motifs. The challenge for future studies will be to identify the binding partners for Kif5b and the motifs that Kif5b recognizes as cargo for vesicular transport. If Kif5b recognizes a motifs or a class of binding partners that is shared among cardiac Kv cardiac channels, then there is a good chance that these channels will prove also to be cargoes of Kif5b.   A survey of the roles of Rab proteins in cardiac Kv channel trafficking was also presented in this thesis. These experiments provide a fundamental grounding for further probing of the underlying mechanisms for the regulation of Kv channel functional expression. Identifying the Rab proteins involved in the trafficking pathways would help to understand and provide   103 mechanistic insight into how upstream signalling can regulate channel expression and thus action potential duration. For instance, investigating the mechanism by which stress induces increases in KCNQ1/KCNE1 currents, Seebohm et al. (2007) demonstrated a link between trafficking of KCNQ1/KCNE1 channel protein to PI(3,5)P2-dependent vesicle recycling. This recycling, they showed, is regulated by the stress-dependent kinase SGK1. The authors further showed that the SGK1-mediated increase of PI(3,5)P2 promotes exocytosis of KCNQ1/KCNE1 to the plasma membrane via a Rab11-dependent pathway. Another example is the recently reported finding that cholesterol regulates Kv1.5 current by modulating its trafficking through the Rab11- associated recycling endosome (Balse et al. 2009). It will be an exciting future direction to determine whether there are pathophysiological conditions such as heart failure and ‘diabetic heart’ linked with channel functional expression regulation via Rab-mediated trafficking of Kv4.2 and other channels.   It was mentioned above, that the trafficking of exogenously expressed Kv1.5 in ventricular myocytes was unaffected by co-expression of a Sar1 dominant negative. It would be of great interest to investigate whether native Kv1.5 in atrial myocytes traffics similarly. Since Sar1-independence suggests that the channel traffics via a non-conventional pathway that does not require COPII in ER-to-Golgi transport, it would be extraordinarily interesting to explore the trafficking machinery of that pathway, including determining which small GTPases and SNARE proteins are involved in this non-conventional ER-to-Golgi trafficking.  4.5 Concluding remarks   Most of the studies to date on Kv channel trafficking were done in neuronal background and there are relatively few studies on trafficking of cardiac Kv channels. The goal of the   104 experimental work in this thesis was to provide an understanding to the trafficking machineries employed by cardiac Kv channels. The contributions made in this thesis enhance our knowledge of the players involved in the trafficking pathway of Kv channels in cardiomyocytes. In particular, the roles of kinesin and several small GTPases in regulating cardiac Kv channel plasmmalemmal expression were elucidated. Because Kv channels are essential for cardiac action potential repolarization and changes of their activities can and do result in arrhythmias, a better understanding of how the Kv channel plasmalemmal expression can be modulated, may help us develop better and safer therapeutic regimes to treat different types of arrythmias.                  105            A                            B                  Figure 4.1 Functional expression of Kv1.5 and Kir2.1 is dependent on Rab1 A, effects of Rab1WT and Rab1DN on sustained current density of Kv1.5-transfected adult rat ventricular myocytes. Cells were depolarized from -120 to +90 mV in 10 mV, 500 ms steps and repolarized to -80 mV between pulses. B, effects of Rab1WT and Rab1DN on endogenous inward sustained current density of adult rat ventricular myocytes. Cells were depolarized from -130 to -80 mV in 10 mV, 500 ms steps and repolarized to -80 mV between pulses. Data are represented as mean ± S.E.M. Current densities from control cells (mCherry), Rab1WT + mCherry and Rab1DN + mCherry transfected cells are plotted against voltage. All experiments were performed 12–24 h post-transfection. ∗P < 0.05, comparing Rab1DN + mCherry to mCherry alone. * * * * * * * * ** membrane voltage (mV) -100 -50 0 50 100 cu rr en t d en si ty  (p A /p F) 0 50 100 150 200 250 300 Kv1.5 (n=11) Kv1.5+ Rab1DN (n=13) Kv1.5+ Rab1WT (n=6) cu rr en t d en si ty  (p A /p F) -16 -14 -12 -10 -8 -6 -4 -2 -130 -120 -110 -100 -90 -80 -70 -60 -50 membrane voltage (mV) cu rr en t d en si ty  (p A /p F) m-Cherry (n=10) Rab1WT + m-Cherry (n=10) Rab1DN + m-Cherry (n=12) * * * * cu rr en t d en si ty  (p A /p F)   106       A                B                 Figure 4.2 Functional expression of Kv1.5 and Kir2.1 is dependent on Sar1 A, effects of Sar1WT and Sar1DN on sustained current density of Kv1.5-transfected adult rat ventricular myocytes. Cells were depolarized from -120 to +90 mV in 10 mV, 500 ms steps and repolarized to -80 mV between pulses. B, effects of Sar1WT and Sar1DN on endogenous inward sustained current density of adult rat ventricular myocytes. Cells were depolarized from -130 to -80 mV in 10 mV, 500 ms steps and repolarized to -80 mV between pulses. Data are represented as mean ± S.E.M. Current densities from control cells (mCherry), Sar1WT + mCherry and Sar1DN + mCherry transfected cells are plotted against voltage. All experiments were performed 12–24 h post-transfection. ∗P < 0.05, comparing Sar1DN + mCherry to mCherry alone. -16 -14 -12 -10 -8 -6 -4 -2 -130 -120 -110 -100 -90 -80 -70 -60 -50 membrane voltage (mV) cu rr en t d en si ty  (p A /p F) Sar1T39N + m-Cherry (n=11) m-Cherry (n=19) Sar1WT + m-Cherry (n=11) cu rr en t d en si ty  (p A /p F) membrane voltage(mV) -100 -50 0 50 100 cu rr en t d en si ty  (p A /p F) 0 50 100 150 200 250 Kv1.5+Sar1T39N (n=8) Kv1.5 (n=7) Kv1.5+Sar1WT (n=8) cu rr en t d en si ty  (p A /p F)   107 4.6 References  Allan BB, Moyer BD, Balch WE (2000). Rab1 recruitment of p115 into a cis-SNARE complex: Programming budding COPII vesicles for fusion. Science 289, 444-448.  Balse E, El-Haou S, Dillanian G, Dauphin A, Eldstrom J, Fedida D, Coulombe A, Hatem SN (2009). Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes. Proc Natl Aacad Sci U S A 106, 14681-14686.  Beckers CJM, Balch WE (1989). Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus. J Cell Biol 108, 1245- 1256.  Boehmer C, Laufer J, Jeyaraj S, Klaus F, Lindner R, Lang F, Palmada M (2008). Modulation of the voltage-gated potassium channel Kv1.5 by the SGK1 protein kinase involves inhibition of channel ubiquitination. Cell Physiol Biochem 22, 591-600.  Cai D, McEwen DP, Martens JR, Meyhofer E, Verhey KJ (2009). Single molecule imaging reveals differences in microtubule track selection between kinesin motors. PLoS Biol 7, e1000216.  Cai Y, Singh BB, Aslanukov A, Zhao H, Ferreira PA (2001). The docking of kinesins, KIF5B and KIF5C, to Ran-binding protein 2 (RanBP2) is mediated via a novel RanBP2 domain. J Biol Chem 276, 41594–41602.  Choi WS, Khurana A, Mathur R, Viswanathan V, Steele DF, Fedida D (2005). Kv1.5 surface expression is modulated by retrograde trafficking of newly endocytosed channels by the dynein motor. Circ Res 97, 363-371.  Chu PJ, Rivera JF, Arnold DB (2006). A role for Kif17 in transport of Kv4.2. J Biol Chem 281, 365–373.  Dou Y, Balse E, Dehghani Zadeh A, Wang T, Goonasekara CL, Noble GP, Eldstrom J, Steele DF, Hatem SN, Fedida D (2010). Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method. Am J Physiol Cell Physiol 298, C1343-1352. Delisle BP, Underkofler HA, Moungey BM, Slind JK, Kilby JA, Best JM, Foell JD, Balijepalli RC, Kamp TJ, January CT (2009). Small GTPase determinants for the Golgi processing and plasmalemmal expression of human ether-a-go-go related (hERG) K+ channels. J Biol Chem 284, 2844-2853. Fadool DA, Levitan IB (1998). Modulation of olfactory bulb neuron potassium current by tyrosine phosphorylation. J Neurosci 18, 6126–6137.    108 Gu C, Zhou W, Puthenveedu MA, Xu MX, Jan YN, Jan LY (2006). The microtubule plus-end tracking protein EB1 is required for Kv1 voltage-gated K+ channel axonal targeting. Neuron 52, 803-816.  Hamm-Alvarez SF, Kim PY, Sheetz MP (1993). Regulation of vesicle transport in CV-1 cells and extracts. J Cell Sci 106, 955-966.  Hasdemir B, Fitzgerald DJ, Prior IA, Tepikin AV, Burgoyne RD (2005). Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway. J Cell Biol 171, 459-46.  Henke G, Maier G, Wallisch S, Boehmer C, Lang F (2004). Regulation of the voltage gated K+ channel Kv1.3 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid inducible kinase SGK1. 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Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway. J Biol Chem 277, 11401-11409.  Zadeh AD, Xu H, Loewen ME, Noble GP, Steele DF, Fedida D (2008). Internalized Kv1.5 traffics via Rab-dependent pathways. J Physiol 586, 4793-4814.                      110 APPENDIX 1 UBC ANIMAL CARE CERTIFICATE Copy of the UBC animal care committee approval letter is included.   111     112   

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