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Localization and trafficking of Kv channels in cardiac myocytes through the use of novel transfection… Wang, Tiantian 2010

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LOCALIZATION AND TRAFFICKING OF Kv CHANNELS IN CARDIAC MYOCYTES THROUGH THE USE OF NOVEL TRANSFECTION METHODS by Tiantian Wang BSc. Huazhong University of Science and Technology, 2007  A THESIS SUBMITTED IN PARTIAL FULFULLMENT 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 © Tiantian Wang, 2010  Abstract Voltage-gated K+ channels (Kv channels) play important roles in the repolarization and the termination of electrical excitation in cardiomyocytes, but very little is known about how their surface expression and localization are regulated. In this thesis, I report the characterization of Kv1.5 localization as well as the trafficking pathways utilized by Kv4.2 in ventricular myocytes. We have developed a new gene gun transfection method that, for the first time, allows the ready and convenient transfection of acutely isolated adult rat cardiac myocytes. Using this system combined with electrophysiology and confocal imaging experiments, we unequivocally demonstrate that tagged Kv1.5 is efficiently localized to the intercalated disk in ventricular myocytes. Furthermore, Kv1.5 deletion mutations known to reduce the surface expression of the channel in heterologous cells do not affect the channel localization to this structure.  The ventricular myocyte transfection system combined with electrophysiological and imaging techniques has also been used identify the small GTPases that regulate the trafficking of cardiac Kv4.2. We demonstrate that the small GTPases Sar1, Rab1, Rab5, Rab4, Rab11and Rab7 are involved in specific steps in the forward and retrograde trafficking of Kv4.2 in rat ventricular myocytes. This work has provided critical insights into the trafficking of cardiac potassium channels and allows us to better understand the modulation of their function in the heart.  ii  Table of contents Abstract……………………………………………………………………………..ii Table of contents……………………………………………………………………iii List of figures……………………………………………………………………….iv List of abbreviations……………………………………………………………………vi Acknowledgements……………………………………………………………………viii Co-Authorship statement………………………………………………………………..ix Chapter 1:  General introduction..............................................................................1 References………………………………………………………16  Chapter 2:  Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes Introduction……………………………………………………27 Materials and methods…………………………………………30 Results…………………………………………………………35 Discussion……………………………………………………..43 References……………………………………………………..56  Chapter 3:  Trafficking of endogenous Kv 4.2 in adult ventricular myocytes Introduction…………………………………………………….61 Materials and methods…………………………………………64 Results………………………………………………………….69 Discussion……………………………………………………76 References……………………………………………………….90  Chapter 4:  Final discussion and conclusions……………………………………96  Appendix  References……………………………………………………103 ………………………….………………………………………………106  iii  List of figures Figure 1.1  Schematic representation of a Kv channel subunit……………………..14  Figure 1.2  Schematic representations of the roles of Sar1, Rab1, Rab5, Rab4, Rab11 and Rab7 in membrane protein forward trafficking, endocytosis, recycling and degradation......................................................................................15  Figure 2.1  Lipofectamine-mediated transfection of adult ventricular myocytes with Kv1.5 dramatically increases sustained current……………………….46  Figure 2.2  A novel biolistic transfection of isolated adult rat myocytes...................48  Figure 2.3  Localization of tagged Kv1.5 in biolistically transfected ventricular myocytes………………………………………………………………49  Figure 2.4  Biolistic transfection of Kv1.5 in rat ventricular cardiomyocytes increases the sustained potassium current………………………………………….50  Figure 2.5  Localization of wild-type and mutant Kv1.5 channels to the intercalated disc in biolistically transfected rat ventricular myocytes.........................52  Figure 2.6  Effects of mutations in transfected Kv1.5 on sustained currents in rat ventricular myocytes…………………………………………………….54  Figure 3.1  Lipofectamine-mediated transfection of adult ventricular myocytes……79  Figure3.2  Normal expression of Ito requires intact Sar1 function…………………80  Figure 3.3  Normal expression of Ito requires intact Rab1 function…………………81  Figure 3.4  Expression of an inducible Kv4.2 in HEK293 cells is significantly reduced by coexpression of Rab1DN and Sar1DN……………………82  Figure 3.5  Overexpression of Rab11WT substantially increases Ito in transfected ventricular myocytes……………………………………………………83  Figure 3.6  Brefeldin A treatment prevents Rab11WT overexpression effect on Ito.........................................................................................................84  iv  Figure 3.7  Interference with Rab5 function increases Ito in transfected ventricular myocytes……………………………………………………………….85  Figure 3.8  Rab4DN expression also increases I to in transfected ventricular myocytes………………………………………………………………86  Figure 3.9  Internalized Kv4.2 colocalizes with Rab4 in ventricular myocytes……87  Figure 3.10  Rab7 function appears irrelevant to maintenance of endogenous Ito……88  Figure 3.11  Incubation with proteasome inhibitor significantly increases the magnitude of Ito……………………………………………………….…89  v  List of abbreviations Kv  Voltage gated potassium channel  Kir  Inwardly rectifying potassium channel  Na+  Sodium ion  K+  Potassium ion  KATP  ATP-sensitive potassium channel  hERG  Human ether-a-go-go  CFTR  Cystic fibrosis transmembrane conductance regulator  Ito  Transient outward potassium current  Ikur  Ultrarapid delayed rectifier potassium current  ER  Endoplasmic reticulum  TGN  Trans-golgi network  SNARE  Soluble N-ethylmaleimide-sensitive factor attachment protein receptor  COP  Coat protein complex  KChIP  Potassium channel-interacting proteins  KChAP  Potassium channel-associated protein  Mg2+  Magnesium ion  4-AP  4-aminopyridine  mM  Millimoles per liter  ml  Milliliter  mg  Milligram  µl  Microliter  ng  Nanogram  °C  Degrees celsius  nm  Nanometer  vi  µM  Micrometer  W  Watt  kHz  Kilohertz  ms  Millisecond  s  Second  mV  Millivolt  MΩ  Megaohm  M  Moles per liter  Hz  Hertz  HEPES  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid  NaC1  Sodium chloride  KC1  Potassium chloride  MgC12  Magnesium chloride  CaC12  Calcium chloride  NaHCO3  Sodium bicarbonate  MgSO4  Magnesium sulfate  Ca(NO3)2  Calcium nitrate  NaOH  Sodium hydroxide  vii  Acknowledgements I would like to thank my supervisor Dr. David Fedida for his guidance over the last two years. He has kept me driven over the last few years to maintain a high quality of work and his encouragement has kept me going through the difficult experimental periods. I would also like to thank my supervisory committee, Dr. Eric Accili and Dr. Chris Ahern, for their input throughout these experiments.  I would like to make a special thanks to Dr. David F. Steele for all his support over last two years for my projects. His suggestions, comments and concerns were always appreciated as it allowed the successful completion of several studies. I would also like to thank Dr. Ying Dou, Dr. Alireza Dehghani Zadeh, Dr. Charitha L. Goonasekara and Yvonne Cheng for their experimental work on Chapters 2 and 3.  viii  Co-authorship statement  Chapter 2. Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes This project was identified and designed by my research supervisor, Dr. David Fedida, and Dr. David F. Steele. Research was performed by Ying Dou, Elise Balse, Alireza Dehghani Zadeh, Tiantian Wang, Charitha L. Goonasekara and Geoffrey P. Noble. Data analysis was performed by Ying Dou, Tiantian Wang and Alireza Dehghani Zadeh. Manuscript preparation was performed by Ying Dou, Tiantian Wang and David F. Steele.  Chapter 3. Trafficking of endogenous Kv 4.2 in adult ventricular myocytes This project was identified by my supervisor, Dr. David Fedida, and Dr. David F. Steele. Research was performed by Tiantian Wang, Yvonne Cheng, Charitha L. Goonasekara and Ying Dou. Data analysis was performed by Tiantian Wang and Yvonne Cheng. Manuscript preparation was performed by Tiantian Wang, Yvonne Cheng and David F. Steele.  ix  Chapter 1 Introduction Ion channels are integral membrane proteins that allow the movement of ions across cell membranes. They play important roles in the physiological functioning of cells and are key elements of multiple signal transduction pathways. Voltage gated potassium channels (Kv) channels open in response to cell membrane voltage depolarization, responsible for the modulation of the duration and amplitudes of the cardiac action potentials. The normal functioning of ion channels depends not only on the regulation of their biophysical properties but also on the fine-tuning of their subcellular localization and their numbers on the cell surface. Newly synthesized channel proteins must be exported from the ER and traffic to the plasma membrane, often to specific microdomains there. Kv channel density at the cell surface is also regulated by a delicate modulation of endocytotic and recycling pathways. The mechanisms of channel targeting and trafficking are only beginning to be elucidated. The study of anterograde and retrograde cardiac Kv channel trafficking will significantly improve our understanding of cardiac functioning and undoubtedly open new avenues for clinical applications as well. Kv channel overview Voltage-gated K+ channels are the prototypical voltage-gated ion channels. Coassembling as homotetrameric or heterotetrameric channels of so-called α-subunits, each α-subunit contains a voltage sensor and contributes to the central pore. The standard Kv channel α subunit has six transmembrane-spanning α-helical segments (S1–S6) with cytoplasmic N- and C-terminal domains and a pore loop between S5 and S6 bearing the K+ selectivity filter signature TxGYG (55, 63, 67) (Figure 1.1). The S4 helix contains positively charged residues (R/K) at approximately every third position and serves as the  1  voltage sensor. Although expression of the pore-forming α subunits is usually sufficient to generate an ion current, recapitulation of the physiological features of the native K+ current frequently requires co-expression of accessory β subunits (49). In the heart, Kv channels play prominent roles in regulating the heart rate and the resting membrane potential, as well as shaping the action potential. They are important targets for the actions of neurotransmitters, hormones, drugs and toxins known to modulate cardiac function (15, 48). In cardiac myocytes, the plateau phase of the action potential involves numerous K+ currents, and the delicate balance of their expression levels, timing of activation and voltage-dependent properties characterize the shape and time course of cardiac action potentials. Differing substantially in their kinetics of activation and inactivation, specific channels underlie specific currents in the heart. Kv4.x channels, for example, activate and inactivate rapidly and underlie the transient outward potassium current (Ito) (10, 27-28), whereas Kv1.5, responsible for the atria-specific Ikur (ultrarapid delayed rectifier K+ current) (47, 76), inactivates much more slowly. hERG (human ether-a-go-go-related gene) activates and inactivates rapidly, then passes most of its current as it recovers from inactivation (56, 70). Kv channel trafficking The functions of channels depend critically also on their numbers at the cell surface, which are modulated jointly by the forward trafficking and endocytic pathways. The channels must be synthesized in the endoplasmic reticulum (ER), assembled and processed appropriately then trafficked and targeted to the membrane or membrane subdomains where they function. The roles of chaperones, anchoring proteins, cytoskeletal systems and their associated motors in this trafficking of newly synthesized channels are just beginning to be elucidated.  2  Once synthesized, potassium channels must first traffic from the endoplasmic reticulum to the Golgi apparatus. Considerable variation has been described in the pathways employed by different channels to reach the Golgi. Coat protein complex II (COPII) complexes are known to mediate the anterograde trafficking of at least some channels by interacting with channel ER export signals. The human ether-a-go-go related (hERG) K+ channel is exported from ER via COPII-coated vesicles (16), and enters a Rab11-associated endosomal compartment prior to being processed in Golgi. The surface expression of Kir3.1 and KATP channels are also dependent on COPII mediated ER exit mechanism(s) (54, 68). Other channels have been shown to reach the plasma membrane via a COPII-independent and/or even a Golgi-independent manner, in at least some cell types. In Neuro2A cells, blocking the COPII pathway does not block the ER exit of Kv4.2 channel when associated with its interacting protein KChIP1 (22, 31). In BHK cells and CHO cells, the cystic fibrosis transmembrane conductance regulator (CFTR) reaches the cell surface by direct transport from ER by COPII carriers (75) via a pathway that does not depend on syntaxin5 (82), a t-SNARE required for newly synthesized proteins trafficking through the Golgi in all eukaryotes. At least a large fraction of CFTR, thus, bypasses Golgi. CFTR deposition in the plasma membrane is, however, blocked by overexpression of syntaxin13, a t-SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) that resides in late/recycling endosomes, suggesting that recycling through a late Golgi/endosomal system is a prerequisite for CFTR maturation. Chaperones and accessory proteins such as β-subunits, KChIPs (potassium channelinteracting proteins) and KChAP (potassium channel-associated protein) have been reported to bind to newly synthesized channels, promoting their forward trafficking from the ER to Golgi (36, 62, 77). Once out of the ER, most newly synthesized channels pass through the Golgi apparatus where glycosylation is completed and preliminary sorting to the plasmalemma 3  or intracellular organelles occurs. Channel localization is quite specific: for example, Kv1.5 is targeted mainly to the intercalated disc in atrial and ventricular myocytes (21). Kv4.2 is similarly found at the intercalated disc (7), and in T-tubules (66), whereas KCNQ1 (KvLQT1) is present in T-tubules and also in the peripheral sarcolemma (53). We have little idea, however, how this targeting of ion channels is effected. We do know, however, that MAGUKs (membrane-associated guanylate kinases) proteins play important roles in at least some of this targeting and/or anchoring of ion channels at specific membrane domains. Among the various MAGUK proteins, PSD-95 (postsynaptic density 95) and SAP97 (synapse-associated protein 97) have been shown to bind to Kv channels and/or affect surface expression (20, 34). Surface expression of both Kv1.4 (34) and Kv1.5 (20), is increased by overexpression of PSD-95 in heterologous cells. Unlike PSD-95, however, binding to SAP97 causes retention of most Kv1 channels in the ER, at least in heterologous cells (69). As a sole exception, SAP97 overexpression causes the clustering and immobilization of Kv1.5 channels in the plasma membrane and increases its corresponding current (19, 24, 46). One possible reason for this very different effect of SAP97 overexpression on Kv1.5 is that, unlike other Kv channels, Kv1.5 does not bind to SAP97 via its canonical C-terminal PDZ-biding domain. SAP97 overexpression increases the expression of even a Kv1.5 deletion mutant lacking the Cterminal PDZ-binding domain to the same degree as the wild-type channel (20, 43). Thus SAP97 very probably regulates Kv1.5 surface expression via an indirect mechanism. A number of cellular components have been implicated in the actual trafficking of ion channels. The coordinated delivery of channel-bearing vesicles appears to depend on the translocation of the vesicles along cytoskeletal elements. Most intracellular transport occurs via the microtubule network, and the microtubule cytoskeleton has been demonstrated to be essential to Kv channel trafficking. Disruption of microtubule  4  transport by nocodazole causes significant increase in Kv1.5 and Kv4.2 currents in both heterologous cells and rat cardiac myocytes (13, 40). Two families of motor proteins, kinesins and dyneins, carry traffic along microtubules (32, 73). There are 14 defined families of kinesin motors based on phylogenetic analyses (37). Most kinesin motors transport cargo towards the plus end of the microtubules located in the cell periphery, and specific isoforms have been shown to play important roles in the anterograde transport of Kv channels. Kinesin isoform Kif17 has been shown to be interact with Kv4.2 and affect its localization in neurons (14). Expression of a dominant-negative Kif17 construct in the neurons blocked surface expression of the channel (14). Our lab has shown that Kif5b, a Kinesin I isoform expressed in heart, is required for the forward trafficking of Kv1.5 (83). Overexpression of Kif5b increased Kv1.5 currents in both HEK293 cells and H9c2 cardiomyoblasts. It did so additively with several manipulations that reduce channel internalization, suggesting that Kif5b is involved in the delivery of the channel to the cell surface. Interestingly, expression of the dominant negative isoform of the motor also increased Kv1.5 current density in cells pre-expressing the channel. This Kif5b dominant negative effect has been shown non-additive with block of channel endocytosis, suggesting that it indirectly interferes with the internalization of Kv1.5. This is consistent with numerous reports in the literature that interference with kinesins indirectly interferes also with dynein function (8, 29). Confirming an essential role for Kif5b in Kv1.5 delivery to the plasma membrane, we also demonstrated that Kif5b overexpression almost completely blocks traffic of newly synthesized channel to the cell surface. Dynein mediates cargo transport towards the mircotubule minus end, i.e., the Microtubule Organizing Centre (MTOC) (60). Our lab has demonstrated that dynein interacts with Kv1.5 and is involved in its retrograde trafficking (13). Blocking of dynein motor function by overexpression of p50/dynamitin causes a greater than 2- fold increase 5  in Kv1.5 currents when the channel is heterologously expressed. p50, an important component of the dynactin complex, is thought to link the cargo-interacting subunits to the dynein motor itself. Overexpression of this protein causes the dynactin complex to dissociate, decoupling the motor from its cargoes (17). Nocodazole treatment, which depolymerizes the microtubule cytoskeleton along which dynein tracks, also increases Kv1.5 currents in HEK cells and the corresponding sustained K+ currents in isolated rat atrial myocytes. Coimmunoprecipitation experiments demonstrated a direct interaction between Kv1.5 and the dynein motor complex in both heterologous cells and rat cardiac myocytes, supporting the role of this complex in Kv1.5 trafficking. An intact SH3binding domain in the Kv1.5 N-terminus was shown to be required for nomal retrograde trafficking to occur. Gene gun transfection of isolated rat adult myocytes Because transfection of adult cardiac myocytes has to date proved an intractable problem, previous findings on cardiac ion channel trafficking have been derived through the use of model expression systems. Though liposome-mediated transfection works in rat neonatal myocytes, several currents (Ito, INa and ICa) in these cells differ dramatically from those in adult myocytes (51, 74). Viral methods using lentivirus, adenovirus, and adenoassociated virus are potentially efficient, but require sophisticated containment facilities and, generally,prolonged culture of the myocytes, which add potential to immunologic and cytotoxic complications (1, 88). Direct injection of naked plasmid DNA into the heart has also been tested for in vivo gene transfer (41), but the efficiency of this method is very low. Compared with the methods listed above, so-called biolistic, or ‘gene gun’, mediated transfection is potentially more simple and convenient. Gene gun-mediated transfection is a mechanical method that, practically, is conducted in two steps. The first  6  step is to make the DNA “sticky” so that it adheres to biologically inert particles, such as tungsten or gold. Once adhered, this DNA-particle complex is accelerated such that it pierces the plasmalemma of the target cells, effectively introducing the DNA into the cells. This approach allows DNA to penetrate directly through cell membranes into the cytoplasm or even nuclei, and to bypass the endosome/lysosome, thus avoiding enzymatic degradation. In early biolistic devices, a vacuum chamber was required. After placing the target sample in the chamber, the overlying air was evacuated and the DNA-coated gold or tungsten particles were fired into the target. The earliest versions used gunpowder to propel the particles; later versions used a system of rupture disks and helium gas. The chamber gene gun system had a few limitations, and the vacuum had the potential to cause damage to the cells. Nowadays, the hand held gene gun is considered more widely applicable. The major difference compared with early devices is that the hand held gene gun system requires no vacuum, removing limitations to the target and its size. In this system, microcarriers are attached to the inside of a length of plastic tubing. The tubing containing the gold/DNA complex is then cut to the appropriate size to form a cartridge and loaded into the gene gun. Firing of the gun involves releasing a pulse of helium gas, which propels the gold/DNA complex from the inner surface of a cartridge into the target cells or tissues. Because this hand held gene gun can be used for in a much wider range of gene transfer applications including tissues and whole animals as well as cultured cells, it has tremendous potential for gene therapy (38, 80). The gene gun technology has various advantages over other methods, and is potentially very useful in myocyte transfection. Unlike lipofectamine transfection, the gene gun method is not dependent on specific biochemical features of the cell surface or on the growth rate of the cells. In addition, it requires only small amounts of DNA and takes only a few seconds to perform a single transfer. Unlike the viral transduction methods generally used to express introduced proteins in myocytes, there is no need for 7  complicated cloning strategies to incorporate the gene of interest into a large viral vector. Instead, any gene in a mammalian expression vector can be introduced into the myocytes. This method also eliminates the need for sophisticated containment facilities and prolonged culture of the myocytes. Gene gun transfection has been proven efficient in various cell lines and tissues. It has proved highly efficient for in vitro transfections in cultured mammalian cell lines, including MCF-7 cells, CHO cells, and HEK293 cells (71, 81). In in vivo transfections, skin, liver, and muscle have been successfully transfected after surgical exposure of the tissue. 10% to 20% of skin epidermal cells in the bombarded area reportedly express the foreign gene after 24-48 hr transfection, and 1% to 5% of similarly bombarded muscle cells do so (78, 81). Gene expression has been demonstrated to last up to 14 days in mouse skin and liver, and the introduced human dystrophin gene was shown to be expressed in mouse skeletal muscle for at least 60 days after bombardment (85). Robust expression was also detected in neurons and glia cells in brain slices 16 hr after transfection and continued to increase over the following 24-72 hours (39). A recent study in rat brain cultures indicated that gene gun transfection was 160-fold, 189-fold and 450fold more effective than lipofection, electroporation and calcium phosphate precipitation, respectively, as assayed by introduced luciferase activity (79). There are much fewer reported studies on biolistic transfections in cardiomyocytes. Serum-free cultured rat neonatal myocytes have been successfully transfected with a plasmid containing the βGal reporter via gene gun (18). 48 hours after bombardment, the β-Gal expression in the cells was quantified by ELISA, and compared with other transfection methods. A transfection efficiency of about 10% was achieved in the bombarded cells, which was much higher the the liposome- or calciumphosphate precipitation-based methods. The gene gun method has also proved of limited use in transfecting surface myocardium in living heart with a non-viral vector containing EGFP (44). After tranfection, the EGFP 8  expression was observed from day 1 to 3 weeks, and the highest expressions were observed at day 3 after bombardment. However, in this study, expression was only detectable only at superficial caridiac muscle areas, and the surface cardiac tissues were damaged to different extents after bombardment, as revealed in histologic analysis. Up to now, the gene gun system has not been successfully adapted for transfections of isolated adult myocytes useful for electrophysiological or imaging studies. Therefore, the development a high-efficiency biolistic transfection method as described in this thesis could greatly facilitate the general study of gene expression and protein trafficking in rat cardiac myocytes.. Small GTPases in Kv channel trafficking Small GTPases are monomeric G proteins with molecular masses of 20–40 kDa that play diverse roles in the regulation of intracellular processes. Typically, these proteins have a great deal sequence homology with each other and harbor several conserved domains, including consensus amino acid sequences responsible for interaction with guanosine diphosphatase (GDP) and GTP, as well as a region interacting with downstream effectors. These GTPases function as molecular switches that alternate between two conformational states: the GTP-bound active form and the GDP-bound inactive form. An upstream signal stimulates the dissociation of GDP from the GDPbound form, and the Rab protein then binds GTP, leading to a conformational change favoring effector binding. The interaction between the small G protein and its effectors then initiate a series of cellular signaling cascades. Eventually, the GTP is metabolized by an inherent GTPase activity, often as modulated by accessory proteins and effectors(26). More than 100 small G proteins have been identified in eukaryotes (11, 30). The members of this superfamily have been sub-classified into at least 5 families on the basis of structural homologies: Ras, Rho, Rab, Sar1/Arf, and Ran (11, 65). These small  9  GTPases regulate diverse processes in eukaryotic cells, and generic definitions for family functions have been developed (50, 64-65): Ras subfamily members primarily regulate gene expression. Rho subfamily members regulate both cytoskeletal organization and gene expression. Rab and Sar1/Arf family members regulate intracellular vesicle trafficking. Ran family members regulate nucleocytoplasmic transport during G1, S, and G2 phases of the cell cycle and microtubule organization during M phase. Rab proteins exist in all eukaryotic cells, from yeast to human, and constitute the largest branch of the small G protein superfamily. In mammalian cells, more than 60 Rab proteins have been identified (26, 33, 65) . They are recognized for their key roles in vesicle trafficking (86), including budding, delivery, tethering and fusion (reviewed in (26)). Many Rab proteins show a distinct subcellular localization, making them ideal candidates to govern the specificity of vesicle trafficking, most likely by cooperatively operating with other proteins (25, 86)(Figure 1.2). Organelle- and Rab-specific membrane domains in the endocytic pathway have been intensively characterized, and several Rab proteins have been implicated in the trafficking of ion channels. Rab5, which localizes to early endosomes and the plasma membrane, regulates endocytosis of clathrin-coated vesicles (CCVs) and endosome fusion. Endocytosis by Rab5 has been shown to be important in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) (23), and the glucose transporter GluT4 (9, 45). Recently Rab5 has been reported to act as a primary effector of the rapid endocytosis of KCNQ1/KCNE1 and Kv1.5 (61, 84). Inhibiting Rab5-mediated endocytosis by expression of a GDP-locked dominant-negative Rab5 significantly increases the surface expression of Kv1.5 in both HEK293 cells and a myoblast (H9c2) cell line.  10  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 dominant negative Rab4 mutant restrains surface expression of CFTR (58) and sodiumselective amiloride-sensitive epithelial sodium channel (ENaC)(59), by retaining the channels in the cytoplasm while recycling. A GDP-locked dominant negative Rab4 mutant significantly increases Kv1.5 surface expression, however, probably by indirectly blocking Rab5-dependent endocytosis (84). Another Rab GTPase, Rab11, localizes to the trans-Golgi network (TGN), postGolgi vesicles and the perinuclear recycling endosome. Some internalized channels may be delivered into Rab11-associated recycling endosomes, and slowly recycled back to cell surface (3, 12, 61, 84). Rab11 has been implicated in the forward trafficking of newly synthesized channel proteins as well. In the neuroendocrine cell line, PC12, Rab11 localizes to the TGN and with TGN-derived vesicles, associating with both consititutive secretory vesicles and regulated secretory granules (72). Overexpression of Rab11 greatly enhances the surface expression of transient receptor potential channel TRPC (12). In our lab, we have shown that a Rab11DN induces increased Kv1.5 surface expression in H9c2 myoblasts but only by 48 hr post-transfection. Unlike Rab5 and Rab4, which associate with internalized Kv1.5 within the first several hours post-labelling, colocalization of Rab11 with Kv1.5 was evident only after prolonged periods of internalization. Perhaps Kv1.5 is marked for transport to Rab11-positive vesicles only after multiple Rab4dependent recycling events. Rab7 is associated with the late endosome and trafficking of cargo to the lysosome for degradation. Our lab has demonstrated that Rab7 WT overexpression reduces Kv1.5 surface expression in H9c2 myoblasts (84), very probably because increased Rab7 expression allows a larger fraction of the internalized channel to be shunted to the degradation pathway. This hypothesis is supported by our finding that 11  inhibition of lysosomal function by ammonium chloride prevents the decrease in channel expression associated with wild-type Rab7 overexpression in the H9c2 cells. Substantial evidence supports the hypothesis that most Rab proteins regulate the targeting/docking/fusion processes and that only some of them regulate the budding process, which is primarily regulated by Sar1/Arf small G proteins. Trafficking through COPII vesicles from ER to Golgi is dependent on the Sar1 GTPase, which initiates the formation of the COPII coat-associated protein complex (4-6). The GDP-to-GTP transition triggers the exposure of the N-terminal amphipathic α-helix element of Sar1 which then inserts into the ER membrane (57). ER-bound Sar1-GTP recruits the COPII coat protein Sec23-Sec24 complex (Sec23/24), forming a so-called “prebudding complex”(35). Rab1 recruits tethering factors into the cis-SNARE complex and facilitates fusion between the ER budded vesicles and Golgi compartments (2). The small GTPase ADP ribosylation factor 1 (ARF1) is required for the formation of COPI vesicles, which mediates retrograde movement of components in the Golgi and back to the ER, as well as anterograde movement of certain components (52). It has been reported that the secretory protein H-Ras is trafficked independently of Sar1 (87) and cyctic fibrosis transmembrane conductance regulator (CFTR) trafficking does not require Rab1 (82), suggesting alternate ER-to-Golgi trafficking or even, in the case of CFTR, bypass the Golgi altogether (42). Trafficking pathways of different K channels may be regulated by different small GTPases. Surface expression of Kir3.1 and Kir6.2 channels depend on Sar1-dependent anterograde protein trafficking (54, 68). Sar1 also regulates the ER export of hERG, as the expression of Sar1DN inhibits hERG functional expression (16). However, the Golgi processing of hERG is not blocked by an ARF1 DN. Surprisingly, dominant negative Rab11 significantly inhibits hERG transport through the Golgi, indicating that hERG might enter endosomal recycling prior to being processed in Golgi (16). Unlike hERG, 12  the ER-to-Golgi trafficking of Kv4.2/ KChIP1 in Neuro2A cells have been shown to require normal function of ARF1, but to be independent of Sar1 (22, 31). Unlike in neurons, though, 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 not been established to date. Further work that delineates the trafficking pathways for various K channels will be very important in understanding the cellular mechanisms that regulate their functional expression. Conclusion Voltage gated potassium channels play important roles in regulating cardiac muscle excitability by controlling action potential duration and frequency. The expression of Kv channels on the plasma membrane is regulated by a dynamic interplay between anterograde and retrograde trafficking pathways. The regulation of Kv channel trafficking in the heart are only beginning to be elucidated, and, until now, most work has been conducted using heterologous cells, as the transfection of adult cardiomyocytes had proved intractable. We have developed a biolistic transfection method for reliable, highefficiency transfection of acutely isolated adult rat ventricular myocytes, and have applied this method to the study of Kv1.5 targeting and localization in ventricular myocytes (Chapter 2). Also, since small GTPases are intimately involved in the regulation of vesicle trafficking, we have investigated the roles of Rab and Sar1 GTPases in the anterograde and retrograde trafficking of Kv4.2 in ventricular myocytes (Chapter 3). These studies have added substantially to our understanding of Kv channel trafficking and targeting in cardiomyocytes.  13  Figure 1.1. Schematic representation of a Kv channel subunit. Each channel is composed of four subunits. 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Am J Physiol Heart Circ Physiol 279: H429-436, 2000.  26  Chapter 21 Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method Introduction A great deal has been learned about the trafficking of cardiac ion channels in heterologous expression systems (reviewed in (25)), and much has been learned about the roles of motifs within K+ channels that affect trafficking of the channels to the cell surface (12, 15, 31, 32). Several Rab GTPases (18, 23, 29) and dynamin (6, 20) have been implicated in Kv channel trafficking and the dynein motor has been proven important in the regulation of the functional expression levels of several Kv channels.(6, 13) All of these findings, however, have relied on model expression systems, generally with only supportive and inferential evidence from cardiomyocytes themselves (6, 13, 18, 23, 29). This reliance on model systems has been a necessity because the transfection of adult cardiomyocytes has proved intractable, and, as for adult neurons, the introduction of cloned genes into adult cardiac myocytes has proven, to date, to be practical only with retroviruses. Standard liposome-mediated protocols work for transfection of rat neonatal myocyte preparations but the currents expressed in these cells are quite different from those of adult cardiomyocytes (22, 28). Viral transduction systems are effective in adult cells but require sophisticated containment facilities and either prolonged culture of the myocytes, during which time substantial dedifferentiation can occur (3, 30) or technically difficult in vivo transduction protocols followed by myocyte isolation (19, 26). On the other hand, so-called biolistic, or ‘gene gun,’ methods have proved of limited use in                                                              1  A version of this chapter has been published. 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(6):C1305-7.  27  transfecting surface myocardium in living heart with a non-viral vector (16) but has not been  successfully  adapted  for transfection of isolated myocytes useful for  electrophysiological or imaging studies. Balse, et al. (4), have recently reported success with an adaptation of a lipofectamine-mediated transfection protocol (7, 27) for the transfection of adult atrial cardiomyocytes with standard mammalian expression vector constructs. However, while ionic currents conducted across the plasma membrane of these cells are broadly similar to those of acutely isolated myocytes, due to the necessity of a lengthier time in culture, these transfectants lack the normal rod-shaped myocyte morphology, so are therefore of limited use for studies of targeting of ion channels to plasmalemmal domains. As shown below, this limitation is true, also, of ventricular myocytes transfected by this method. While transfection of adult ventricular myocytes is clearly effective by this method, the dedifferentiation that occurs concomitantly including the loss of striation and intercalated disc structures renders the method of limited use for the study of trafficking and localization of potassium channels in the myocytes. As our intent is to study this trafficking and localization, we tested whether another method, biolistic transfection, could be adapted for use in cardiomyocytes and, if so, whether myocytes transfected in this manner would retain the currents and structures of freshly isolated cells. We report the successful adaptation of biolistic transfection methods for reliable, high-efficiency transfection of acutely isolated adult rat ventricular myocytes. Expression is robust within 24 hours of transfection, a time frame during which, in control and EGFP-transfected myocytes, the currents remain essentially unchanged from the time of isolation. Transfection of these ventricular myocytes with Kv1.5 results in a selective increase in Isus, as expected in myocytes overexpressing Kv1.5. Confirming that the increase in Isus is indeed underlain by Kv1.5, the induced Isus is sensitive to 4aminopyridine (4-AP). We further demonstrate the utility of this system in the study of 28  the functional expression and localization of a number of Kv1.5 mutants in cardiomyocytes.  29  Materials and methods Plasmid constructs Human Kv1.5 was N-terminally tagged with EGFP or mCherry or double tagged with a T7 tag at the N-terminus and an HA tag within the S1-S2 linker of the channel in pcDNA3 as described previously (29). EGFP and mCherry in pcDNA3 were used as control plasmids. The Kv1.5 deletion mutations used were ∆N135, ∆ETDL, ∆N209, and ∆SH3 (1). Antibodies Monoclonal Mouse-anti-HA antibody (Clone 12CA5) was purchased from Roche. Rabbit-anti-HA was from Zymed Laboratories. Mouse-anti-T7 was from Novagen. Rabbit-anti-Kv1.5 (Clone Y436) was generated by Yenzyme Antibodies. Alexa Fluor 488 and Alexa Fluor 594 were from Molecular Probes. Myocyte isolation 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  30  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), 100 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, 1 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. Lipofectamine-mediated myocyte transfection For transfection, freshly isolated rat ventricular myocytes in ~ 0.2 mL Storage Buffer (base Ca2+-free solution (see above) plus 20 mmol/L taurine, 1% BSA, 100 µmol/L Ca2+) were plated on laminin-coated coverglasses at high density in M199 medium containing 1% FBS, 0.1X ITS (Insulin-Transferrin-Selenium) (GIBCO) plus streptomycin and penicillin (Invitrogen) to final concentrations of 100 µg/mL and 100 U/mL, respectively. The myocytes were then incubated overnight in a 1% CO2 incubator at 37oC. The following day, the culture medium was changed to M199 containing 5% FBS, 0.1X ITS without antibiotics and transfected using a liposomal approach. Briefly, 4µg of DNA (per dish) and 10µl of Lipofectamine (Invitrogen) were separately prepared in Opti-MEM I medium (500µl final volume in each) and incubated 5 minutes at room temperature. The two solutions were mixed, incubated 20 minutes at room temperature and then added to the culture dish. Transfection was allowed for 4 hours in a 1% CO2 incubator. Cells were gently washed twice with M199 medium containing 5% FBS, 0.1X ITS and streptomycin/penicillin and incubated 48 hours in a 5% CO2 incubator before experiments. 31  Biolistic myocyte transfection Biolistic gold beads coated with plasmid DNAs were prepared as directed in the BioRad Helios Gene Gun System instruction manual with the exception that no polyvinylpyrrolidone was employed. Briefly, 25 mg of 0.6 micron gold beads were suspended in 50 mmol/L spermidine and 1 to 2 µg of plasmid DNA per mg of beads was added and precipitated onto the beads by dropwise addition of 1 mmol/L CaCl2 to ~330 mmol/L. After 10 minutes at room temperature, the beads were washed 4 times with 100% ethanol that had been dried successively over 2 beds of silica gel. The beads were then suspended in 3 mL of 100% ethanol, sonicated in a Branson 1210 sonicating bath, and loaded into plastic tubing using the BioRad Tubing Prep Station and a BioRad EconoPump peristaltic pump. For transfection, freshly isolated rat ventricular myocytes in ~ 0.2 mL Storage Buffer (base Ca2+-free solution (see above) plus 20 mmol/L taurine, 1% BSA, 100 µmol/L Ca2+) were plated on laminin-coated cover glasses at high density. After 5 to 15 minutes, the overlaying Storage Buffer was replaced with 1 mL M199, pH7.4, supplemented with 2 mmol/L EGTA, 0.6 µg/mL insulin, 5 mmol/L creatine, 2 mmol/L DL-carnitine, 2 mmol/L glutamine, 5 mmol/L taurine, 50 U/mL penicillin and 50 µg/mL streptomycin and the cells were incubated at 37oC for 1 hour in a 5% CO2 incubator. Immediately prior to bombardment, the media was quickly removed. The myocytes were immediately bombarded with the DNA coated beads using a BioRad Helios Gene gun held ~1 cm above the cover glass on which the myocytes were plated. Firing pressures were generally between 90 and 110 psi helium. After bombardment, the cells were immediately resupplied with 1 mL of the supplemented M199 media and incubated overnight at 37oC in the 5% CO2 incubator.  32  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 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 -120 to +90 mV in 10 mV increments followed by 100 ms pulse at +60 mV. In the prepulse experimental protocol, a 75-ms pulse at -40 mV was applied before the 500 ms test potentials to inactivate the transient outward current. Immunostaining and confocal 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. 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. In experiments in which fluorescent proteins were employed, cells were mounted in DABCO/Glycerol (90% glycerol, 2.5% w/v DABCO– PBS solution) at this stage. In other experiments, 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 Alexa488 were excited using the 488 nm line of an argon laser set 33  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). Data statistics Results are expressed as mean ± S.E.M. Statistical analyses were conducted using Student’s t-test (paired) or by One Way ANOVA, as appropriate.  34  Results Lipofectamine-mediated transfection of isolated adult rat ventricular myocytes As an initial approach to adult rat cardiomyocyte transfection, we adapted the lipofectamine-mediated method employed for the transfection of adult atrial myocytes by Balse, et al. (4). To test the effectiveness and effects of the procedure on ventricular myocytes, adult rat ventricular myocytes were isolated and cultured overnight under a low (1%) CO2 atmosphere. The following morning, they were transfected by a lipofectamine procedure with either an N-terminally T7-tagged, externally HA-tagged Kv1.5 construct in pcDNA3 (for imaging experiments) or, for electrophysiological studies, with a Kv1.5 construct plus an mCherry construct, both in pcDNA3. Similarly to atrial myocytes (4), ventricular myocytes transfected by this method express the introduced Kv1.5 at high levels (Figure 1). However, also like atrial myocytes, substantial dedifferentiation is evident in these transfected myocytes. Even after only two days in culture, cells are rounded and lack striation and other myocyte-specific structures such as the intercalated disc (Figure 1A). After longer culture times, the cells become flattened with extensive processes (Figure 1B). Unlike fresh ventricular myocytes where endogenous Kv1.5 localizes almost entirely to the intercalated disc (5, 8, 17) (Figure 3A, upper right panels), Kv1.5-HA expression is apparent across the entire plasma membrane in these cells (Figure 1A and B). Functional expression of the transfected channel was robust. At +80 mV, sustained current densities were 9.5±0.6 pA/pF in control, mCherry transfected ventricular myocytes and 109.2±22.8 pA/pF in those transfected with Kv1.5 plus mCherry (Figure 1 C and D). Confirming that the increased sustained currents were due to Kv1.5 overexpression, these currents were reduced to 70.3±14.5 pA/pF, a 40% reduction, by administration of 100 µM 4-aminopyridine (4-AP) (Figure 1D; p<0.01).  35  Although in some of the mCherry-transfected control cells currents typical of cardiomyocytes were present, in over 60% of these transfectants, as in Figure 1C, little or no Na+ current and only small transient outward currents were seen upon depolarization from -80 mV. These changes occurred also in untransfected myocytes and are thus due to the prolonged culture times required to achieve transgene expression rather than to the lipofectamine-mediated transfection itself. Biolistic bombardment efficiently transfects freshly isolated adult rat ventricular myocytes To determine whether a biolistic protocol was capable of transfecting freshly isolated adult rat ventricular myocytes, we bombarded these cells with gold particles of sizes ranging from 0.6 to 1.6 microns coated with a pcDNA3 construct encoding mCherry fluorescent protein. Freshly isolated adult male rat ventricular myocytes were plated on laminin-coated cover glasses and incubated for one hour at 37oC in an M199 based media (see Materials and Methods). To minimize dedifferentiation in culture, no fetal bovine serum (FBS) was used. Whereas FBS was necessary for the survival of myocytes transfected by the lipofectamine-mediated method to the 24-48 hours necessary to detect gene expression in that system, expression in biolistically transfected myocytes is detectable in less than 24 hours. Over this time frame, myocytes remain viable and retain their morphological characteristics without the FBS (see below). After the one hour incubation, the myocytes were bombarded with gold particles coated with pcDNA3/mCherry or pcDNA3/EGFP (1 µg DNA/mg gold beads) using a Bio-Rad Helios gene gun (Figure 2A) and incubated overnight in the same media. The following morning they were assayed for transfection by fluorescence imaging and electrophysiological methods.  36  At pressures commonly used for other cell types, e.g., 400 p.s.i., cardiomyocytes did not survive no matter which size gold particle was used (data not shown). Optimization of pressure, distance and gold particle size, however, yielded mCherry expression in myocytes in practical numbers when 0.6 micron gold particles were used. No transformants were recovered in experiments employing 1.0 and 1.6 micron gold. Bombardment at 90-110 psi with 0.6 micron gold particles coated with 1 µg DNA/mg gold beads and a gene gun nozzle to myocyte distance of ~1 cm yielded the best results. Although roughly 90% of initially viable myocytes were killed in this pressure range, transfection efficiencies among surviving myocytes were quite high. An average of 28.2 ± 5.7% of surviving myocytes were transfected as assayed in several experiments by mCherry fluorescence. No transfectants were detectable when particles were fired at 60 psi. Pressures greater than ~130 psi killed essentially all of the bombarded myocytes. Myocytes transfected under the optimized conditions retained typical myocyte morphology 24 hours after bombardment. Transfected with expression vectors encoding EGFP (Figure 2B upper panels) or encoding mCherry (Figure 2B middle panels), the transfected myocytes remained rod shaped, striated and with intercalated discs intact. Thus, this is the first successful transfection method for acutely isolated adult rat cardiomyocytes which yields transfectants with essentially normal myocyte morphology. Transfected Kv1.5 is targeted to the intercalated disc in biolistically transfected rat ventricular myocytes Having successfully adapted gene gun technology to the transfection of adult cardiomyocytes, we employed this technique in the study of Kv1.5 trafficking and localization in these cells. Kv1.5 has been reported to localize largely to the intercalated disc in rat and canine ventricular myocytes (2, 8, 17), although some argue that this apparent colocalization may be an artifact of non-specific binding of antibody to this  37  “sticky” region. To test whether or not the channel is truly targeted to that domain, we performed experiments in which we transfected freshly isolated adult male rat ventricular myocytes with a Kv1.5 construct, in pcDNA3, N-terminally tagged with mCherry (Kv1.5-Cherry). When examined under the confocal microscope, transfected cells were readily identified. Like wild-type Kv1.5 detected with anti-Kv1.5 (2, 8, 17), the Kv1.5-Cherry fluorescence was concentrated at the intercalated disc regions of the cells and was quite faint elsewhere (Figure 3A, left panels). Little fluorescence was evident in the cell interior or at peripheral locations of the plasmalemma. This pattern was similar to that of endogenous Kv1.5. When freshly isolated ventricular myocytes were stained with an antibody directed to the C-terminus of Kv1.5, the strongest staining for the endogenous channel was at the intercalated disc (Figure 3A, right panels). While these data clearly indicated that exogenous channels were targeted to the intercalated disc, it was not possible to discriminate between channel inserted in the plasma membrane or those located in a subsurface compartment. To test whether tagged Kv1.5 in transfected myocytes is expressed at the cell surface, experiments with a surface tagged channel were conducted. Freshly isolated myocytes were transfected by the same method with a pcDNA3 construct in which Kv1.5 was tagged with an HA epitope in the extracellular S1-S2 linker and a T7-tag at the intracellular N-terminus (29). Staining of live cells for Kv1.5-HA (with mouse anti-HA, detected with Alexa594-conjugated goat anti-mouse after fixation) clearly demonstrated cell surface expression of the tagged Kv1.5 in the transfected cells (Figure 3B). While in a few cells, some channel was evident at peripheral locations, expression was highest at the intercalated disc in all cases. Figure 3B shows a cell in which peripheral localization of the channel was highest among all myocytes transfected with Kv1.5-HA in these initial experiments. Even in this case, the majority of Kv1.5 is localized to intercalated discs. 38  Transfection with Kv1.5 substantially alters myocyte current characteristics To determine whether the transfected Kv1.5 was functional in the ventricular myocytes, freshly isolated myocytes were co-transfected, as described above, with a Kv1.5 construct in pcDNA3 plus another pcDNA3 construct encoding the either EGFP or mCherry fluorescent protein (1 µg each per mg gold). The construct encoding the fluorescent protein (FP) allowed transfected myocytes to be readily identified for electrophysiology. As illustrated in Figure 4, current profiles were altered as expected for myocytes over-expressing Kv1.5. Whereas peak transient outward current at + 80 mV was unchanged by transfection with Kv1.5 (at 21.2 ± 2.4 pA/pF in Kv1.5 + FPtransfected myocytes vs 21.1 ± 3.5 pA/pF in myocytes transfected with FP alone), the sustained current was increased to 11.7 ± 1.0 pA/pF in the Kv1.5 + FP transfected myocytes from 7.9 ± 0.7 pA/pF in myocytes transfected with FP alone (p<0.05; Figure 4A and C). When the myocytes were subjected to a -40 mV prepulse for 75 ms to inactivate Ito, the functional expression of Kv1.5 could be detected even at early depolarization times (Figure 4B). Peak current at +80 mV in these prepulsed Kv1.5transfected cells was 13.8 ± 1.8 pA/pF versus 10.3 ± 0.9 pA/pF in control, FP-transfected myocytes (p<0.05; Figure 4B and D). Sustained current densities in these prepulsed myocytes were 6.9 ± 0.7 pA/pF in the FP-transfected and 9.7 ± 1.3 pA/pF in the Kv1.5 + FP-transfected cells, respectively. Confirming that the increase in current density was due to expression of the transfected Kv1.5, the sustained currents in Kv1.5-transfected- but not in FP-transfected myocytes were significantly reduced (p<0.05) by 100 µM 4aminopyridine (Figure 4E); sustained currents in myocytes transfected with FP alone showed no such sensitivity to 4-AP.  39  Molecular determinants of Kv1.5 targeting to the intercalated disc of adult rat myocytes Having established that tagged Kv1.5 is functionally expressed and trafficked to the intercalated disc in transfected rat ventricular myocytes, we next investigated the roles in channel trafficking played by several putative mechanisms already described using heterologous expression systems. A canonical PDZ-binding domain located at the Cterminus of the channel has been proposed to be involved in the localization of Kv1.5 at the intercalated disc, where several anchoring proteins containing PDZ domains are located, including ZO-1 (10), ZO-2 (24), ALP (21), and SAP97 (9). We studied the expression of an EGFP-tagged Kv1.5 in which the PDZ binding domain was deleted (Kv1.5∆ETDL-EGFP). As illustrated in Figure 5B, deletion of the PDZ binding domain had no effect on the targeting of the transfected channel. Like full-length Kv1.5 (Figure 5A), the channel localized almost entirely to the intercalated disc. Deletion of the N-terminal-most SH3 binding domain of the channel prevents internalization of the channel in HEK293 cells (6). We hypothesized that Kv1.5 localization at the intercalated disc may result from selective internalization from lateral membrane in the ventricular myocytes. When transfected with a EGFP-tagged SH3 deletion mutant, Kv1.5∆SH3(1)-EGFP, however, rat ventricular myocytes trafficked the mutant channel, like wild-type, to the intercalated disc (Figure 5C). Since there was no disruption of localization to the intercalated disc by the ∆ETDL and ∆SH3 mutants, we moved on to studies on the localization of tagged Kv1.5 channels with larger deletions in their N-termini, one known to have no effect on expression in heterologous cells, another known to not to traffic to the plasma membrane in such cells and another in which surface expression in the heterologous cells is reduced.  40  In this way we would get a handle on whether forward trafficking is at least under the same constraints in ventricular myocytes as in heterologous cells. An HA-tagged deletion of the N-terminal-most 119 amino acid residues, Kv1.5∆N119-HA, was first tested. In heterologous cells, this mutant traffics normally to the plasma membrane (Fedida laboratory, unpublished observation). As illustrated in Figure 5D, when transfected into ventricular myocytes, this channel did indeed traffic normally to the cell surface, specifically to the intercalated disc. A larger deletion, Kv1.5∆N209, results in lower expression than the wild-type channel in heterologous expression systems. This pattern was recapitulated in the ventricular myocytes transfected with a GFP- and HA-tagged Kv1.5∆N209. Expression was detectable as GFPfluorescence at the intercalated disc, but only when brightness and contrast were considerably enhanced (Figure 5E, upper right panels). This was confirmed by staining for the external HA tag. Intercalated disc localization of the GFP-tag and HA-tag overlapped precisely (compare Figure 5E upper panels 2 and 4). We tested also a channel mutant, Kv1.5∆135, which localizes entirely to the cell interior and fails to produce currents when transfected into HEK293 cells (Fedida laboratory unpublished observation). This channel is deleted into the mid T1-domain and likely fails to assemble properly in the HEK cells. When mCherry-tagged Kv1.5∆135 was transfected into ventricular myocytes it failed almost completely to traffic to the intercalated disc. In a few cases, slight punctate staining was evident at this structure (Figure 5F) but in the great majority of transfectants no surface expression of the channel was evident. Electrophysiological analysis of deletion mutant expression in transfected ventricular myocytes was consistent with the imaging data. Transfection with Kv1.5∆SH3 increased the sustained current densities in rat ventricular myocytes to 10.8±1.5 pA/pF at + 80 mV (Figure 6A and B) a value not statistically different from the 11.8±1.4 pA/pF seen in myocytes transfected with wild-type Kv1.5. Densities in both were significantly 41  higher (p<.05) than the 7.7±0.4 pA/pF of control, mCherry alone-transfected myocytes at +80 mV (Figure 6B and G). Neither transfection with Kv1.5∆N209 (Figure 6C and D) nor with Kv1.5∆N135 (Figure 6E and F) had any effect on the sustained currents. Current densities at +80 mV were 8.8±0.4 pA/pF and 8.6±0.5 pA/pF in myocytes transfected with Kv1.5∆N209 and Kv1.5∆N135, respectively, values not significantly different from the 7.7±0.4 pA/pF of control, mCherry-transfected myocytes and significantly lower (p<0.05) than the 11.8±1.4 pA/pF densities of wild-type Kv1.5-transfected myocytes. Of these mutants, only Kv1.5∆SH3 conferred 4-AP sensitivity on the sustained currents in the myocytes. 100 µM 4-AP significantly reduced current densities at +80 mV in Kv1.5∆SH3-transfected myocytes (p<0.05; Figure 6H). Current densities for myocytes transfected with the partial T1-deletion mutants Kv1.5∆N209- and Kv1.5∆N135 were, like myocytes transfected with mCherry alone, not affected by 4-AP application. Thus, as in heterologous systems, an intact T1 domain appears to be necessary for normal trafficking of Kv1.5 in rat ventricular myocytes.  42  Discussion We have employed a new transfection protocol to introduce plasmid-encoded tagged Kv1.5 constructs into adult rat ventricular myocytes and have studied determinants of the surface localization of the Kv1.5 channel using this method. This biolistic transfection method has several major advantages. First, it shows a high efficiency: over 20% of surviving myocytes bombarded with the DNA-coated beads expressing tagged Kv1.5 as assayed by immunocytochemistry. Second, unlike the viral transduction methods generally used to express introduced proteins in myocytes, there is no need for complicated cloning strategies to incorporate the gene of interest into a large viral vector. Any gene in a mammalian expression vector can be introduced into the myocytes. Finally, one major advantage of this method is the rapid expression of the transgene – within 24 hours – avoiding myocyte dedifferentiation in culture. Indeed, the expression of the transgene is rapid enough that the localization and effects of the introduced protein can be assayed in cells retaining the morphological and electrophysiological properties of freshly isolated myocytes. This method also eliminates the need for sophisticated containment facilities and prolonged culture of the myocytes or sophisticated surgical infection protocols. Taken together, this method represents a major breakthrough in the study of ion channel expression in their intact and native environment. Indeed, using this method we provide for the first time unequivocal evidence that Kv1.5 localizes to the intercalated disc in adult cardiac myocytes. This was previously proposed using mainly immunohistochemical approaches for the study of endogenous channel expression in the myocardium (2, 8, 17), but still questioned due to the reportedly sticky nature of the intercalated disc. Using antibody to the channel, Abi-Char, et al. (1), recently demonstrated apparent Kv1.5 partial localization at cell-cell contacts in cultured neonatal rat myocytes, but were unable to detect such localization in neonatal myocytes transfected with a GFP-Kv1.5 fusion construct. This failure to detect GFP-Kv1.5 43  localization at the intercalated discs in these cells may be due to differences in channel expression patterns in neonatal myocytes from those of the adult or to the apparent dedifferentiation of the myocytes in culture. Dedifferentiation is much less problematic in myocytes transfected by our biolistic method. As evident in Figures 2, 3 and 5, adult ventricular myocytes transfected by our method retain morphology, 24 hours after transfection, very similar to that of freshly isolated cells. Currents recorded in fresh and GFP- or mCherry-transfected control cells are also very similar at both time points (Figure 4A). Thus, results obtained with these cells are more representative of in vivo expression patterns than are neonatal myocytes cultured for several days. Our results also indicate that the N-terminal part of the Kv1.5 channel is a crucial determinant for its localization at the level of the intercalated disc. The PDZ-interacting domain located at the C-terminus of the channel appears not to be necessary for this localization. However, it is possible that once addressed to the intercalated disc, anchoring proteins containing PDZ domains play an important role in the organization of Kv α-subunits in large protein complexes, including second messengers (7). We did not find mistargeting to the lateral membrane by any of the channel mutants we tested. Of course, there may be specific motifs outside the regions of the channel that we have tested that are indeed required for correct surface localization. Further work is clearly required to answer this question. The significance of the localization of Kv1.5 to the intercalated disc remains unknown. One possibility is that the intercalated disc functions somewhat analogously to the nervous system’s nodes of Ranvier, as suggested by Maier, et al. (14). In this model, concentration of ion channels at the intercalated disc speeds conduction by allowing depolarization to jump in a saltatory fashion from intercalated disc to intercalated disc. Kucera, Rohr and Rudy (11) have modeled the effects that the high density of NaV1.5 channels in the intercalated disc may have on conduction between cells. However, they  44  found that under most plausible conditions, a high density of functional sodium channels at these sites impaired rather than enhanced conduction. In summary, we have adapted a biolistic procedure to the transfection of acutely isolated rat cardiac myocytes. Transfection efficiencies among surviving myocytes are high and expression of the introduced genes is rapid and robust. Further, the targeting of a tagged potassium channel, Kv1.5, introduced by this method recapitulates that of the endogenous channel. This method has substantial advantages over viral and lipofectamine-mediated methods and will facilitate the study of gene and protein expression as well as of protein trafficking in cardiac cell systems.  45  Figure 2.1. Lipofectamine-mediated transfection of adult ventricular myocytes with Kv1.5 dramatically increases sustained current. (A) Bright field and fluorescence images of a rat ventricular myocyte transfected with N-terminally T7-tagged, externally HA-tagged Kv1.5 construct in pcDNA3 imaged after two days in culture. Surface T7(N)Kv1.5-HA was detected by labeling the cells with anti-HA prior to permeabilization followed by staining with Alexa-488 conjugated secondary antibody. The cells were then fixed, permeabilized and total channel was detected by labeling with anti-T7 antibody followed by staining with secondary antibody conjugated to Alexa-594. Scale bar = 20 µM (B) Bright field and fluorescence images and overlay of a similarly transfected rat cardiomyocyte after four days in culture. The myocyte was transfected with Kv1.5-HA and surface expression was detected as in (A). Scale bar = 10 µM (C) Representative current traces from a ventricular myocyte transfected by the lipofectamine-mediated  46  method with Kv1.5 plus mCherry. Traces shown are from –120 mV in 10-mV steps to +90 mV, from a holding potential of –80 mV, followed by a 100-ms pulse to +60 mV. 4AP traces were after administration of 100 µM 4-aminopyridine to the bath. (D) Mean steady-state current density-voltage relationships from mCherry-transfected and Kv1.5Cherry + mCherry-transfected rat ventricular cardiomyocytes stimulated as described in (A). ** p<0.01 (paired t-test, comparing Kv1.5+Cherry ± 100 µM 4-AP).  47  Figure 2.2. A novel biolistic transfection of isolated adult rat myocytes. (A) A schematic of the protocol employed for transfection of freshly isolated adult rat ventricular myocytes. (B) Top: Widefield fluorescence and bright field overlay image of adult rat ventricular myocytes transfected with EGFP acquired with a 10x objective. The transfected myocyte glows green. Middle: A rat ventricular myocyte transfected with mCherry acquired with a 60x objective. Bottom: An untransfected myocyte imaged as for mCherry. Except where noted in (B) upper panels, scale bars = 20 µM.  48  Figure 2.3. Localization of tagged Kv1.5 in biolistically transfected ventricular myocytes. (A) Left Panels: Bright field and fluorescence image of a rat ventricular myocyte transfected with N-terminally mCherry-tagged Kv1.5 by the biolistic method. Fluorescence is from mCherry; cells were fixed but not stained. Smaller panel on right is a close up view of the boxed area in the middle panel. Right Panels: Fluorescence image of a rat ventricular myocyte labeled for endogenous Kv1.5. Cells were fixed, permeabilized and stained with rabbit anti-Kv1.5 followed by Alexa-594 conjugated goat anti-rabbit secondary antibody. Smaller panel on left is a close up view of the boxed area in the right panel. (B) Bright field and fluorescence images of a ventricular myocyte transfected with T7(N)-Kv1.5-HA. Cells were fixed but not permeabilized and stained with anti-HA and detected with Alexa-594 conjugated secondary antibody. Optical slices across the myocyte in 1 micron intervals are shown in the fluorescence images at left. Scale bars = 20 µM  49  Figure 2.4. Biolistic transfection of Kv1.5 in rat ventricular cardiomyocytes increases the sustained potassium current. (A) Representative currents from fresh (upper left), overnight cultured (upper right), FP-transfected (lower left) and Kv1.5 + FP transfected (lower right) rat ventricular cardiomyocytes. Traces shown are 0 mV and +60 mV, from a holding potential of –80 mV. The stimulation protocol is shown at top. (B) Representative currents from FP-transfected (left panel) and Kv1.5 + FP-transfected (right panel) rat ventricular myocytes after a prepulse to -40 mV to inactivate Ito. Traces shown are from –120 mV in 10-mV steps to +90 mV, from a holding potential of –80 mV with a 75-ms prepulse to -40 mV. The stimulation protocol is shown at top. (C) Mean peak and steady-state (ss) outward current density-voltage relationships from FPtransfected and Kv1.5 + FP-transfected rat ventricular myocytes stimulated as indicated in panel A. * indicates significant difference (p<0.05) between Kv1.5-FP ss to FP ss (One Way ANOVA) (D) Mean peak and steady-state current density-voltage relationships from FP-transfected and Kv1.5 + FP-transfected rat ventricular cardiomyocytes stimulated with 50  a 75-ms prepulse to -40 mV as in panel B. Upper * indicates significant difference (p<0.05) between Kv1.5-FP prepulse peak to FP prepulse peak; lower * indicates significant difference (p<0.05) between Kv1.5-FP prepulse ss to FP prepulse ss (One Way ANOVA). (E) 4-AP sensitivity of sustained currents in control, FP-transfected and Kv1.5 + FP-transfected rat ventricular myocytes. The ratio of sustained current density at + 80 mV after treatment with 4-AP to the sustained current density prior to 4-AP treatment is indicated ± S.E.M. * p<0.05 (paired t-test).  51  Figure 2.5. Localization of wild-type and mutant Kv1.5 channels to the intercalated disc in biolistically transfected rat ventricular myocytes. (A) Localization of transfected GFP-tagged full length Kv1.5 in rat ventricular myocytes. Fluorescence is from GFP; cells were fixed but not stained. Panel at right is a close up view of the boxed area in the middle panel. (B) Localization of transfected GFP-tagged Kv1.5∆EDTL in rat ventricular myocytes. Fluorescence is from GFP. (C) Localization of transfected GFPtagged Kv1.5∆SH3(1) in rat ventricular myocytes. Fluorescence is from GFP. (D) Localization of transfected externally HA-tagged Kv1.5∆N119 in rat ventricular myocytes. Kv1.5 was detected with anti-HA and an Alexa-488 conjugated secondary antibody in cells were that fixed but not permeabilized. (E) Localization of Kv1.5∆N209 in transfected rat ventricular myocytes. GFP- and HA-doubly tagged Kv1.5∆N209 was detected with anti-HA and an Alexa-488 conjugated secondary antibody in cells were that fixed but not permeabilized (panel 2). GFP fluorescence at the same acquisition settings 52  used in all other experiments is shown in the third panel. Panel 4 shows the same image as panel 3, but enhanced in brightness and contrast such that the intercalated disclocalized signal is visible. A bright field image of the cell is in panel 1. (F) Localization of transfected mCherry-tagged Kv1.5∆135 in rat ventricular myocytes. Fluorescence is from mCherry. Scale bars = 20 µm. Smaller panels in each group are close up views of the boxed areas of the respective fluorescence images.  53  Figure 2.6. Effects of mutations in transfected Kv1.5 on sustained currents in rat ventricular myocytes. (A) Representative currents from rat ventricular cardiomyocytes co-transfected with Kv1.5∆SH3 and mCherry. Traces shown are from –80 mV in 10-mV steps to +80 mV, from a holding potential of –80 mV. (B) Mean steady-state current density-voltage relationships from ventricular myocytes transfected with mCherry (control), Kv1.5 + mCherry, and Kv1.5∆SH3 + mCherry stimulated as indicated in A. * indicates significant difference (p<0.05) between control ss and both Kv1.5 ss and Kv1.5∆SH3 ss (One Way ANOVA). (C) Representative currents from rat ventricular cardiomyocytes co-transfected with Kv1.5∆N209 and mCherry stimulated as indicated in A. (D) Mean steady-state current density-voltage relationships from ventricular myocytes transfected with mCherry (control), Kv1.5 + mCherry, and Kv1.5∆N209 + mCherry stimulated as indicated in A. * indicates significant difference (p<0.05) between Kv1.5 ss and both Kv1.5∆N209 ss and control ss (One Way ANOVA). (E) Representative currents 54  from rat ventricular cardiomyocytes co-transfected with Kv1.5∆N135 and mCherry stimulated as indicated in A. (F) Mean steady-state current density-voltage relationships from ventricular myocytes transfected with mCherry (control), Kv1.5 + mCherry, and Kv1.5∆N135 + mCherry. * indicates significant difference (p<0.05) between Kv1.5 ss and both Kv1.5∆N135 ss and control ss (One Way ANOVA). (G) Representative currents from rat ventricular cardiomyocytes co-transfected with mCherry alone stimulated as indicated in A. (H) 4-AP sensitivity of sustained currents in control, mCherry-transfected ventricular myocytes and in ventricular mycoytes transfected with the mutants described in panels A through F. The ratio of sustained current density at + 80 mV after treatment with 4-AP to the sustained current density prior to 4-AP treatment is indicated ± S.E.M. *p<0.05 (paired t-test).  55  References 1. Abi-Char J, El Haou S, Balse E, Neyroud N, Vranckx R, Coulombe A and Hatem SN. The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes. Am J Physiol Heart Circ Physiol 294: H1851-H1861, 2008. 2. Abi-Char J, Maguy A, Coulornbe A, Balse E, Ratajczak P, Samuel JL, Nattel S and Hatem SN. Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes. J Physiol 582: 1205-1217, 2007. 3. Aikawa R, Huggins GS and Snyder RO. Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction. J Biol Chem 277: 18979-18985, 2002. 4. Balse E, El-Haou S, Dillanian G, Dauphin A, Eldstrom J, Fedida D, Coulombe A and Hatem SN. Cholesterol modulates the recruitment of Kv1.5 channels from Rab11associated recycling endosome in native atrial myocytes. Proc Natl Acad Sci U S A 106: 14681-14686, 2009. 5. Barry DM, Trimmer JS, Merlie JP and Nerbonne JM. Differential expression of voltage-gated K+ channel subunits in adult rat heart: Relation to functional K+ channels. Circ Res 77: 361-369, 1995. 6. Choi WS, Khurana A, Mathur R, Viswanathan V, Steele DF and Fedida D. Kv1.5 surface expression is modulated by retrograde trafficking of newly endocytosed channels by the dynein motor. Circ Res 97: 363-371, 2005. 7. El Haou S, Balse E, Neyroud N, Dilanian G, Gavillet B, Abriel H, Coulombe A, Jeromin A and Hatem SN. Kv4 Potassium Channels Form a Tripartite Complex With  56  the Anchoring Protein SAP97 and CaMKII in Cardiac Myocytes. Circ Res 104: 758-769, 2009. 8. Eldstrom J, Van Wagoner DR, Moore ED and Fedida D. Localization of Kv1.5 channels in rat and canine myocyte sarcolemma. FEBS Letts 580: 6039-6046, 2006. 9. Godreau D, Vranckx R, Maguy A, Rucker-Martin C, Goyenvalle C, Abdelshafy S, Tessier S, Couétil J-P and Hatem SN. Expression, regulation and role of the MAGUK protein SAP-97 in human atrial myocardium. Cardiovasc Res 56: 433-442, 2002. 10. Kieken F, Mutsaers N, Dolmatova E, Virgil K, Wit AL, Kellezi A, Hirst-Jensen BJ, Duffy HS and Sorgen PL. Structural and Molecular Mechanisms of Gap Junction Remodeling in Epicardial Border Zone Myocytes following Myocardial Infarction. Circ Res 104: 1103-U219, 2009. 11. Kucera JP, Rohr S and Rudy Y. Localization of Sodium Channels in Intercalated Disks Modulates Cardiac Conduction. Circ Res 91: 1176-1182, 2002. 12. Li DQ, Takimoto K and Levitan ES. Surface expression of Kv1 channels is governed by a C-terminal motif. J Biol Chem 275: 11597-11602, 2000. 13. Loewen ME, Wang ZR, Eldstrom J, Zadeh AD, Khurana A, Steele DF and Fedida D. Shared requirement for dynein function and intact microtubule cytoskeleton for normal surface expression of cardiac potassium channels. Am J Physiol Heart Circ Physiol 296: H71-H83, 2009. 14. Maier SK, Westenbroek RE, Schenkman KA, Feigl EO, Scheuer T and Catterall WA. An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart. Proc Natl Acad Sci U S A 99: 40734078, 2002. 57  15. Manganas LN, Wang Q, Scannevin RH, Antonucci DE, Rhodes KJ and Trimmer JS. Identification of a trafficking determinant localized to the Kv1 potassium channel pore. Proc Natl Acad Sci U S A 98: 14055-14059, 2001. 16. Matsuno Y, Iwata H, Umeda Y, Takagi H, Mori Y, Miyazaki J, Kosugi A and Hirose H. Nonviral gene gun mediated transfer into the beating heart. Asaio J 49: 641644, 2003. 17. Mays DJ, Foose JM, Philipson LH and Tamkun MM. Localization of the Kv1.5 K+ channel protein in explanted cardiac tissue. J Clin Invest 96: 282-292, 1995. 18. McEwen DP, Schumacher SM, Li Q, Benson MD, Iniguez-Lluhi JA, Van Genderen KM and Martens JR. Rab-GTPase-dependent Endocytic Recycling of KV1.5 in Atrial Myocytes. J Biol Chem 282: 29612-29620, 2007. 19. Muller OJ, Katus HA and Bekeredjian R. Targeting the heart with gene therapyoptimized gene delivery methods. Cardiovasc Res 73: 453-462, 2007. 20. Nesti E, Everill B and Morielli AD. Endocytosis as a Mechanism for Tyrosine Kinase Dependent Suppression of a Voltage Gated Potassium Channel. Mol Biol Cell 15: 4073-4088, 2004. 21. Pashmforoush M, Pomies P, Peterson KL, Kubalak S, Ross JR, Hefti A, Aebi U, Beckerle MC and Chien KR. Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy. Nat Med 7: 591-597, 2001. 22. Quignard JF, Grazzini E, Guillon G, Harricane MC, Nargeot J and Richard S. Absence of calcium channels in neonatal rat aortic myocytes. Pflugers Archiv-Eur J Physiol 431: 791-793, 1996.  58  23. Seebohm G, Strutz-Seebohm N, Birkin R, Dell G, Bucci C, Spinosa MR, Baltaev R, Mack AF, Korniychuk G, Choudhury A, Marks D, Pagano RE, Attali B, Pfeufer A, Kass RS, Sanguinetti MC, Tavare JM and Lang F. Regulation of endocytic recycling of KCNQ1/KCNE1 potassium channels. Circ Res 100: 686-692, 2007. 24. Singh D, Solan JL, Taffet SM, Javier R and Lampe PD. Connexin 43 interacts with zona occludens-1 and-2 proteins in a cell cycle stage-specific manner. J Biol Chem 280: 30416-30421, 2005. 25. Steele DF, Eldstrom J and Fedida D. Mechanisms of cardiac potassium channel trafficking. J Physiol 582: 17-26, 2007. 26. Svensson EC, Marshall DJ, Woodard K, Lin H, Jiang F, Chu LI and Leiden JM. Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinant adeno-associated virus vectors. Circulation 99: 201-205, 1999. 27. Thum T, Galuppo P, Wolf C, Fiedler J, Kneitz S, van Laake LW, Doevendans PA, Mummery CL, Borlak J, Haverich A, Gross C, Engelhardt S, Ertl G and Bauersachs J. MicroRNAs in the human heart - A clue to fetal gene reprogramming in heart failure. Circulation 116: 258-267, 2007. 28. Walsh KB and Parks GE. Changes in cardiac myocyte morphology alter the properties of voltage-gated ion channels. Cardiovasc Res 55: 64-75, 2002. 29. Zadeh AD, Xu HJ, Loewen ME, Noble GP, Steele DF and Fedida D. Internalized Kv1.5 traffics via Rab-dependent pathways. J Physiol 586: 4793-4813, 2008. 30. Zhou YY, Wang SQ, Zhu WZ, Chruscinski A, Kobilka BK, Ziman B, Wang S, Lakatta EG, Cheng HP and Xiao RP. Culture and adenoviral infection of adult mouse  59  cardiac myocytes: methods for cellular genetic physiology. Am J Physiol Heart Circ Physiol 279: H429-H436, 2000. 31. Zhu J, Watanabe I, Gomez B and Thornhill WB. 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, 2001. 32. Zhu J, Watanabe I, Gomez B and Thornhill WB. Trafficking of Kv1.4 potassium channels: interdependence of a pore region determinant and a cytoplasmic Cterminal VXXSL determinant in regulating cell-surface trafficking. Biochem J 375: 761768, 2003.  60  Chapter 32 Trafficking of endogenous Kv4.2 in adult ventricular myocytes 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, 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 (41) and, like several other potassium channels (17), the numbers remaining at the plasma membrane are modulated via a dynein-dependent process (9). 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 (18, 41), and that of another potassium channel, KCNQ1 (27). These small GTPases regulate diverse processes in eukaryotic cells, and are very important in regulating intracellular membrane trafficking (23, 31, 32). These Rab proteins are intimately involved in the regulation of vesicle trafficking in all eukaryotic cells (42), including budding, delivery, tethering and fusion (reviewed in (14)), and they play significant roles in channel internalization and                                                              2  A version of this chapter will be submitted for publication. Wang T, Cheng Y, Goonasekara CL, Dou Y, Steele D and Fedida D (2010) Trafficking of endogenous Kv4.2 in adult ventricular myocytes  61  recycling. Rab5, localized to the early endosomes, acts as a primary effector of the rapid endocytosis of KCNQ1/KCNE1 and Kv1.5 (27, 41). 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 Rab5dependent endocytosis (41). After internalization, Kv channels may also be delivered into Rab11-associated recycling endosomes, and slowly recycled back to cell surface (1, 3, 27). Rab7 WT overexpression reduces Kv1.5 surface expression in H9c2 myoblasts, very probably by enhancing the lysosomal degradation of the channel (41). 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 (30)), 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 (33, 36), others have been shown to traffic independently of these vesicles, at least in some cell types (11, 15). Trafficking through COPII vesicles is dependent on the Sar1 small GTPase, which regulates the formation of the COPII coat-associated protein complex (4, 5). Rab1 recruits tethering factors into the cis-SNARE complex and facilitates fusion between ER budded vesicles and Golgi compartments (2). However, it has been reported that secretory protein H-Ras is trafficked independently of Sar1 (43) and cystic fibrosis transmembrane conductance regulator (CFTR) trafficking does not require Rab1 (40), 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 (11, 15). Unlike in neurons, cardiac Kv4.2 forward trafficking is dependent on KChIP2; the involvement of 62  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 (26, 39). 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 degradaton or that shunting to that compartment is rare or slow.  63  Materials and methods 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, 12.42 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, about 1 ml of the enzyme-containing solution was periodically decanted into a 15 mL conical tube and filled with storage 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 and 50 µg/mL streptomycin. Media 64  with 1% FBS, 50 U/mL penicillin and 50 µg/mL streptomycin, 1/1000 GIBCO™ InsulinTransferrin-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 4hrs, 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. HEK 293 cell preparation and transfection HEK293 cells were cultured and transfected as described previously (1). 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. Plasmid constructs Plasmid constructs were generally as described previously (41). 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 (2). In generating a tetracycline-inducible Kv4.2, mCherry fused to the N-terminus of Kv4.2 was inserted into pcDNA4-TO. Plasmid DNA was prepared for transfection using the Qiagen Plasmid Midi Kit (Qiagen Inc, Valencia, CA).  65  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 -120 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 6h 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 6h 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 6h prior to electrophysiological recordings. 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 66  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 Alexa488 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). 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-specific binding was blocked by incubation in 2% BSA in PBS for 30 min at room temperature. Following this, the cells were labelled with Alexa594 conjugated goat anti-mouse antibody (Molecular Probes) for 1 h to detect Kv4.2-HA. Samples were imaged and analysed as described above.  67  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 cotransfected 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. 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.  68  Results 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 M199based 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 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 untransfected 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 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 1C and 1D), both of which were essentially identical to those of freshly isolated myocytes. 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 heterologous expression system (15). To test whether Sar1 influences Kv4.2 functional expression in ventricular myocytes, electrophysiological experiments were performed on transfected myocytes. Ventricular myocytes were cotransfected 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 69  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. Rab 1DN 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 (20). 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 (21). The GDP-locked Rab1 S25N (Rab1DN) has been reported to efficiently block trafficking of the Kir3.1/Kir3.4 complex to the plasma membrane (24). We tested whether this Rab1 dominant-negative would similarly affect the trafficking of Kv4.2 in ventricular myocytes. As illustrated in Figure 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 (41). Thus, HEK293 cells were 70  simultaneously transfected with inducible Kv4.2-Cherry ± either Sar1DN or Rab1DN tagged with EGFP. On the next day, Kv4.2 expression was induced with tetracycline and, 12 to 24 hours later, patch clamping was performed. As shown in Figure 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. 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 (8, 10, 34). It mediates both the trafficking of many newly synthesized proteins to the same location and recycling of many endocytosed proteins to the plasma membrane (3, 12, 18, 27, 41). As shown in Figure 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 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 dynamin inhibitory peptide (DIP), a 71  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 5C and D). As Rab11 is involved in both recycling of endocytosed channels (3, 18, 27, 41) and in the trafficking of newly synthesized proteins to the cell surface (35), 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 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 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. 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 of other potassium channels. Rab5 is required for clathrin-mediated 72  endocytosis and early endosome formation (6). 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 (41). We tested electrophysiologically whether Rab5 was involved, also, in the trafficking of Kv4.2. As shown in Figure 7, transfection with Rab5 DN 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 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 (29, 38). When applied to ventricular myocytes, DIP treatment significantly increased peak current density at +90 mV from the 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 7C and D). Thus, it is highly likely that both Rab5 DN and DIP are interfering with Kv4.2 trafficking by inhibiting endocytosis of the channel. 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 (41). We therefore investigated whether Rab4 is involved in endogenous Kv4.2 73  trafficking. As shown in Figure 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 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 (41). 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 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 Kv4.2 construct (a kind gift of Dr. Robert Bähring, University of Hamburg, Germany) and Rab4WT-GFP. The next day, the 74  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 9, there was no colocalization of Kv4.2 and Rab4 at 0 minute (Figure 9, top panels) but, by 45 minutes, Rab4 colocalization with internalized Kv4.2 was readily apparent (Figure 9, middle panels). By six hours internalization time, this colocalization was quite extensive (Figure 9, bottom panels). Thus, internalized Kv4.2 does indeed traffic through the Rab4-positive compartment, very probably recycling rapidly to the cell surface. Kv4.2 degradation likely occurs in the proteosome 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 (41). 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 (7, 19). 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 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 the proteosome, the proteosome inhibitor MG132 increased peak current densities by roughly 40% from 25±3.4 to 34.3±3.3 pA/pF (Figure 11).  75  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 (41). 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. Proteosomal 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 (11, 15).  In the HeLa cells,  Kv4.2/KChIP1 traffic independently of Sar1, traveling from the ER to Golgi instead via vesicles uncoated with COPII (11) and, in a majority of cells, reaching the plasmalemma even in the presence of a Sar1 dominant negative that completely blocked VSVG traffic to that membrane (15). Hasdemir, et al. (15), hypothesize that Kv4.2 traffics from the ER to Golgi via ‘KChIP vesicles’ that, in the case of KChIP1 at least, are demonstrably independent of Sar1 function. KChIP1 is predominant in brain (22) but not in the heart (26). Thus, a prime possibility is that KChIP2-dependent trafficking of Kv4.2 is very 76  different than that driven by KChIP1. KChIP2 (3 splice variants) is predominant in cardioymyotes (26). Perhaps KChIP2 traffics very differently than KChIP1. Indeed, Flowerdew, et al. (11) 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 (41). The dominant negative did, however, block the increase in Kv1.5 associated with cholesterol depletion in atrial myocytes (3). 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 (31)). 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 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 (28) and the amiloride-sensitive sodium channel, ENaC (25). 77  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 (41) and to be degraded also by the proteasome (16), 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 (37) and hERG channels (13), 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.  78  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 posttransfection 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 -120 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.  79  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 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.  80  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 1. B. Current density versus voltage plot for data for mCherry-, mCherry + Rab1WT-, and mCherry + Rab1DN (T39N) transfected myocytes, respectively. Data are represented as mean ± SEM. * p<0.05, comparing Sar1DN + mCherry to control, mCherry-transfected myocytes.  81  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. Current density versus voltage were plotted from data recorded 12-24 hours post induction. * p<0.05, relative to Kv4.2 control.  82  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 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 + Rab11DN, incubated for 6 hours with dynamin inhibitory peptide (DIP) as indicated. Current protocols were as described in Figure 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.  83  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 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.  84  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 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 Rab5WT + mCherry to control, mCherry-transfected myocytes. C. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post transfection with mCherry or Rab5-WT + mCherry + Rab5DN, incubated for 6 hours with dynamin inhibitory peptide (DIP) as indicated. Current protocols were as described in Figure 1. D. Current density versus voltage plot for myocytes thus treated. Data are represented as mean ± SEM.  85  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 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 Rab4WT + mCherry to control, mCherry-transfected myocytes. C. Representative currents from rat ventricular cardiomyocytes recorded 12-24 hours post transfection with mCherry or Rab4-WT + mCherry + Rab4DN, incubated for 6 hours with dynamin inhibitory peptide (DIP) as indicated. Current protocols were as described in Figure 1. D. Current density versus voltage plot for myocytes thus treated. Data are represented as mean ± SEM.  86  Figure 3.9: Internalized Kv4.2 colocalizes with Rab4 in ventricular myocytes. Ventricular myocytes were transfected with externally HA-tagged Kv4.2 plus EGFPtagged Rab4, then, 24 hours later incubated with mouse anti-HA at 37oC for the times indicated prior to fixation then staining with Alexa594 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.  87  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 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.  88  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 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.  89  Reference s 1.  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Yoo JS, Moyer BD, Bannykh S, Yoo HM, Riordan JR and Balch WE. Nonconventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway. J Biol Chem 277: 11401-11409, 2002. 41. Zadeh AD, Xu HJ, Loewen ME, Noble GP, Steele DF and Fedida D. Internalized Kv1.5 traffics via Rab-dependent pathways. Journal of Physiology-London 586: 47934813, 2008. 42. Zerial M and McBride H. Rab proteins as membrane organizers (vol 2, pg 107, 2001). Nature Reviews Molecular Cell Biology 2: 216, 2001. 43. Zheng H, Mckay J and Buss JE. H-ras does not need COP I- or COP II-dependent vesicular transport to reach the plasma membrane. J Biol Chem 282: 25760-25768, 2007.  95  Chapter 4 Final discussion and conclusions The data presented in this thesis are the results from two interrelated projects. The first project involved the development of a biolistic (gene gun) method that allows highly efficient transfection of isolated rat adult cardiac myocytes with standard mammalian expression vectors, and the application of that method to the study of Kv1.5 localization in ventricular myocytes. The second project used a new transfection method to investigate the roles of several small GTPases in the forward and retrograde trafficking of Kv4.2 in cardiac myocytes. This work has allowed us to further our understanding of how Kv channel localization and surface expression are regulated in cardiomyocytes, and has provided important insights into how these channels modulate cardiac functioning. Gene gun project Gene gun-mediated (biolistic) transfection has proven a powerful technique in the transfection of a wide range of cell and tissue types, but, to date, has proved of limited use in the cardiovascular field. Here we have developed, for the first time, a reliable and convenient biolistic method for the transfection of acutely isolated rat ventricular myocytes. Employing this method, we unequivocally demonstrated that tagged Kv1.5 is efficiently localized to the intercalated disk in ventricular myocytes. We further demonstrated that Kv1.5 deletion mutants known to reduce the surface expression of the channel in heterologous cells similarly reduce its surface expression in ventricular myocytes, but do not affect the targeting of the channel to the intercalated disk. It is possible that this is because the specific motifs required in Kv1.5 localization are not within the regions of the channel that we have tested. Alternatively, this may be because the mutant channels co-assemble with endogenous Kv1 channels, and the coassembled wild-type subunits effectively complement the trafficking defect that would exist in a 96  homomultimeric mutant channel. The biolistic transfection method that we have developed has the potential of becoming a very powerful tool to study the mechanism and regulators in channel localization and trafficking in cardiac myocytes. Regulation of channel trafficking by small GTPases In this study, the involvement of Rab1, Rab5, Rab4, Rab11 and Sar1 GTPases in the trafficking of endogenous Kv4.2 in ventricular myocytes is reported for the first time. We find that in cardiac myocytes, Kv4.2 is exported from ER via a conventional COPII pathway dependent on Sar1. Block of the normal functioning of Sar1 or Rab1 significantly reduced the surface expression of Kv4.2. These results are very different from those reported in another study that found that Kv4.2/KChIP1 traffic out of ER independent of Sar1 in transfected HeLa cells (6). One possibility is, because KChIP2 is predominant in cardiomyocytes, KChIP2-mediated Kv4.2 trafficking pathways are different from KChIP1. Alternatively, the trafficking machinery and regulators between cell types may differ in as yet unrecognized ways. For example, a distinct set of v- and tSNAREs appears to be involved in TGN-to-plasma membrane transport in different cell types. In neural cells, the t-SNAREs syntaxin 1 and the v-SNARE synaptobrevin (also called VAMP2) are involved in the regulated delivery of synaptic vesicles (13). At least three other syntaxin homologs and several VAMP homologs function in the delivery of various cargo to the plasma membrane in other cell types (1). Similarly, the involvement of small GTPases may also vary in different cell types. We have implicated, also, four prototypical Rab GTPases in the trafficking of Kv4.2. Rab 5 and Rab4 were previously reported to be involved in the endocytosis and rapid recycling of Kv1.5 in HEK cells and the H9c2 myoblast cell line (19). Similarly, block of Rab5 mediated endocytosis by overexpression of a Rab5DN mutant significantly increases Kv4.2 surface expression in ventricular myocytes. Rab4DN also increases the  97  functional expression of Kv4.2, very probably by indirectly interfering with the channel endocytosis. Our lab has previously shown that internalized Kv1.5 colocalizes with wildtype Rab4 (19). We also showed in immunocytochemistry experiments that Kv1.5 endocytosis is significantly reduced upon expression of Rab4DN (19). These results suggest that it’s highly possible that Rab4 is involved in rapid recycling of both Kv1.5 and Kv4.2. Unlike in Kv1.5 trafficking, Rab11DN did not affect Kv4.2 functional expression, while Rab11WT overexpression significantly increased the Kv4.2 currents. Pre-treatment with BFA successfully prevented the Rab11WT-associated increase of Kv4.2 surface expression, indicating that Rab11 overexpression enhances the forward trafficking of newly synthesized channels to plasma membrane. This is very different from the role that Rab11 plays in the recycling of Kv1.5 and KCNQ1/KCNE1(17), suggesting either a diversity of channel trafficking pathways or divergence in the pathways employed in ventricular myocytes from those in other cell types. Finally, we found that the degradation of cardiac Kv4.2 is likely not dependent on Rab7 dependent delivery to the lysosome. Neither Rab7DN nor WT overexpression had any effect on Kv4.2 functional expression. The results with the proteasome inhibitor MG132 indicate that channel more likely undergoes ubiquitin–dependent proteasomal degradation, similar to the degradation of CFTR (18) and hERG channels (5). Future directions The mechanisms that regulate the expression of potassium channels at the sarcolemma of adult rat ventricular myocytes and the intracellular pathways through which these channels are trafficked have been investigated. The determinants of these processes are only beginning to be elucidated, and our work suggests that there are many  98  future experiments to be attempted and several future studies related to the work in this thesis are outlined below.  Kv1.5 localization to the intercalated disk The gene gun transfection technique has allowed us to unequivocally demonstrate that Kv1.5 localizes to the intercalated disk in rat ventricular myocytes. However, the significance of the localization of Kv1.5 to the intercalated disk remains unknown. In ventricular myocytes, Ikur, the current underlain by Kv1.5 is notably absent. In atrial myocytes, where Ikur is quite prominent, Kv1.5 localizes to both intercalated disk and to lateral membrane (11). One reasonable speculation is that the localization of Kv1.5 at intercalated disk prevents it from being functional, and only the laterally expressed Kv1.5 actually conducts potassium currents. This speculation is supported by the lack of 4-AP sensitive current in rat adult ventricular myocytes, in which endogenous Kv1.5 is known to be expressed at the intercalated disk. It is possible that actin cytoskeleton could affect channel anchoring and functioning at intercalated disk. It has been shown that Kvβ1 subunit actions on Kv1 channels are modified by actin filament disruption induced by cytochalasins (8). Recent investigations also show a direct binding between Kv1.5 and αactinin-2, a member of the spectrin-family of actin crosslinking proteins (9). Both disruption of the actin cytoskeleton with cytochalasin D and incubation with an α-actinin2 antisense oligonucleotide significantly increased Kv1.5-dependent potassium currents in Kv1.5-expressing HEK cells (9). Therefore, it is possible that actin cytoskeleton anchors Kv1.5 at the intercalated disk or alters the kinetics of channels present there or both. Further study of the interaction between Kv1.5 and the cytoskeleton in adult cardiac myocytes could point the way to new regulatory mechanisms of this channel.  99  It is also likely that there are some function-altering β subunits, specifically expressed in ventricular myocytes, that may also contribute to the localization of Kv1.5 to intercalated disk. It will be important to determine the subunit composition of the Kvl.5 protein-containing complexes in ventricular myocytes, and whether these β subunits colocalize with the Kv1.5 channel in rat ventricle but not in atrium. Exploring the effects and mechanisms of Kv1.5 localization to intercalated disk will be very important in understanding how the organization of Kv channel surface expression is associated with the channel function or even how it is perturbed in cardiac diseases. Characterization of Kv channel trafficking pathways Previous studies on the trafficking of Kv1.5 demonstrated the involvement of several Rab GTPases (2, 12, 19). The study of the Kv4.2 trafficking reported here has similarly revealed the involvement of several small GTPases in the forward trafficking, internalization, and recycling of that channel. It will be interesting to investigate the mechanism(s) behind the trafficking through each of these GTPase-defined compartments; we will learn much about the mechanism by which cardiomyocytes regulate the functional expression of its resident ion channels. For example, the cAMP-dependent protein kinase (PKA) and Src-family protein tyrosine kinases (PTKs) have been reported to affect the functional expression of hERG and Kv1.5 (3, 7, 10, 15). Determining how these kinases and other signalling molecules modulate Rab-mediated channel trafficking pathways will provide insights into the mechanisms of electrical remodelling in a variety of heart diseases, such as atrial fibrillation. In addition, our results show that Rab11DN has different effects on surface expression Kv1.5 and Kv4.2, indicating that trafficking pathways are likely to be used differentially by different channels. Using different trafficking pathways for different channels might be a way of fine-tuning specific potassium channel activity in response to various physiological and pathological conditions. Different Kv channels may interact with different cytoplasmic adaptor 100  proteins or SNARE complexes, which lead them to different sub-cellular compartments. Another level of diversity for Kv channels may be in their associations with distinct accessory β-subunits, which act as chaperones, thereby regulating channel cell-surface expression and current kinetics. Due to their molecular diversity, channels may also respond to different cellular signals, which could target internalized channel to recycling or degradation pathways. Therefore, the study of Kv channel interacting proteins, as well as the signalling pathways involved in the channel trafficking, will greatly enhance our understanding of whether targeting of channels to specific post-internalization pathways are necessarily specific or whether some or all choices are stochastic. For example, the shunting of internalized transferrin might be at least partially stochastic(14). All of this will improve our understanding of heart function in health and, importantly, also in disease. The mechanisms underlying normal and abnormal cardiac rhythms are complex and incompletely understood. Through the study of uncommon inheritable arrhythmia syndromes, including the long QT and Brugada syndromes, it is becoming more and more appreciated that the mechanisms by which heart cells process ion channel proteins and traffic them must have important implications in cardiac arrhythmia diseases. Once understood, manipulation of channel trafficking pathways may provide novel treatments for cardiac dysfunctions. Protein trafficking defects reduce the delivery of channels to the cell plasma membrane and have emerged as a common LQT2 disease mechanism. Previous investigations have found that some LQT2-associated mutant HERG channels are incorrectly folded and therefore earmarked for intracellular retention by cellular quality control mechanisms (16, 20). Interestingly, some mutant channels defective in trafficking can be “pharmacologically rescued,” for instance by incubating with HERG channel blocking drugs, which may help to stabilize intermediate states of mutants of that channel and thus promote its exit from the ER (4). The rescue methods 101  increase the probability that a mutant protein will reach its native conformation, and therefore have important therapeutic implications for treating cardiac diseases. Understanding how all channels traffic will undoubtedly open up yet unimagined mechanisms to be exploited in these treatments.  102  References 1.  Advani RJ, Bae HR, Bock JB, Chao DS, Doung YC, Prekeris R, Yoo JS, and  Scheller RH. Seven novel mammalian SNARE proteins localize to distinct membrane compartments. J Biol Chem 273: 10317-10324, 1998. 2.  Balse E, El-Haou S, Dillanian G, Dauphin A, Eldstrom J, Fedida D,  Coulombe A, and Hatem SN. Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes. Proc Natl Acad Sci U S A 106: 14681-14686, 2009. 3.  Chen J, Sroubek J, Krishnan Y, Li Y, Bian J, and McDonald TV. PKA  phosphorylation of HERG protein regulates the rate of channel synthesis. Am J Physiol Heart Circ Physiol 296: H1244-1254, 2009. 4.  Ficker E, Obejero-Paz CA, Zhao S, and Brown AM. The binding site for  channel blockers that rescue misprocessed human long QT syndrome type 2 ether-agogo-related gene (HERG) mutations. J Biol Chem 277: 4989-4998, 2002. 5.  Gong Q, Keeney DR, Molinari M, and Zhou Z. Degradation of trafficking-  defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway. J Biol Chem 280: 19419-19425, 2005. 6.  Hasdemir B, Fitzgerald DJ, Prior IA, Tepikin AV, and Burgoyne RD. Traffic  of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway. J Cell Biol 171: 459-469, 2005. 7.  Holmes TC, Fadool DA, Ren R, and Levitan IB. Association of Src tyrosine  kinase with a human potassium channel mediated by SH3 domain. Science 274: 20892091, 1996. 8.  Jing J, Peretz T, Singer-Lahat D, Chikvashvili D, Thornhill WB, and Lotan I.  Inactivation of a voltage-dependent K+ channel by beta subunit. Modulation by a  103  phosphorylation-dependent interaction between the distal C terminus of alpha subunit and cytoskeleton. J Biol Chem 272: 14021-14024, 1997. 9.  Maruoka ND, Steele DF, Au BP, Dan P, Zhang X, Moore ED, and Fedida D.  alpha-actinin-2 couples to cardiac Kv1.5 channels, regulating current density and channel localization in HEK cells. FEBS Lett 473: 188-194, 2000. 10.  Mason HS, Latten MJ, Godoy LD, Horowitz B, and Kenyon JL. Modulation  of Kv1.5 currents by protein kinase A, tyrosine kinase, and protein tyrosine phosphatase requires an intact cytoskeleton. Mol Pharmacol 61: 285-293, 2002. 11.  Mays DJ, Foose JM, Philipson LH, and Tamkun MM. Localization of the  Kv1.5 K+ channel protein in explanted cardiac tissue. J Clin Invest 96: 282-292, 1995. 12.  McEwen DP, Schumacher SM, Li Q, Benson MD, Iniguez-Lluhi JA, Van  Genderen KM, and Martens JR. Rab-GTPase-dependent endocytic recycling of Kv1.5 in atrial myocytes. J Biol Chem 282: 29612-29620, 2007. 13.  McMahon HT, and Sudhof TC. Synaptic core complex of synaptobrevin,  syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem 270: 2213-2217, 1995. 14.  Lakadamyali M, Rust MJ, and Zhuang X. Ligands for clathrin-mediated  endocytosis are differentially sorted into distinct populations of early endosomes. Cell 124: 997-1009, 2006. 15.  Nitabach MN, Llamas DA, Araneda RC, Intile JL, Thompson IJ, Zhou YI,  and Holmes TC. A mechanism for combinatorial regulation of electrical activity: Potassium channel subunits capable of functioning as Src homology 3-dependent adaptors. Proc Natl Acad Sci U S A 98: 705-710, 2001. 16.  Paulussen A, Raes A, Matthijs G, Snyders DJ, Cohen N, and Aerssens J. A  novel mutation (T65P) in the PAS domain of the human potassium channel HERG results in the long QT syndrome by trafficking deficiency. J Biol Chem 277: 48610-48616, 2002.  104  17.  Seebohm G, Strutz-Seebohm N, Birkin R, Dell G, Bucci C, Spinosa MR,  Baltaev R, Mack AF, Korniychuk G, Choudhury A, Marks D, Pagano RE, Attali B, Pfeufer A, Kass RS, Sanguinetti MC, Tavare JM, and Lang F. Regulation of endocytic recycling of KCNQ1/KCNE1 potassium channels. Circ Res 100: 686-692, 2007. 18.  Ward CL, Omura S, and Kopito RR. Degradation of CFTR by the ubiquitin-  proteasome pathway. Cell 83: 121-127, 1995. 19.  Zadeh AD, Xu H, Loewen ME, Noble GP, Steele DF, and Fedida D.  Internalized Kv1.5 traffics via Rab-dependent pathways. J Physiol 586: 4793-4813, 2008. 20.  Zhou Z, Gong Q, Epstein ML, and January CT. HERG channel dysfunction in  human long QT syndrome. Intracellular transport and functional defects. J Biol Chem 273: 21061-21066, 1998.  105  Appendix  106  107  

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