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Regulation of the cell surface expression of voltage-gated potassium channels Choi, Woo Sung 2006

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REGULATION OF THE C E L L SURFACE EXPRESSION OF VOLTAGE-GATED POTASSIUM CHANNELS by WOO SUNG CHOI D . V . M . , Konkuk University, 1996 M . S c , Konkuk University, 2001 A THESIS SUBMITTED IN PARTIAL F U L L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Physiology) THE UNIVERSITY OF BRITISH C O L U M B I A August 2006 © Woo Sung Choi, 2006 ABSTRACT Functional expression of voltage-gated potassium (Kv) channels in the plasmalemma is essential to setting the repolarization following the action potential and to the regulation of excitation in cardiac myocytes, endocrine cells and neurons. One way in which expression of these channels is regulated is by their trafficking to/from the cell surface. This trafficking process is poorly understood. The purpose of the studies presented in this dissertation was to investigate the roles of one of the molecular motors, dynein, and of hormonal stimulations with incretins in the regulation of the cell surface expression of K v channels. The studies described here utilized a range of techniques from patch clamp recording, confocal fluorescence imaging, enzyme activity assay and radioimmunoassay along with biochemical and molecular biological approaches. The studies of the role of dynein indicate that disruption of dynein motor function either by over-expression of dynamitin/p50 or by nocodazole treatment increased Kv l .5 surface expression in both rat atrial myocytes and a heterologous cell model system. This was shown to be due to a reduction of the internalization rate of the channel. Endocytosis of Kvl .5 was shown to be dynamin-dependent, resulting in localization of the channel in early endosomes. A proline-rich domain in the Kvl .5 N-terminus was shown to be essential for this process. Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) also modulate expression of K v channels in the plasma membrane. The results presented in this dissertation demonstrate that dynamin-dependent endocytosis induced by GIP stimulation is involved in Kv l .4 internalization in human pancreatic p-cells and that PKA-dependent phosphorylation of T601 of the Kv l .4 C-terminus is essential for the endocytosis. Both GIP and GLP-1 inhibit delayed rectifier K + current through internalization of delayed rectifier i i K v channels, a process which is likely to be P K A phosphorylation-dependent. In addition, these regulations of K v channel expression are involved in insulin secretion of human and mouse pancreatic P-cells and insulin-secreting cells, (3-INS1 cells. These studies extend our understanding of the transport mechanisms by which K v channels are internalized, and of the controls on the level of K v channel surface expression. i i i TABLE OF CONTENTS Abstract ii Table of Contents iv Table of Figures viii Abbreviations x Acknowledgemets xii Co-Authorship Statement xiii CHAPTER 1 General Introduction 1 1.1 Overview 2 1.2 Voltage-gated K + (Kv) channels 3 1.2.1 Basic structure of Kv channels 4 1.2.2 Kv channels in cardiac myocytes 5 1.2.3 Roles of Kv channels in insulin secretion 7 1.3 Molecular motors 9 1.3.1 Basic structure 10 1.3.2 Stepping of the molecular motors 11 1.3.3 Cargo association and regulation of motor activity 12 1.4 Kv channels trafficking 14 1.4.1 Synthesis and transport into ER 14 1.4.2 From ER to Golgi 15 1.4.3 From Golgi to cell surface 17 1.4.4 Distribution at the Cell Surface 18 1.4.5 Internalization 19 1.4.6 Sorting of endocytosed proteins 20 1.4.7 Phosphorylation 21 1.5 Incretin hormones • 23 1.5.1 Physiological aspects of GIP and GLP-1 24 1.5.2 Receptor signalling of GIP and GLP-1 24 1.5.3 Effect of incretin on Kv channel 25 1.6 Scope of thesis investigation 25 1.7 References 30 iv CHAPTER 2. Kvl.5 Surface Expression Is Modulated by Retrograde Trafficking of Newly Endocytosed Channels by the Dynein Motor 43 2.1 Introduction 44 2.2 Materials and methods 46 2.2.1 Cell preparation and transfection 46 2.2.2 Plasmid constructs 46 2.2.3 Electrophysiological experiments and solutions 47 2.2.4 Myocyte isolation and electrophysiology 48 2.2.5 Imaging 48 2.2.6 Image analysis 50 2.2.7 Western blotting and densitometry: 51 2.2.8 Co-immunoprecipitation experiments ..52 2.2.9 Proteinase K experiments 52 2.3 Results .- 53 2.3.1 Disruption of dynein function increases Kv l .5 surface expression 53 2.3.2 Kv l .5 is present in early endosomes 54 2.3.3 Nocodazole treatment mimics dynein inhibition in HEK-293 Cells and cardiomyocytes 56 2.3.4 Kvl .5 coimmunoprecipitates with the dynein motor complex 58 2.3.5 A proline rich region in Kvl .5 is essential for the regulation of channel expression by dynein 59 2.4 Discussion 61 2.4.1 Dynein motor function affects Kvl .5 surface expression 61 2.4.2 Endocytosis and the role of a Kvl .5 consensus SH3 binding domain 62 2.4.3 Implications for forward trafficking 64 2.4.4 Relevance to the heart 65 2.5 Conclusions 66 2.6 Acknowledgements 67 2.7 References 80 CHAPTER 3. A Novel Mechanism for the Suppression of a Voltage-gated Potassium Channel by Glucose-dependent Insulinotropic Polypeptide: Protein Kinase A-dependent Endocytosis 83 3.1 Introduction T.....84 3.2 Experimental procedures 86 3.2.1 Generation of a GIPR-HEK-293 cell line 86 3.2.2 cDNA constructions of Kv l .4 plasmids and transient transfections in GIPR-HEK-293 cells 86 3.2.3 Enzyme activity assay of P K A 87 3.2.4 K v l . 4 protein purification and in vitro phosphorylation 87 3.2.5 Western blot analysis 88 3.2.6 Islet isolation and cell culture 88 3.2.7 Electrophysiological studies 88 3.2.8 Proteinase K digestion experiments 89 3.2.9 Confocal microscopy 89 3.2.10 Islet embedding in agar and confocal microscopy 90 3.2.11 Insulin secretion 90 3.2.12 Statistical analysis 91 3.3 Results -. 92 3.3.1 GIP decreases peak current amplitude of K v l . 4 in GIPR-HEK-293 cells ..92 3.3.2 GIP activates P K A in GIPR-HEK-293-Kv cells and GIP-stimulated P K A activation is involved in the decrease of K v l . 4 peak current amplitude 93 3.3.3 GIP-stimulated P K A activation resulted in the phosphorylation of K v l . 4 and decreases in Kv l .4 peak current amplitude 93 3.3.4 GIP-induced decreases in Kv l .4 peak current amplitude result from channel endocytosis ...95 3.3.5 Phosphorylation is involved in GIP-induced retrograde trafficking of Kvl .4 96 3.3.6 Kv channel expression in human islets 97 3.3.7 Phosphorylation-dependent internalization of K v l . 4 participates in the effect of GIP on insulin secretion 98 3.4 Discussion 100 3.5 Acknowledgements 105 3.6 References 115 CHAPTER 4. Discrete Effects of Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) on p-cell Delayed Rectifier K + Channels 119 4.1 Introduction 120 4.2 Research design and methods 123 4.2.1 Islet isolation and cell culture 123 4.2.2 Western blot analysis 123 4.2.3 Immunocytochemistry 123 4.2.4 Electrophysiological recordings 124 4.2.5 Measurement of GSIS 124 4.2.6 Proteinase K digestion experiments 124 VI 4.2.7 Generation of GIPR-HEK-293 and GLP-1 R-HEK-293 cell lines and transient transfections of K v plasmids... 125 4.2.8 Data analysis 125 4.3 Results 127 4.3.1 Expression of delayed rectifier K v channels in rodent and human islets.. 127 4.3.2 Multiple delayed rectifier K v channels contribute to the generation of IDR in human islets 127 4.3.3 GIP and GLP-1 inhibit human islet cell I D R 128 4.3.4 Suppression of K v channels resulted in the decreased insulino tropic effects of incretins in human islets 130 4.3.5 GIP and GLP-1 inhibit mouse islet cell IDR and K v channels participate in the insulinotropic activity of GIP and GIP-1 130 4.3.6 GIP and GLP-1 regulate Kv2.1, Kv l .5 and Kv3.2 macroscopic current in HEK-293 cells expressing incretin hormone receptors 132 4.3.7 Suppression of P K A resulted in the blocking of incretins effects on delayed rectifier K v channels 133 4.3.8 GIP and GLP-1 reduce K v channel surface expression 133 4.4 Discussion 135 4.5 Acknowledgements..... 140 4.6 References 152 CHAPTER 5. General Discussion and Future Directions 156 5.1 Endocytosis mechanism of Kv l .4 and Kvl .5 158 5.2 Recycle or degradation? 159 5.3 Complexity of vesicle transport: Motor coordination 160 5.4 Effect of dynamitin over-expression in Kvl .4 , Kv4.2 and hERG channels 162 5.5 How to overcome a dilemma of motor coordination? 163 5.6 Summary 164 5.7 References 173 APPENDIX I Animal care certificates 175 APPENDIX II Tissue for research information form 177 vii LIST OF FIGURES Figure 1.1 Typical action potentials recorded from cells in the ventricle, SA node, and atrium 28 Figure 1.2 Glucose-stimulated insulin secretion (GSIS) and action potentials recorded from pancreatic P-cell in mouse and rat 29 Figure 2.1 p50/dynamitin coexpression significantly increases Kv l .5 current levels and surface expression 68 Figure 2.2 Kvl .5 is internalized via an endocytotic pathway 70 Figure 2.3 Nocodazole pretreatment increases K v l . 5 , but not transient currents in HEK-293 cells and in rat atrial myocytes 71 Figure 2.4 Nocodazole pretreatment increases Kvl .5 surface expression in rat atrial myocytes 73 Figure 2.5 Nocodazole-induced increase in atrial myocyte current is caused by I K U R 74 Figure 2.6 Kvl .5 coimmunoprecipitates with the dynein intermediate chain in HEK-293 cells and myocytes .....75 Figure 2.7 An N-terminal segment of Kvl .5 is required for sensitivity to p50 over-expression 76 Figure 2.8 The N-terminal most Kvl .5 SH3-binding domain regulates Kvl .5 surface expression 77 Figure 2.9 Line scan analyses of Kvl .5-EEA1 colocalization 78 Figure 2.10 Co-immunoprecipitation of Kvl .5 with p50 79 Figure 3.1 GIP decreases peak ionic current amplitude of K v l . 4 in GIPR-HEK-293 cells 106 Figure 3.2 GIP activates P K A in GIPR-HEK-293-Kv cells and P K A activation is involved in the decrease in K v l . 4 ionic currents 108 Figure 3.3 GIP-stimulated P K A activation resulted in the phosphorylation of Kv l .4 and decreases in ionic current 109 Figure 3.4 GIP treatment resulted in the endocytosis of K v l . 4 protein and decrease in ionic current I l l Figure 3.5 Phosphorylation is involved in GIP-induced retrograde trafficking of K v l . 4 . . 112 viii Figure 3.6 K v channel expression in human islets 113 Figure 3.7.Phosphorylation-dependent internalization participates in the effect of GIP on insulin secretion 114 Figure 4.1 Expressions of delayed rectifier K v channel in rat and human islets 141 Figure 4.2 Functional expression of Kv2.1, Kv l .5 and Kv3.2 in human islets 142 Figure 4.3 Effects of incretins on I D R of human islet cells 143 Figure 4.4 Effects of T E A on GSIS of human islet cells 144 Figure 4.5 Effects of incretins on I D R of mouse islet cells 145 Figure 4.6 Kv channels participate in the insulinotropic activity of GIP and GIP-1 146 Figure 4.7 Effect of P K A inhibitor on incretin-mediated inhibition of human and mice islet IDR 148 Figure 4.8 GIP and GLP-1 reduce Kv Channel Surface Expression 149 Figure 4.9 Effects of GIP on Kv2.1, Kv l .5 and Kv3.2 in HEK-293 cells 150 Figure 4.10 Effects of GLP-1 on Kv2.1, Kv l .5 andKv3.2 in HEK-293 cells 151 Figure 5.1 Endocytosis of Kv l .4 protein resulted from PKA-dependent phosphorylation 165 Figure 5.2 p50 over-expression significantly decreases Kv l .4 , Kv4.2, and hERG current levels 166 Figure 5.3 Over-expression of p50 does not alter the macroscopic kinetics of Kv l .4 168 Figure 5.4 Nocodazole pretreatment decreases Kv l .4 currents in HEK-293 cells 169 Figure 5.5 The N-terminus of Kv l .4 is essential for the response to p50 over-expression 170 Figure 5.6 K v l . 4 N-terminus endows Kvl .5 with the opposite result of p50 over-expression 171 Figure 5.7 Dynamitin (p50) over-expression affects bidirectional trafficking 172 ix ABBREVIATIONS Amino Acid Alanine Arginine Asparagine Aspartate Cysteine Glutamine Glutamate Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Serine Threonine Tryptophan Tyrosine Valine 3 Letter Code A l a A r g A s n Asp Cys G i n G l u G l y His He Leu Lys Met Phe Ser Thr Try Tyr V a l 1 Letter Code A R N D C Q E G H I L K M P S T W Y V 4-AP, 4-aminopyridine AP, Action potential CaMK, Calcium/calmodulin-dependent protein kinase cAMP, cyclic A M P CHO, Chinese hamster ovary C M V , Cytomegalovirus DIC, Dynein intermediate chain domain D M M , 2-(3,4-Dimethylphenyl)-3-(4-methoxyphenethyl) metathiazan-4-one DMSO, Dimethyl sulfoxide EEA1, Early endosome associated protein 1 ER, Endoplasmic reticulum GFP, Green fluorescent protein GIP, Glucose-dependent insulinotropic polypeptide GIPR, GIP receptor GLP-1, Glucagon-like peptide-1 GLP-1 R, GLP-1 receptor GPCR, G protein-coupled receptor GSIS, Glucose-stimulated insulin secretion HEK-293, Human embryonic kidney-293 hERG, Human ether-a-go-go channel IA, A-type currents (=It0) IC50, 50 % inhibitory concentration IDR, Delayed rectifier potassium current IKUR, Ultra rapid potassium current I t 0, Transient outward K + currents x KATP, ATP-sensitive potassium channel Kca, Calcium-activated potassium channel KChIP, Potassium channel interacting protein K R B H , Krebs-Ringer buffer with HEPES Kv, Voltage-gated potassium M A P K , Mitogen-activated protein kinase mry-DIP, Myristoylated dynamin inhibitory peptide N M D A , N-methyl-D-aspartate p50, Dynamitin PDE, Phosphodiesterase PDZ, PSD-95/DLG/ZO-1 PI3K, Phosphoinositide 3,4,5'-triphosphate P K A , Protein kinase A PKB, Protein kinase B PKC, Protein kinase C PLC, Phospholipase C P M A , Phorbol 12-myristate 13-acetate RIA, Radioimmunoassay SAP97, Synapse associated protein-97 SH3, Src homology 3 domain T l , Tetramerization TdP, Torsade de Pointes T E A + , Tetraethylammonium ion T M , Transmembrnae V1/2, Half activation voltage VDCCs, Voltage-dependent C a 2 + channels VDF, Vancouver diabetic fatty WT, Wild-type ACKNOWLEDGEMENTS I would like to express my gratitude to all those who gave me the possibility to complete this thesis. I specially thank to my graduate supervisory committee (Dr. Edwin Moore, Dr. Steven Kehl, Dr. Michael Walker and Dr. David Fedida) and Dr. Christopher Mcintosh for their advice in the whole period of my program. I am deeply indebted to Dr. Su Jin Kim whose help, stimulating suggestions and encouragement helped me in all the time of research for and writing of this thesis. I owe particular thanks to Dr. David Steele and Dr. Zhuren Wang whose help, co-working and answering to my gigantic number of questions. I also want to thank my colleagues in the Fedida Lab for all their help and assistance. Special thanks are owed to my family, whose have supported me throughout my program. xii CO-AUTHORSHIP STATEMENT CHAPTER 2: Kvl.5 Surface Expression Is Modulated by Retrograde Trafficking of Newly Endocytosed Channels by the Dynein Motor Woo Sung Choi*, Anu Khurana, Rajesh Mathur, Vijay Viswanathan, David F. Steele and David Fedida * Contributions by Woo Sung Choi: Responsible for project management, experimental design, patch clamp recording, primary cell isolation and preparation for electrophysiological experiments and imaging experiments, some of. the cloning, mutagenesis, transfections, data analysis, some of the figure preparation, writing and editing the manuscript (Percent Contribution: 60 %). CHAPTER 3. A Novel Mechanism for the Suppression of a Voltage-gated Potassium Channel by Glucose-dependent Insulinotropic Polypeptide: Protein kinase A-dependent endocytosis Su-Jin K i m # , Woo Sung Choi #*, John Song Mou Han, Garth Warnock, David Fedida, and Christopher H. S. Mcintosh * Contributions by Woo Sung Choi: Responsible for project management, experimental design, patch clamp recording, transfections, some of the cloning, mutagenesis, radio immuno assay, data analysis, some of the figure preparation, writing and editing the manuscript (Percent Contribution: 45 %). * Both authors contributed equally to this work. CHAPTER 4. Discrete effects of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) on P-cell delayed rectifier K + channels Woo Sung Choi #*, Su-Jin K i m # , Garth Warnock, Christopher H.S. Mcintosh and David Fedida * Contributions by Woo Sung Choi: Responsible for project management, experimental design, patch clamp recording, transfections, radioimmunoassay, data analysis, some of the figure preparation, writing arid editing the manuscript (Percent Contribution: 45 %). * Both authors contributed equally to this work. xiii CHAPTER 1. GENERAL INTRODUCTION 1.1 OVERVIEW Transport of ions across cellular membranes requires the participation of specific transport proteins called ion pumps, ion exchangers, or ion channels. Ion channels are permeable pores that provide pathways for diffusion across the bilayer. Functional surface expression of voltage-gated potassium (Kv) channels in excitable cells such as cardiac myocytes and endocrine cells is essential to the maintenance and control of cell excitability. Kv channels expressed in the plasma membrane function to generate macroscopic potassium (K + ) currents necessary for the repolarization phase of the action potential. Macroscopic currents are regulated by two processes: 1. biophysical and biochemical modulation, for example phosphorylation, of gating and/or ion permeation that affect ion channel activity in the plasma membrane, and 2. biosynthesis and trafficking of channel proteins (Delisle et al, 2004). Kv channel expression begins with gene transcription and biosynthesis of the protein (Deutsch, 2003); further levels of control include trafficking to the cell surface, and regulated retrieval and degradation. The plasma membrane is a dynamic structure with transport vesicles continuously fusing with and being excised from it. Plasma membrane bound proteins, for example, Kv channel proteins may be inserted via forward trafficking similar to exocytosis, and retrieved by endocytosis and retrograde trafficking processes to be recycled or degraded. The trafficking of K + channels now is recognized as a particularly important mechanism in the regulation of channel expression and function (Ficker et al 2003; Kuryshev et al, 2005). While several groups have investigated motifs that affect trafficking in Kv channels, little is known about the actual mechanisms of channel trafficking. To gain insights into these mechanisms, the principle focus of the studies described herein has been on the 2 molecular mechanisms behind the trafficking of Kv channels with a particular emphasis on the endocytic process and retrograde trafficking. 1.2 VOLTAGE-GATED K+ (Kv) CHANNELS K v channels are integral membrane proteins that conduct K + ions across the plasmalemma in response to changes in the potential across that membrane. The cc-subunits of K v channels contain 6-transmembrane segments, and function as a tetramer in the cell membrane. There are at least two types of outward K + currents generated by K v l . X -K v l l . X (Coetzee et al, 1999; Ottschytsch et al, 2002): transient outward K + currents (A-type current) with fast-inactivation, and delayed rectifier outward K + currents (steady-state current) which exhibit slow-inactivation after depolarization (Fedida et al, 1998; Hille, 2001). Fast inactivation of Kv channels is frequently described as a ball-and-chain mechanism, while a conformational change of the external mouth of the pore is thought to be involved in the slow-inactivation mechanism (Yellen, 1998). K v channels are widely distributed and play essential roles in many physiological processes such as the rhythmic beating of the heart, impulse conduction in neurons, and the secretion of hormones. K + channel mutations are known to cause diseases of the brain (epilepsy, episodic ataxia), ear (deafness), heart (arrhythmias), muscle (myokymia, periodic paralysis), kidney (hypertension), pancreas (hyperinsulinemic hypoglycemia, neonatal diabetes), and developmental abnormalities of neural crest-derived tissues (Andersen's syndrome). Here I focus on the distribution and the physiological functions of Kv channels in cardiac myocytes and pancreatic P-cells. 3 1.2.1 Basic structure of Kv channels A l l Kv channels share a similar architecture: they consist of four identical oc-subunits, each of which contributes to the formation of a gated central pore. Each subunit includes six transmembrane domains, SI to S6, including the voltage sensor (S4), a P-loop 'pore domain' and intracellular N - and C-terminal segments (Li et al, 1992; Sato et al, 1990; Yellen, 1998). The crystal structures of the related channels KcsA from Streptomyces lividans, (Doyle et al 1998) and KvAP from Aeropyrum pernix (Jiang et al, 2003a) are known, as is the structure of the membrane spanning domains of K v l . 2 (Long et al, 2005). Like Kv channels, KvAP is a tetramer of 6-transmembrane-domain subunits; KcsA is also a tetramer but each subunit has only two transmembrane segments and these correspond to the S5-P-S6 region of the K v channels (Cuello et al 1998). The crystal structure of KvAP shows that transmembrane segments SI, S2 and part of S3 surround the pore region, S5 to S6, and what appears to be a 'voltage sensor paddle' consisting of S4 and the rest part of S3 (S3b) (Jiangs al, 2003b). The N - and C-terminal regions of Kv channel proteins are located in the cytoplasm where they serve several regulatory roles, including affecting subunit assembly and influencing gating (Hille, 2001; Schulteis et al, 1998). In Kvl .4 , like many Shaker-type channels, end of the N-terminal domain is responsible for the process known as 'N-type inactivation' (Hoshi et al, 1990). N-type inactivation is a fast-inactivation mediated by occlusion of the pore by the ball-like structure at the extreme end of the N-terminal domain. The T l domain resident in all Kv channel N-termini promotes tetramerization of the Kv a-subunits (Kva) and provides a platform for the binding of the Kv (3-subunits (KvP) (Lu et al, 2001; Gulbis et al, 2000). K v channel C-termini are involved in a variety of processes 4 ranging from channel gating and voltage sensitivity to the binding of the membrane-associated guanylate kinases (Jerng and Covarrubias, 1997). 1.2.2 Kv channels in cardiac myocytes Heart muscle contraction is driven by electrical activity in cardiac myocytes. The electrical excitability of these cells is controlled by coordination of diverse ion channels conducting Na + , Ca 2 + , CI", and K + . K+-selective channels carry outward K + currents within specific physiological membrane potential ranges. K + currents are involved in the repolarization process and in maintenance of the resting membrane potential near the K + ion reversal potential. Particularly, transient outward currents (I t0) and ultra-rapid delayed rectifier currents (Ijcur) are mediated by Kv4.2/Kv4.3 in mouse, rat and human and/or Kv l .4 in rabbit and Kvl .5 in mouse, rat, dog and human, respectively (Snyders 1999; Fedida 1998). Figure 1.1 shows typical action potential waves form in ventricular and atrial myocytes, and pacemaker cells in heart. I t 0 influences the early repolarization phase and generates the notch of the cardiac action potential. In adult rats, I t 0 is thought to be mediated mostly by Kv4.2 and Kv4.3 because the recovery time course from inactivation of Kv l .4 is slower than that observed for I t 0 (Nerbonne 1998). In contrast, Kv l .4 appears to underlie expression of I t 0 in neonatal rodents (Matsubara et al, 1993; Petersen and Nerbonne, 1999). Studies using dominant-negative Kv4.2 subunits have confirmed a role for this channel as the basis of cardiac I t 0 (Johns et al, 1997; Bahring et al, 2001). However, the rate of recovery from inactivation of the native cardiac I t 0 is still faster than that of Kv4.2 or Kv4.3 (Franqueza et al, 1999). These may support the contention that Kv4.2 and Kv4.3 are co-expressed with the 5 potassium channel interacting proteins (KChlPs) in the heart. Indeed, co-expression of KChlPs with Kv4.2 and Kv4.3 accelerates the rates of both inactivation and recovery from inactivation, as well as increasing outward K + current density (Sanguinetti, 2000; L i and Adelman, 2000). The plateau phase of the cardiac action potential is the outcome of a precise equilibrium between inward and outward currents. The shift of net cardiac current to the outward direction causes the repolarization phase in cardiac myocytes (Bennett et al, 1993). Torsade de Pointes (TdP), literally meaning "twisting of points" in an electrocardiogram, is characterized by a gradual change in the amplitude and twisting of the QRS complexes around the isoelectric line. TdP is strongly associated with a prolonged QT interval, which corresponds with depolarization and repolarization processes of ventricular myocytes. Therefore, both ventricular and atiral arrhythmias are thought to arise from a disorder of repolarization or an abnormally prolonged cardiac action potential by a malfunction of outward K + currents from delayed rectifier K + channels: IK S , IIO, and IicUr, which are mediated by K v - L Q T l - M i n K complex, hERG-MiRP complex, and K v l . 5 , respectively (Kass and Moss, 2003; Moss and Kass, 2005; Brendel and Peukert, 2003). Recently, delayed rectifier K + channels have been a target for antiarrhythmic drugs designed to prevent certain types of cardiac arrhythmias (Brendel and Peukert, 2003; Zolotoy et al, 2003; Lee et al, 2003; Gerlach 2003). Kv l .5 is particularly highly expressed in human, rat and dog atrial myocytes and is an important component of delayed rectifier K + currents in the repolarization phase (Fedida et al, 1993; Eldstrom et al, 2003; Fedida et al. 2003). Contribution of Kvl .5 to Ijtur has been confirmed by blocker analysis with 4-aminopyridine (4-AP) and a more specific blocker C9356, prolonging action potential duration in human 6 and canine atrial myocytes (Wang et al, 1993; Fedida et al, 2003). Therefore, the regulation of Kvl .5 is an important target in the treatment of atrial fibrillation (Van Wagoner et al, 2000). 1.2.3 Roles of Kv channels in insulin secretion. Glucose-stimulated insulin secretion (GSIS) from pancreatic p-cells is the outcome of a delicate balance between metabolic and electrical events. GSIS as a rapid response to an increase of the extracellular glucose level is generally attributed to an ATP-sensitive K + (KATP) channel-dependent pathway (Figure 1.2; Bell and Polonsky, 2001; Nichols and Koster, 2002; Minami et al, 2004). Glucose entering through the glucose transporter 2 (GLUT2) is converted into glucose 6-phosphate (G6P) by glucokinase. Metabolism of G6P in mitochondria results in an increased cytoplasmic ATP/ADP ratio. The increased ATP causes K A T P channels (IK(ATP)) to close, leading to membrane depolarization, thereby activating voltage-gated C a 2 + channels leading to increased intracellular C a 2 + concentration ([Ca2+]i) and subsequently resulting in insulin secretion. Membrane depolarization also leads to activation of K v channels. K v channel activity in pancreatic P-cells has been reported as a regulator of C a 2 + oscillations in response to glucose stimulation via modulation of the action potential. This suggests the association between Kv channel activation and insulin secretion (Edwards and Westion, 1995; Philipson, 1999). Depolarization of pancreatic P-cell appears with membrane potential oscillations in response to elevated glucose (Figure 1.2B and C; Manning Fox et al, 2006). Although N a + channels and T-type Ca channels are present in p-cells, most of them are inactivated because of the resting membrane potential, approximately -60 mV. Therefore, L-type C a 2 + 7 channels primarily contribute for inward current during the action potential. As mentioned above, Kv channels are important in the repolarization phase of the action potential (Hille, 2001). Outward flow of K + ions through activated Kv channels repolarizes the cell and results in the termination of insulin secretion in pancreatic P-cells. Therefore, i f Kv channel currents are inhibited, action potential duration is prolonged, causing an increase in the magnitude and duration of insulin secretion. Whole cell recordings from pancreatic p-cells revealed that outward K + currents are sensitive to external tetraethylammonium (TEA) and the Kd values of TEA are in the 5-7 m M range in mouse, rat and human cells (Ashcroft and Rorsman, 1989; Bokvist et al, 1990; Smith et al, 1990; Herrington et al, 2005). Indeed, TEA blockade of K + currents leads to prolongation of the action potential duration, resulting in significant increase of intracellular Ca signalling and GSIS in pancreatic P-cells (MacDonald and Wheeler, 2003; Tamarina et al, 2005; Kuznetsov et al, 2005; Herrington et al, 2006). K currents in mouse pancreatic p-cells are well studied. Delayed rectifier and Ca -activated K + channels have been detected via patch-clamp techniques and single-channel conductance values of the Ca -independent K current from Kv channels are approximately 10 pS (Rorsman and Trube, 1986; Smith et al, 1990; Edwards and Westion, 1995). In pancreatic P-cells, the resting membrane potential is approximately -70 to -60 mV when extracellular glucose is in the 0-2 mM range (Ashcroft and Aschroft, 1990; Cook et al, 1991; Dufer et al, 2004); under these conditions most K v channels are closed. More recently, the biophysical properties of Kv currents from rat, human pancreatic islet cells and insulin-secreting cell lines have been characterized, as have the molecular components of these Kv currents (Su et al, 2001; MacDonald and Wheeler, 2003; MacDonald et al, 8 2003; Herrington et al, 2005). Multiple subtypes of K v channels have been detected in pancreatic islet and/or P-cells and insulin secreting cell lines in electrophysiological and biochemical investigation. Numerous studies have shown both delayed rectifier and A-type K + currents (Edwards and Westion, 1995; Gopel et al, 2000; MacDonald et al, 2001; Herrington etal, 2005). Kvl .4 , Kvl .6 , Kv2.1, and Kv4.2 protein expression and/or mRNA transcripts have been detected in rat pancreatic islets and MIN6 cells, an insulinoma cell line, by Western blot and by real-time PCR (MacDonald et al, 2001; MacDonald et al, 2002). The functions of Kv2.1 and Kvl .4 were confirmed by over-expression of dominant negative for each subunit, resulting in approximately 60 % and 25 % inhibition of delayed rectifier and A-type K + current, respectively (MacDonald et al, 2001; MacDonald and Wheeler, 2003). The expressions of Kv2.1, Kv3.2, Kv6.2 and Kv9.3 in human and Rhesus monkey pancreatic P-cells were also confirmed with real-time PCR and confocal imaging (Yan et al, 2004). In addition, INS-1 cells, an insulin-secreting cell line, contain Kvl .4 , K v l . 5 , Kv2.1, Kv2.2, Kv3.1 and Kv3.2 mRNAs, and insulin secretion induced by 100 uM tolbutamide was increased by 4-aminopyridine (4-AP) and T E A by 65 % and 41 %, respectively (Su et al., 2001). 1.3 MOLECULAR MOTORS Three classes of cytoplasmic motors are known: myosins, kinesins and dyneins. Myosins track along actin filaments, whereas kinesins and dyneins move along microtubules. A l l three motors have ATPase activity and convert chemical energy, ATP, to physical movement. There are at least 18 myosin families, 10 different classes of kinesins and 2 groups of dyneins (Schliwa and Woehlke, 2003). 9 1.3.1 Basic structure Molecular motors are usually described as consisting of three different parts: motor domain, neck linker, and tail domain. The motor domain contains the binding sites for an ATP molecule and for the molecules (actin or tubulin) on which the motor tracks. Although the sequence homology of the ATP binding pockets in kinesin and myosin motors is restricted to only several key residues, the high-resolution crystal structures of these pockets are almost identical (Vale, 1996). The two motors, however, have clearly distinguishable neck linker structures and modes of function. The neck linker amplifies the conformational change of the motor domain from ATP hydrolysis to the power stroke. This domain of the kinesin motor is relatively short and flexible, and results in the typical stepping "hand-over-hand" motion of kinesins (Vale, 2003; Yildiz et al, 2004). On the other hand, myosins have a long and rigid structure, so that the conversion region acts as a lever arm that swings through an angle of up to 70° (Geeves and Holmes, 1999; Houdusse et al, 2000). Conversely, the design of dynein motors is fundamentally different from kinesins and myosins in several ways. First of all, the dynein motor domain is the largest among the three cytoplasmic motors. Secondly, it is composed of six AAA-ATPase modules, comprising a ring structure. An A A A protein contains an ATPase domain of -220 amino acids, playing important roles in many physiological events (Vale, 2000). A mechanism for force generation of dynein has been suggested as the rotation of the A A A -ATPase ring resulted from coordinated multiple conformational changes. Small conformational changes between the first A A A domain and its neighbours are amplified by docking the head ring onto a linker that connects the tail and the head ring. The resulting 10 rotation of the head ring, in a counter-clockwise direction, swings the stalk (Koonce and Tikhonenko, 2000; Burgess etal, 2003). 1.3.2 Stepping of the molecular motors There are two fundamentally different behaviours of motors in their stepping: processive and non-processive stepping (Higuchi and Endow, 2002). The best example of a non-processive motor is muscle myosin II which drives muscle contraction. In this case, the myosin II motors work as a team without changing their location. Instead, they make the actin filament move. Although myosin II has a dimeric structure, the two heads do not cooperate, and the dwell time on the actin track takes only approximately 5 % of the time of an ATP hydrolysis (Mermall et al, 1998). On the other hand, conventional kinesin I provides a good example of processive movement in the so-called "hand-over-hand" model, whereby in one ATP-hydrolysis cycle the free head makes a step to move towards a new binding site passing by the bound head. The time of interaction between motor heads and microtubule occupies 50 % of an ATP-hydrolysis cycle in this case (Endow and Barker, 2003; Yildiz et al, 2004). Some myosins, such as myosin V and myosin VI , also show processive stepping (Tyska and Mooseker, 2003). Dynein exhibits processive movement operated by a hand-over-hand mechanism and occasional backward steps (Gross et al, 2002; Toba et al, 2006). Interestingly, recent studies suggest that rather than a single motor multiple dynein motors transport vesicles (McGrath, 2005). The stepping sizes of molecular motors resulting from the hydrolysis of one ATP molecule have been reported to be 36 nm for myosin V , 8 ran for kinesin I and 16 nm for cytoplasmic dynein (Burgess et al, 2003; De La Cruz et al, 1999; Toba et al, 2006). The 11 rate of movement of dynein, 0.8 um per second, is much faster than that of kinesin which is 0.5 um/sec (Toba et al, 2006; Helfand et al, 2004). Dynein and kinesin microtubule-dependent motors travel in opposite directions on the microtubule: dyneins move toward the minus end (toward the centrosome, i.e. the retrograde direction), whereas, kinesins move toward plus end of microtubule (away from the centrosome, i.e., anterograde) (Howard and Hyman, 2003). 1.3.3 Cargo association and regulation of motor activity Cytoplasmic molecular motors are essential to cellular organelle and vesicle transport. The motors associate with their cargoes via linkage to the phospholipid bilayer or integral membrane proteins. For example, KIF1 family kinesins have been shown to interact with phosphatidylinositol 4,5-biphosphate (PIP2)-containing lipid rafts through a pleckstrin homology (PH) domain present in the KIF1 tail domain (Klopfenstein et al, 2002). In neurons, kinesin I light chains bind amyloid precursor protein (APP); malfunction of the motor is thought to contribute to the development of some neuro-degenerative diseases (Kamal et al 2001). Cytoplasmic dynein in photoreceptor cells directly interacts with rhodopsin, an integral membrane protein, via its Tctex-1 light chain (Tai et al, 1999). Inhibition of this linkage leads to retinitis pigmentosa (Terada and Hirokawa, 2000). The most common mode of association between motors and integral membrane proteins is through a linker protein or a linker protein complex. For example, the low-density lipoprotein receptor family interacts with conventional kinesin via an interaction of the kinesin light chains and Jun kinase-interacting proteins (JIPs) (Verhey et al, 2001). Some cytoskeletal motors have been reported to be involved in retrograde transport with 12 Rab proteins, small GTPases expressed in intracellular organelle membrane. For example, Rabkinesin-6, a kinesin-like protein (KLP), from mouse interacts with Rab6 expressed in the Golgi complex, regulating both inter-Golgi transport and retrograde transport from the Golgi to ER. Rabkinesin-6 should be a plus-directed microtubule motor, so that interaction of this motor with Rab6 may inhibit retrograde trafficking (Martinez et al, 1997; Echard et al, 1998).. In melanocytes, Rab27a binds to myosin V via a Rab-binding partner, melanophilin (Wu et al, 2002). Cytoplasmic dynein recruits an accessory protein complex, called dynactin, to associate with its vesicular cargo (Schroer, 1994). Although precisely how the dynein-dynactin complex associates with these cargoes is not understood, the protein p l50 g l u e d , which binds to dynein intermediate chains, dynamitin (p50), important for the interaction of dynein with dynactin, and Arpl a short filament of the actin-related protein, that interacts with vesicle-resident spectrin, are all implicated in this association (Muresan et al, 2001). The dynein-dynactin complex is also implicated in the movement of material within the endocytic pathway. Disruption of the dynactin complex by dynamitin (p50) over-expression was found to perturb endosomal trafficking (Valetti et al, 1999). Dynamitin over-expression is an effective method for disrupting dynein function because it prevents association of the motor with its cargoes (Burkhardt et al, 1997). Dynactin is dissociated from the motor and dynein can thus carry no cargo at all. Dynactin is known to allow cytoplasmic dynein to drive long-range movements of vesicle cargo on microtubules, and is required also for mitosis (Schroer and Sheetz, 1991; Schroer, 2004). Dynactin has also been shown recently to contribute to kinesin II activity (Deacon et al. 2003). Although its role in 13 kinesin II-based movement has not yet been fully established, dynactin likely plays a similar role for this kinesin as it does for dynein. 1.4 Kv CHANNELS TRAFFICKING Most K v channels function in the plasma membrane. Newly synthesized K v channel proteins must be transported from ER through the Golgi to the cell surface. In addition, the expression of K v channels may be controlled through internalization of plasma membrane resident channels followed by degradation or reuse. Understanding of the mechanisms of Kv channel trafficking is essential to the understanding of channel expression under physiological as well as disease conditions (Curran et al, 1995; Neyroud et al, 1997; Bierverte^fl/., 1998). 1.4.1 Synthesis and transport into ER The generation of a channel begins when newly synthesized mRNA associates with cytosolic ribosomes and tRNA and undergoes translation. As with most eukaryotic membrane proteins, this newly synthesized Kv channel protein is also co-translationally inserted into the endoplasmic reticulum (ER). The channel protein is folded and assembled, perhaps during synthesis (Deutsch, 2003) followed by the transport from ER to Golgi and from Golgi to plasma membrane to be expressed functionally. For K v l . 3 , at least, the soluble N-terminal domain of the nascent peptide is known to acquire its tertiary structure as it is completed and exits the ribosome (Kosolapov and Deutsch, 2003). The entire nascent peptide/ribosome complex is then targeted to the ER membrane where synthesis continues. Typically, targeting of ER membrane proteins involves a signal sequence in the 14 N-terminus that binds a signal recognition particle (SRP), which subsequently binds to a receptor on the ER membrane. The acetylcholine receptor channel, for example, has such a cleavable signal sequence. K v channels, however, lack this signal and use the second transmembrane domain for targeting instead (Tu et al, 2000). Kv channels contain sites on their amino- (N-) and carboxyl- (C-) termini, pore domain, and intracellular loop that are required for the response to cellular signalling and which affect sub-cellular translocalization. K v channel N - and C-termini are in the cytosol in both the ER and the plasma membrane (Deutsch, 2003; Trimmer, 2004). For exit from the ER, proper folding is required; otherwise, the protein is retained in the ER by chaperone proteins such as calnexin and calreticulin (Manganas and Trimmer, 2004). K v channel N -termini have highly conserved ' T l domains,' (Li et al, 1992) which are important for a-subunit assembly and contain the binding domain for KvP-subunits (Xu et al, 1995). If the T l domain is deleted, surface expression of the channel is significantly reduced because of substantially reduced tetramer formation (Deutsch, 2002). 1.4.2 From ER to Golgi Several mechanisms are involved in ER exit and subsequent trafficking. These include retention/retrieval signals, anterograde signals, phosphorylation, and association of the channel with scaffolding/anchoring proteins. Subunit composition and stoichiometry are also important in determining surface expression rates. In some K + channels, the ER retention signal has been characterized. Kir6.2 is one of the best-studied examples. In this channel, exposure of a C-terminal R X R motif to the cytosol prevents surface expression (Aguilar-Bryan and Bryan, 1999). Although an R X R ER retention signal has not been 15 specifically identified in other Kv channels, there are RXRs in their N - and C- termini as well as in intracellular loops that may be important for ER retention (Birnbaum et al, 2003). In K v channels, leucine zipper and dileucine motifs that are highly conserved in Kv4 family of diverse species, including human, have been shown to play a role in the subcellular localization of Kv4.2, Kv l .3 and Kv l .4 (Rivera et al, 2003). The electrically silent Kv channel group, for example, Kv6.3 shows a high ER retention rate since its C-terminus inhibits the formation of a functional homotetramer. When the ER retention signal of this channel is swapped with the C-terminus of Kv2.1, the electrical silence is overcome (Ottschytsch et al, 2005). In Kvl .4 , the V X X S L motif in the C-terminus promotes efficient expression of that channel; while Kvl .5 contains a similar but less effective motif, L X X S L (Li et al, 2000). The outer pore region of Kvl .4 has also been found to affect trans-Golgi glycosylation and cell surface expression (Zhu et al, 2001). In patients with persistent hyperinsulinemic hypoglycemia of infancy (HI), a genetic disorder, the K A T P channel contains a mutation on its auxiliary subunit, sulfonyl urea receptor 1 (SUR1), which lacks phenylalanine at 1388, reducing dramatically cell surface expression of the channel (Huopio et al, 2002). Folded proteins are concentrated in transitional ER-budding zones (with no attached ribosomes) where the proteins are packed for transport to c/'s-Golgi. GTP-bound Sari, a GTPase (by sec 12, a Guanyl nucleotide exchange factor), inserts its fatty-acid tail into the ER membrane, followed by recruitment of the sec23/24 dimer and the sec 13/31 heterotetramer. This complex is the basis of the COP II coatomer complex including targeted proteins, leading to pinching off transport vesicle from ER membranes. Multi-16 tubular clusters resulting from fusion of COP II-coated vesicles are maturated and fused with Golgi membrane (review in Lee et al., 2004). 1.4.3 From Golgi to cell surface For transport from Golgi to the cell surface, clathrin-dependent vesicle transport has been suggested as a mechanism. In this mechanism, dynamin-2 recruits cytoplasmic clathrin with the synthesized proteins, resulting in the formation of partially clathrin-coated vesicles. Kinesin motors seem likely to play a role in anterograde trafficking of transport vesicles in trans-Golgi membranes. For example, KIF13A binds to A P I , an adaptor protein, and this binding is implicated in the transport for lysosomal peptidases to lysosomes and mannose-6-phosphate receptor to plasma membrane (Nakagawa et al, 2000). Recently, KIF17 has been shown to interact with Kv4.2 in neurons, suggesting KIF17 may be the motor of the channel for forward trafficking (Chu et al., 2006). Channel expression in the plasma membrane may also be regulated by interactions with auxiliary proteins such as Kvp subunits, scaffolding proteins or KChlPs. Both K v p i . l and Kvp2 promote forward trafficking for cell surface expression of Kv l .2 a-subunit (Campomanes et al, 2002). Co-expression of a PDZ domain-containing protein, SAP97, with Kv l .4 increases ER retention of the channel and significantly reduces its cell surface expression (Kim et al, 1995; Tiffany et al, 2000),whereas, over-expression of SAP97 with Kvl .5 amplifies the channel current density (Eldstrom et al, 2003; Mathur et al, 2006). Scaffolding proteins include PDZ domain-containing proteins and Homo protein (enable vasp homology domain) form a complex with membrane integral proteins and cytoskeleton, affecting the protein expression in the plasma membrane. Plasma membrane expression of 17 Kv4.2 is also enhanced by co-expression of PSD95, a PDZ domain containing protein (Wong et ah, 2002). In addition, KChIP interaction with Kv4 a-subunits dramatically improves surface expression of the channel via the masking of an N-terminal hydrophobic domain, leading to transport of the channel protein from ER to Golgi, via a Sarl-COPII-independent vesicular trafficking pathway (Shibata et al, 2003; O'Callaghan et al, 2003; Hasdemir et al, 2005). 1.4.4 Distribution at the Cell Surface Although the cellular machineries and molecular mechanisms involved in Kv channel surface distribution are not yet clear, there have been several relevant reports of late. In the lipid raft fraction of plasma membrane, the small flask-like structures are known as caveolae. Caveolin, the main protein of caveolae, binds cholesterol with high affinity, the presence of caveolin implies a special subset of plasma membrane with cholesterol or glycosphingolipids (Anderson, 1993). Kv l .5 has been found in lipid raft fractions and interaction of the channel with caveolin-1 appears to be required for internalization of the channel protein in mouse Ltk" cells and pulmonary artery smooth muscle cells (Martens et al, 2001; Cogolludo et al, 2006). Caveolin-2 associates Kv2.1 with soluble N-ethylamide sensitive factor associated protein receptors (SNARE) proteins such as syntaxin-lA, SNAP-25, VAMP-2 in mouse Ltk", HIT-T15, MIN6, INS1 and rat pancreatic islet cells (Martens et al, 2000; Xia et al, 2004). Membrane fusion process in vesicle transport requires a stoichiometric complex of three SNAREs: syntaxin, SNAP-25 and VAMP/synaptobrevin (Rothman, 2002). V A M P resides mainly in the vesicle membrane, whereas synataxin and SNAP-25 reside mainly in the plasma membrane, and every vesicle 18 and every target membrane has its own v-SNARE (like V A M P ) and t-SNARE (like the syntaxin-SNAP-25 dimer), respectively (Chernomordik and Kozlov, 2005). 1.4.5 Internalization Although the mechanism(s) involved in K v channel endocytosis is/are not yet known, the information gleaned from studies of other membrane proteins may guide further research. The well-studied endocytosis mechanisms of other integral proteins such as G-protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) may be particularly instructive. Phosphorylation of GPCRs, by G-protein coupled receptor kinases and/or other serine/threonine-based kinases (e.g. P K A and PKC), inhibits receptor coupling to heterotrimeric G-proteins at the plasma membrane and facilitates the interaction of phosphorylated receptors with P-arrestins, resulting in receptor endocytosis via binding of GPCR-P-arrestin complex to internalization machinery such as clathrin and/or AP2 (Grady et al, 1997; Claing et al, 2002). R T K internalization also requires phophorylation of the receptor by tyrosine kinase, leading to receptor complex interactions with internalization machineries (Brodsky et al, 2001). Dynamin, a large GTPase (lOOkD), plays an important role in scission of transport vesicles from the membrane in clathrin-dependent and caveolae-dependent endocytosis via binding with diverse binding partners such as phosphoinositides, G-proteins, amphiphysin, endophilin, Src, PI3K, phospholipase D and so on (Brodsky et al, 2001). This large GTPase can self-assemble into tetramers, leading to a ring structure on the neck region of invaginated pit in the membrane (Hinshaw, 2000). There, clathrin triskelion molecules assemble into a basket-like structure, a clathrin lattice, including a transport vesicle. This process is facilitated by AP-180/CALM (McMahon, 19 1999). After pinching off from the plasma membrane, the coated vesicle disassembles the coatomers immediately, promoted by hsp70-like chaperon proteins and auxilin (Brodsky et a/., 2001). Transport vesicles containing endocytosed proteins are delivered to the endosomal compartment with cooperation between the cytoskeleton and cytoplasmic molecular motors. Firstly, signalling that initiates the internalization process stimulates rearrangement of actin filaments, propelling transport vesicles away from the plasma membrane as well as activating non-conventional myosins that may be responsible for motility of the transport vesicles (Engqvist-Goldstein and Drubin, 2003). Later, during internalization, the vesicle is transferred to minus-directed microtubule-based motors, i.e., the cytoplasmic dyneins and some kinesins. 1.4.6 Sorting of endocytosed proteins Rab proteins are thought to confer specificity between transport vesicles and target organelles. Rab proteins are small GTP-binding proteins expressed in membranes of subcellular organelles as membrane proteins. For example, Rab5 is expressed in early endosomes and Rab7 and Rab 11 indicate late endosomes and recycling endosomes, respectively (Zerial and McBride, 2001). Rab5 fuses with the membrane of the endocytosed vesicle from the cell surface: Rab5-GDP dissociation inhibitory protein complex inserts into the membrane. Replacement of GTP by GDP activates Rab5 in the vesicular membrane, leading to fusion between the vesicle and endosomal membranes (Brodsky et a/., 2001). 20 The proteins in the endocytosis process are sorted in early endosomes. The early endosome sorts the proteins into the "tubular" part for recycling and into the "vacuole" part of the early endosome for degradation, depending on the phosphorylation state of the proteins, and then the tubular part is dissociated from the vacuole part, to be matured to a recycling endosome, whereas the remaining part matures into the multi-vesicular body (MVB) and late endosome (Sorkin and Von Zastrow, 2002). Recently, two different populations of early endosomes have been reported: dynamic early endosomes marked for degradation and static early endosomes, the contents of which are recycled. Interestingly, the proteins to be endocytosed are sorted with different endocytosis machineries such as AP2 and non-AP2 adaptors in the plasma membrane (Lakadamyali et al, 2006). 1.4.7 Phosphorylation Protein phosphorylation is another potential mechanism for regulating channel surface expression, and K v channels can be phosphorylated by several kinases. Direct phosphorylation of the channel protein by serine/threonine kinases is a well-studied means by which ion channels are regulated. More recently, phosphorylation by tyrosine kinases has also been identified as an important means of ion channel regulation. The delayed rectifier potassium channel K v l . 2 was the first example of a voltage-gated ion channel identified to be so regulated (Huang et al, 1993; Nesti et al, 2004). Since then a range of voltage- and ligand-gated channels have also been found to be regulated by tyrosine kinases, including N M D A receptors, voltage-gated calcium channels, and a variety of potassium channels (Davis et al, 2001; Strock, 2004). Importantly, additional K v l family channels, the best studied of which are Kvl .3 and Kvl .5 have been shown to undergo 21 tyrosine kinase-dependent regulation (Holmes et al, 1996a, 1996b; Fadool et al, 1997; Fadool and Levitan, 1998). The mechanisms by which tyrosine phosphorylaton regulates voltage-gated ion channel expression are unclear, however. It has been reported that Kvl .5 modulation by tyrosine kinases involves the SH3 domain-mediated physical interaction of Src kinase with the channel protein (Holmes et al, 1996b). Interestingly, the mechanism of this modulation appears to be multifaceted because the interaction itself, even in the absence of catalytic Src activity, is sufficient to modulate channel function (Nitabach et al, 2002). Kv l .2 does not appear to interact with Src in this way, suggesting the existence of different mechanisms for K v l . 2 vs. Kv l .5 regulation. Kvl .3 is also regulated by tyrosine kinases, but a study examining the possible internalization of Kvl .3 as a mechanism of channel suppression yielded negative results (Fadool et al, 1997). The effects of serine/threonine phosphorylation of the N-terminal domain on Kv l .4 biophysical properties have also been studied. Calcium/calmodulin-dependent protein kinase has been shown to slow the inactivation of Kvl .4 currents by phosphorylating SI 23 in the cytoplasmic N-terminal (Roeper et al, 1997). Treatment of Kvl.4-expressing Xenopus oocytes with phorbol 12-myristate 13-acetate, a protein kinase C activator, has been shown to lead to a biphasic change in the magnitude of peak current: an initial increase in peak current was followed by a later reduction (Murray et al, 1994). Although in most cases the precise mechanisms by which serine/threonine phosphorylation affects channel function are not clear, the most commonly suggested mechanism is that phosphorylation-induced changes in channel structure alter the channel's biophysical properties (Levitan, 1994). Recently, Ca /calcineurin-dependent dephosphorylation has been reported as an important component in modulating the clustering and cell surface expression of Kv2.1 22 (Misonou et al, 2004; Mohapatra and Trimmer, 2006). In addition, PKC-dependent phosphorylation of KATP channels promotes internalization of the channel protein via dynamin-dependent endocytosis, resulting in downregulation of the channel number in the plasma membrane (Hu et al, 1996; Hu et al, 2003). 1.5 INCRETIN HORMONES Incretin hormones are essential regulators of insulin secretion and glucose homeostasis. To understand the insulinotropic action of incretins it is necessary to define the enteroinsular axis. The enteroinsular axis is a concept developed to explain the regulation of insulin release from pancreatic p-cells by signals from the gastrointestinal tract (Kieffer and Habener, 1999). Insulin release followed by orally administrated glucose is greater than that of intravenously injected glucose, which indicates that postprandial insulinotropic action regulates 50-70 % of glucose-stimulated insulin secretion (Vahl and D'Alessio, 2004). Two incretins have been characterized: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). GIP is secreted from gut K-cells (which are primarily distributed in the duodenum) in response to ingestion of food containing glucose and fat, while L-cells are scattered in distal small intestine and colon and secret GLP-1 (Vahl and D'Alessio, 2004). The secretion of incretins is proportional to the amount of ingested glucose and fat. Released incretins enter the hepatic portal vein and ultimately circulate throughout the body (Hansotia and Drucker, 2005). 23 1.5.1 Physiological aspects of GIP and GLP-1 GIP is composed of 42 amino acids and is metabolized by dipeptidyl peptidase IV (DPP IV). DPP IV is present in membrane-anchored and circulating forms, which cleave GIP at an alanine residue (position 2) to inactivate GIP (3-42). In humans, the half-life of bioactive GIP is 5.0 + 1.2 minutes. Plasma GIP concentration during fasting is about 10 p M and increased to 80-150 p M after food ingestion. The bioactive form of GLP-1 (7-36) becomes inactive (9-36) with DPP IV enzymatic activity. The half-life of GLP-1 (7-36) is 2.3 + 0.4 minutes, and the postprandial concentration reaches to 20-30 p M from a fasting concentration of 5-10 p M in the human (Brown et al, 1969; Meier and Nauck, 2004). Both GIP and GLP-1 activate specific G-protein coupled receptors expressed in pancreatic p-cells, leading to rapid membrane depolarization, insulin secretion vesicle exocytosis, and insulin release. GLP-1 activates the portal glucose sensor by which vagal efferent nerve activity is directed to the brain, liver, pancreas and peripheral tissues, to prepare the body for the incoming nutrient load and to facilitate glucose uptake (Hansotia and Drucker, 2005). 1.5.2 Receptor signalling of GIP and GLP-1 The subset of signalling networks enhanced by both GIP and GLP-1 receptors leads to increased intracellular cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) and transcription factors (Meier and Nauck, 2005). Alternatively, both GIP and GLP-1 receptor signalling facilitates cell proliferation and reduces cell apoptosis in pancreatic p-cells. Recently, a molecular mechanism for the anti-apoptotic effect of GIP receptor activation has been described, showing that free Py-subcomplex of 24 G-protein activates phosphoinositide-3 kinase (PI3K) and protein kinase B (PKB), leading to phosphorylation of foxol which is a transcription factor of the pro-apoptotic Bax gene (Kim et al, 2005). Taken together, both GIP and GLP-1 promote acute glucose-dependent insulin secretion and have long-term effects on P-cell survival. Therefore, the actions of GIP and GLP-1 are prime targets for drugs to treat patients with type 2 diabetes. 1.5.3 Effect of incretin on Kv channel Recently, GLP-1 has been reported to regulate delayed rectifier K + currents (IDR) in pancreatic P-cells, perhaps explaining the insulinotropic effect of the hormone. Exendin 4, a long-acting GLP-1 analogue, reduces IDR and consequently prolongs action potential duration in P-cells (MacDonald et al, 2002). Exendin 4 is known to affect IDR via both PKA-dependent and PI3K-dependent pathways as well as via protein kinase C (PKC)-dependent signalling (MacDonald et al, 2003). As mentioned above, inhibition of outward K + currents results in prolonged action potential duration, enhancing C a 2 + signalling and insulin secretion from pancreatic P-cells and insulinoma cells. Therefore, the modulation of K + currents is suggested as an important mechanism to explain the insulinotropic function of incretins. 1.6 SCOPE OF THESIS INVESTIGATION This thesis focuses on the regulation of K v channel expression at the cell surface, with particular emphasis on endocytosis. The first general hypothesis of this experimental work is that K v channel internalization requires cytoplasmic molecular motors. The second general hypothesis is that external signals can induce K v channel endocytosis. To gain 25 insight into these processes, I have investigated the role of the dynein motor in the trafficking of K v channels, particularly Kv l .5 , and the effect of the activated G-protein coupled receptor signalling on the regulation of K v channel currents from Kvl .4 , K v l . 5 , Kv2.1 andKv3.2. Chapter 2 describes cytoplasmic dynein motor operation in retrograde trafficking of newly endocytosed Kvl .5 channel protein, along with an examination of the molecular mechanisms underlying the endocytosis process in this channel. This examination was performed using a well-established method for disrupting dynein function, dynamitin (p50) over-expression, to dissect the function of a microtubule-based molecular motor, dynein, in internalization of a channel that modulates a sustained K + current in heart muscle cells. In addition, this study includes a deletion analysis of most of. the Kvl .5 cytoplasmic N - and C-termini and an analysis of point mutations lying in the proline-rich region, a consensus Src binding domain, to identify an essential site in the channel for Kvl .5 endocytosis. This study is the first to demonstrate a critical role for the cytoplasmic dynein motor in Kv channel retrograde transport (Choi et al., 2005). The work presented in Chapter 3 and 4 examines the regulation of K v channel currents by G-protein coupled receptor signalling in pancreatic P-cells, effecting insulin secretion. In Chapter 3, the study demonstrates that activated GIP receptor signalling suppresses the transient outward K + current mediated by Kvl .4 through PKA-dependent endocytosis of the channel protein. Using electrophysiological, biochemical and imaging experiments combined with analysis of truncations and point mutations in the channel, this study delineates a molecular mechanism underlying Kvl .4 endocytosis induced by GIP binding to GIP receptors. This study also validates the hypothesis that phosphorylation-26 dependent internalization of Kvl .4 is important to the effect of GIP on insulin secretion, i.e. that Kv channel currents play an important role in pancreatic p-cell excitability. In addition, the electrophysiological examination of human islet cells demonstrates that the reduction in Kv l .4 dependent currents associated with administration of GIP was transient. After elimination of GIP from the external solution, the currents recovered within a few minutes, suggesting that the internalized channel protein is rapidly recycled, to the cell surface (Kim etal, 2005). The final studies, presented in Chapter 4, were undertaken to further our understanding of the regulation of multiple Kv channels by G-protein coupled receptors. The effects of incretin hormones, GIP and GLP-1, on the molecular components responsible for delayed rectifier K + currents were studied in pancreatic P-cells where diverse K v channel subtypes are expressed; we focused on K v l . 5 , Kv2.1 and Kv3.2. The results from this study demonstrate the presence of three K v channels in rat and human pancreatic P-cells and that the expression pattern of these channels changes depending on disease status. In addition, this study demonstrates that both GIP and GLP-1 receptor signalling modulates K + currents from Kvl .5 and Kv2.1 to a similar degree, but with different kinetics, whereas Kv3.2 current is modulated in a different manner (Choi et al, in preparation). 27 Figure 1.1 Typical action potentials recorded from cells in the ventricle (A), SA node (B), and atrium (C). Sweep velocity in B is one-half that in A or C. (From Hoffman BF, Cranefield PF: Electrophysiology of the heart, New York, 1960, McGraw-Hill.) 28 ft ATP/A DP Glucose GK 6P B. Mouse W \f L * l C. Rat Tlme(s) 5 0 10 SO _ 30 * 60 T i l T » ( S ) Figure 1.2 Glucose-stimulated insulin secretion (GSIS) and action potentials recorded from pancreatic p-cell in mouse (B) and rat (C) (the action potential recordings from Manning Fox et al., 2006). 1. Glucose entering through GLUT2 is converted into glucose 6-phosphate (G6P) by glucokinase. 2. Metabolism of G6P in mitochondria results in increase of the cytoplasmic ATP/ADP ratio. 3. ATP closes down IK(ATP) and leads to membrane depolarization, subsequently, [Ca 2 +]i increased by activated voltage-gated C a 2 + channels results in insulin secretion. 4. Membrane depolarization also leads to activation of K v channels, resulting in the termination of insulin secretion because of membrane repolarization in pancreatic p-cells. 5. 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Kvl.5 Surface Expression Is Modulated by Retrograde Trafficking of Newly Endocytosed Channels by the Dynein Motor A version of this chapter has been published. Woo Sung Choi, Anu Khurana, Rajesh Mathur, Vijay Viswanathan, David F. Steele and David Fedida (2005) Kvl .5 surface expression is modulated by retrograde trafficking of newly endocytosed channels by the dynein motor. Circulation Research. Vol . 97, Issue 4, 363-71. 43 2.1 INTRODUCTION Voltage-gated K + channels (Kv channels) are intimately involved in the cellular regulation of excitation in all cardiovascular cells, and their influence on excitability depends, in part, on the surface density (channels/unit area). Surface expression is regulated by changes in gene expression, interactions with accessory subunits, by phosphorylation, and by cellular components that regulate their trafficking to the cell surface (Van Wagoner et al, 1997; Grammer et al, 2000; Takimoto and Levitan, 1994). Trafficking can also provide an explanation for the mechanisms by which drugs may act to achieve their therapeutic actions (Ficker et al, 2004). Although several groups have investigated motifs within K + channels that affect trafficking, little is known about the molecules and machinery involved in these processes in the heart (Li et al, 2000; Manganas et al, 2001; Zhu et al., 2001; Zhu et al., 2003). Surface expression requires movement from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane, and several studies have investigated channel determinants that affect this trafficking process. Motifs in the C termini and pore domains of K v channels have been implicated in their differential surface expression presumably through effects on forward trafficking (Li et al, 2000; Manganas et al, 2001; Zhu et al, 2001). Functional expression of channels can also be regulated by the removal of extant channels from the cell surface, as recently shown for Kv l . 2 . Surface expression is modulated by tyrosine phosphorylation which promotes the endocytosis of the channel (Nesti et al., 2004). Dhani et al. have implicated retrograde transport along the microtubule cytoskeleton in the regulation of C1C-2 chloride channel expression. C1C-2 interacts 44 directly with the dynein motor and inhibition of this motor significantly increased the surface expression of the channel (Dhani et al, 2003). Here we have used a multi-pronged approach to show that dynein is also closely involved in the regulation of Kvl .5 surface expression, probably through its role in transporting newly formed endosomes from the cell surface to the cell interior. Direct interference with dynein function caused a significant increase in Kvl .5 surface expression, as did disruption of the microtubule cytoskeleton or direct interference with endocytosis itself. We have identified specific residues in Kvl .5 that are essential to this process and shown the process to be relevant both in heterologous cell expression systems and in rat cardiac myocytes. 45 2.2 MATERIALS AND METHODS Many methods used in this study have been previously described (Eldstrom et al., 2003). In electrophysiological experiments where controls were compared with cells overexpressing p50, dynamin inhibitory peptide, or compared with nocodazole pretreatment, control and experimental groups were prepared in parallel and the experimenter was blinded to their identity. 2.2.1 Cell Preparation and Transfection Unless otherwise indicated, hKvl.5 channels were studied in HEK-293 cells as described previously (Fedida et al, 2003). Transfections were by a liposome-mediated method. One day before transfection, cells were plated on a coverslip in 3 5-mm dishes at 40-50% confluence. After one day's growth, transfections were performed using 1.5 ug of each relevant plasmid and Lipofectamine 2000™ transfection reagent (Invitrogen) according to the manufacturer's instructions. For nocodazole experiments, cells were incubated with or without 35 uM nocodazole for 6 hr, prior to electrophysiological analysis. In experiments with the dynamin inhibitory peptide, cells were incubated for 30 min with 50 (j,M mry-DIP (myristoylated dynamin inhibitory peptide; Tocris Cookson Ltd., Bristol, UK) prior to electrophysiological analysis. 2.2.2 Plasmid Constructs The construction of hKvl.5 in pcDNA3, Kvl.5AN209, Kvl.5AN91 and Kvl.5AC51 were described previously (Eldstrom et al, 2002; Eldstrom et al, 2003). Human p50 in pEGFP was a gift of Richard Vallee (Columbia University, NY) . Kvl.5A65-46 93 was constructed by a PCR-based method using appropriate primers. The PCR product was cloned into hKvl.5 in pcDNA3 as a HindlllBspEI fragment, replacing the wild-type sequence in that region. A l l channel constructs used in this study were cloned into the pcDNA3 vector and various deletion mutants were prepared by PCR with Hindlll and EcoRV insertions for directed cloning. Site-directed mutant constructs were prepared using the QuickChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA). Following mutagenesis and sequencing, the mutant sequence was subcloned into the parent vector to ensure that no extraneous mutations were present in the constructs used. Plasmid D N A was prepared for transfection using the Qiagen Plasmid Midi Kit (Qiagen Inc, Valencia, CA). 2.2.3 Electrophysiological Experiments and Solutions Solutions and methodology for the recording of ionic currents were as previously reported from our laboratory (Fedida et al, 1999; Wang et al, 1999). The standard bath solution contained (in mM): NaCl, 135; KC1, 5; M g C l 2 , 1; sodium acetate, 2.8; HEPES, 10; CaCb, 1; adjusted to pH 7.4 using NaOH. The standard pipette filling solution contained (in mM): KC1, 130; EGTA, 5; M g C l 2 , 1; HEPES, 10; Na 2 ATP, 4; GTP, 0.1; adjusted to pH 7.2 with K O H . A l l chemicals were from Sigma (Mississauga, ON, Canada). Whole cell current recording and data analysis were done using an Axopatch 200B amplifier and pClamp 8 software (Axon Instruments, Foster City, CA). Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL) and polished by heating. We used pipettes having a resistance of 1-3 MCI. Compensation for capacitance and series resistance was performed manually in all whole cell recordings. HEK-293 cells were depolarized between -70 and +80 mV in 10 mV-steps from a holding 47 potential of-80 mV followed by repolarization pulse to -40 mV to record tail currents. A l l whole cell recordings were performed at room temperature (20-23 °C), and control and experimental group identities were concealed from the experimenters. 2.2.4 Myocyte Isolation and Electrophysiology Rat atrial myocytes were isolated from hearts of male Wistar rats weighing 250-300 g using a conventional method with normal Tyrode's solution (Mitra and Morad, 1985). In nocodazole experiments, after pretreatment with the drug, cells were depolarized between -80 and +80 in 10-mV steps for 800 ms from a holding potential of -80 mV to record ionic current using the solution contained (in mM): NaCl, 135; KC1, 5; MgCb, 1; sodium acetate, 2.8; HEPES, 10; adjusted to pH 7.4 using NaOH. The pipette filling solution contained (in mM): KC1, 130; EGTA, 5; M g C l 2 , 1; HEPES, 10; Na 2 ATP, 4; GTP, 0.1; adjusted to pH 7.2 with K O H . 2.2.5 Imaging Cells were prepared according to previously published methods (Scriven et al, 2000). For studies on the effects of p50 over-expression, HEK-293 cells stably expressing T7-tagged Kvl .5 were seeded onto coverslips and later transfected with p50-GFP and cultured for a further 12 h prior to fixation. The cells were rinsed and fixed with 4% paraformaldehyde for 12 min at room temperature (RT). After three 5-min washes with lxphosphate-buffered saline (PBS; 137 m M NaCl, 2.7 m M KC1, 4.3 m M Na 2 HP04, 1.4 m M KH2PO4), cells were incubated in a permeabilizing/blocking solution (PBS containing 2% B S A and 0.2% Triton X-100) for 30 min at room temperature (RT). A mouse 48 monoclonal antibody to the T7 Tag (1:1000; Novagen) was diluted in blocking solution and incubated with the cells for 2 h at RT. Cells were then washed three times for 5 min in PBS on a rotator before incubation with secondary antibodies, Alexa 594-conjugated goat anti-mouse IgG antibody (1:1000; Molecular Probes) for 1 h on the rotator at RT. Coverslips were then washed three times with PBS prior to mounting with 10 ul of a 90 % glycerol, 2.5 % w/v D A B C O - P B S solution. Images were collected on a Nikon CI laser scanning confocal unit (Nikon DEclipse C I , Melville, NY) and processed using the operation software EZ-C1 for Nikon CI confocal microscope (Nikon). Images were prepared using the Adobe PhotoShop software package. For endosome imaging studies, HEK-293 cells stably expressing Kv l .5 were labeled with antibodies against the C-terminus and against EEA1. For Kvl .5-EEA1 colocalization experiments, stable Kvl.5-T7 lines were plated onto coverslips and treated with cycloheximide supplemented media (cycloheximide, Sigma, 200 ug/ml) for 4 h to arrest protein synthesis. After the first 2 h of treatment at 37°C, cells were placed into a 20°C incubator for the last 2 h of cycloheximide treatment. This temperature allows normal internalization but is not compatible with trafficking out of early endosomes or recycling to the plasma membrane (Dhani et al, 2003; Echeverri et al, 1996; Burkhardt et al, 1997). The cells were rinsed and fixed with 4 % paraformaldehyde for 12 min at room temperature (RT). After three 5-min washes with lx phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 m M KC1, 4.3 m M Na 2 HP0 4 , 1.4 mM K H 2 P 0 4 ) , cells were incubated in permeabilizing/blocking solution (PBS containing 2 % B S A and 0.2 % Triton X-100) for 30 min at RT. Rabbit Polyclonal C-terminal Kvl .5 antibody (1:500) and a mouse monoclonal EEA-1 antibody (1:250; BD Biosciences), were diluted in blocking solution 49 and incubated with the cells at 4°C overnight (Fedida et al, 2003). Cells were then washed three times for 5 min in PBS on a rotator. Secondary antibodies, Alexa 594-conjugated goat anti-mouse IgG antibody and Alexa 488-conjugated goat anti-rabbit IgG antibody (1:1000; Molecular Probes) were then incubated with the cells for 1 h on the rotator at RT. The coverslips were once again washed three times with PBS prior to mounting with 10 ul of a 90 % glycerol, 2.5 % w/v D A B C O - P B S solution. Images were collected using a Deltavision Deconvolution Microscope using a 60 X lens using Softworks software. Images were later viewed and prepared using Adobe PhotoShop software. For studies of the effects of nocodazole on rat atrial myocytes, the myocytes were treated with 35 uM nocodazole (Sigma) for five hours then fixed and immunolabeled as previously described (Fedida et al, 2003). Myocytes were co-stained with anti-Kvl.5 and anti-P-tubulin (Sigma) to verify the depolymerizing actions of nocodazole. 2.2.6 Image Analysis To quantitatively assess Kvl .5 distribution in myocytes, line scanning analysis was performed using Image J software (NIH imaging). For each cell, eight 0.25 um confocal slices were taken along the long axis of the cell, at 45-55 % of the cell depth, approximating the cell middle. 3 lines were drawn across the width of each slice chosen at positions approximately lA, XA and % of the cell length from one end of the cell. The line was then divided into eight equal segments. Pairs of segments (i.e., the outermost eighths, the eighths immediately inside adjacent to those eighths, etc.) were combined to form 'quarters' across the width of the cell. These data from the lines at the four positions along the length of the cell were combined to get an average estimate of the fluorescence 50 distribution in each cell. The fluorescence intensity for each quartile was expressed as a percentage of the total fluorescence intensity in the lines. Image J was used also for line scan analysis of Kvl .5 colocalization with EEA1. Lines were drawn through endosomes across several cells in confocal images and the intensities of Kvl .5 and EEA1 signals at each pixel were plotted on the same graph for comparison. Correlation coefficients, r, between Kv l .5 and EEA1 signals in pixels across each line scan were calculated by the formula r = (2 x - x")(y - y) I VS(x - x') 2E(y - y)2 . To assess the significance of each r value, t was determined according to t = r I V ( l - r2) I (N - 2) and compared to critical values of t. 2.2.7 Western Blotting and Densitometry Western blotting was performed using standard techniques. Following protein estimation by the Lowry method, cell extracts were subjected to P A G E and blotted to PVDF membranes. Antibodies were diluted to appropriate working concentrations in blocking buffer (5 % skim milk powder, 0.1 % Tween-20 in PBS) and incubated with the membranes. Following incubation with appropriate HRP-conjugated secondary antibody, protein bands were visualized by using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) and detected on X-ray film. For densitometry, X-ray film images, were digitized using an Epson Perfection 1200U scanner and saved as .tif files. They were then analyzed using NIH Image J software according to the instructions provided on the NIH webpage. Optical Density calibration for the analysis was performed using a Kodak optical density step tablet. 51 2.2.8 Co-immunoprecipitation Experiments HEK-293 cells stably expressing T7-tagged Kvl .5 were lysed in lysis buffer (25 m M phosphate buffer, 150 mM NaCl, 2.5 m M EDTA, 10 % glycerol and 1 % Triton). 2.5 ug of anti-T7 tag antibody (Novagen) with protein A Sepharose (Sigma) and 3 ug Dynein antibody (Anti-Dynein cytoplasmic; Chemicon International) with protein G Agarose (Roche Diagnostics) were used for immunoprecipitation of T7 tagged Kv l .5 and Dynein intermediate chain respectively. Western blotting was performed using the T7 tag antibody (1:10000) and Dynein antibody (1:3000) and blots were developed using biotin-labeled goat anti-rabbit antibody (Amersham) and Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). 2.2.9 Proteinase K experiments Digestion of surfaced proteins in living HEK-293 cells was conducted as described by Manganas, et al. For Proteinase K-sensitivity experiments, HEK-293 cells expressing T7-tagged Kvl .5 alone or T7-tagged Kvl .5 plus p50 were washed, and then treated with 200ug/mL Proteinase K for 30 min (Manganas, et al, 2001). Following extensive washing to remove residual Proteinase K, cells were lysed and processed for Western analysis. The N-terminally T7-tagged Kvl .5 was detected with anti-T7 monoclonal antibody (Novagen). 52 2.3 RESULTS 2.3.1 Disruption of Dynein Function Increases Kvl.5 Surface Expression Over-expression of p50/dynamitin is established as an effective and specific method for disrupting the dynein motor system (Echeverri et al, 1996; Burkhardt et al, 1997; Valetti et al, 1999). p50, an important component of the dynactin complex, is thought to link the cargo-interacting subunits to the dynein motor itself. Over-expression of this protein causes the dynactin complex to dissociate, decoupling the motor from its cargoes (Echeverri et al, 1996). As shown in Figure 2.1, over-expression of p50 in H E K -293 cells significantly increased Kvl .5 currents on the day after transfection. Peak current densities at +80 mV were approximately doubled from 0.57 ± 0.07 nA/pF to 1.18 ± 0.2 nA/pF, PO.01 (Figure 2.1A). This increase in response to p50 over-expression was not limited to HEK-293 cells, and experiments in CHO cells yielded similar results (Figure 2.IB). Activation and inactivation kinetics were unchanged with increases in current across the whole activation range (data not shown), suggesting an increase in the number of functional channels at the cell surface. To confirm that increased Kvl .5 currents were indeed caused by increased surface expression, Kvl .5 localization was examined by immunocytochemistry/confocal microscopy and channel Proteinase K sensitivity was assayed. In Figure 2.ID, p50 over-expression caused a substantial redistribution of Kvl .5 staining. When expressed alone in HEK-293 cells, Kv l .5 was distributed throughout the cell. Membrane expression was significant, but large amounts of the channel were cytoplasmic (Figure 2. ID, upper panel, center). p50 over-expression dramatically reduced internal staining (lower panels), whereas staining at the cell surface remained intense. This is most clearly demonstrated in the lone 53 cell in the lower middle panel (white arrow), which escaped transfection with p50-GFP, but was transfected with K v l . 5 . Protease-sensitivity experiments yielded results consistent with this. When applied externally to living cells, Proteinase K cleaves exposed protein on the exterior of the cell although cytoplasmic proteins are unaffected. This protease is thus a sensitive test for cell surface expression and is useful also for determining the relative proportions of the channel at the cell surface and in the cytoplasm (Manganas et al, 2001). Uncleaved T7-tagged Kvl .5 migrates at 83 kDa; the digested N-terminal fragment (roughly, the cytoplasmic N terminus plus SI), migrates at -47 kDa. Densitometric analysis of the results shown in Lanes 3 and 4 of Figure 2. IE confirmed that the intensity of the 47 kDa Kvl .5 band relative to the upper band intensity was significantly increased in cells overexpressing p50 (1.52 ± 0.09, n=3, paired t test P<0.05) compared with control cells. That is, p50 over-expression increased Kvl .5 surface residency by -50%. This result is consistent with the 2-fold increase in Kvl .5 expression detected electrophysiologically and with the redistribution of the channel seen in the imaging experiments. Given that p50 is well established as a specific disruptor of dynein motor function, the dynein motor is strongly implicated in the regulation of Kvl .5 surface expression (Echeverri et al, 1996; Burkhardt et al ,1997; Valetti et al., 1999). 2.3.2 Kvl.5 Is Present in Early Endosomes Dynein motor disruption by p50 over-expression might increase Kvl .5 surface expression by interfering with endocytosis, because dynein is required for transport of endosomes from the cell surface to the interior (Valetti et al, 1999). To see whether Kvl .5 54 is trafficked via endosomes, we used confocal microscopy to look for colocalization of Kvl .5 with EEA1, an early endosomal marker. As shown in Figure 2.2A, Kvl .5 and EEA1 colocalized in what appear to be small vesicles, ie, early endosomes, near the plasma membrane. Linescan analysis across 9 groups of endosomes in this cell, as well as endosome-rich regions of several additional cells confirmed this. In all linescans the pixel correlation between Kvl .5 and EEA1 fluorescence was extremely high (PO.0001, Figure 2.9A and IB). In contrast, there was no correlation of Kvl .5 and EEA1 associated pixels in line scans across regions where Kvl .5 was found but endosomal localization was not obviously present (see below). If dynein disruption interferes with the removal of Kvl .5 from the plasma membrane, then direct inhibition of endocytosis should mimic this effect. Dynamin is responsible for pinching off the neck of the budding endocytotic vesicle and dynamin inhibitory peptide (DIP, Tocris, Bristol, UK) has been previously established to be an effective blocker of endocytosis (Nong et al, 2003). HEK-293 cells stably expressing Kvl .5 were incubated overnight with 50 uM DIP or a control scrambled peptide. As shown in Figure 2.2C, DIP incubation doubled Kvl .5 currents, an effect essentially identical to that elicited by p50 over-expression. Peak current densities at +80 mV were 0.36 ± 0.05 nA/pF (n=15) in control cells and 0.76 ± 0.05 nA/pF (PO.01, n=ll) in cells incubated overnight with DIP. Imaging analysis showed that colocalization of Kvl .5 and EEA1 in residual early endosomes was dramatically reduced in DIP-treated cells relative to controls. Linescan analyses across these endosomes showed no significant pixel correlation between EEA1 expression and Kv l .5 (Figure 2.9C) with probability values between 0.065 and 0.420. 55 These data suggest that dynein inhibition may increase Kvl .5 surface expression by interfering with the net removal of channels from the plasma membrane. 2.3.3 Nocodazole Treatment Mimics Dynein Inhibition in HEK-293 Cells and Cardiomyocytes If p50 over-expression exerts its actions through interference with dynein transport along microtubules, then disruption of the microtubule cytoskeleton should have similar effects on Kvl .5 currents. To test this prediction we used the microtubule depolymerizing agent, nocodazole, which strongly inhibits trafficking by dynein (Cole et al, 1996). Incubation of Kvl.5-expressing HEK-293 cells with nocodazole substantially increased Kvl .5 currents (Figure 2.3A and B). Kv l .5 currents increased in a concentration-dependent manner with a maximum effect at 70 uM. p50 over-expression was additive to the nocodazole current increase at lower doses (open triangles, Figure 2.3B) where the drug had a submaximal action. However, current levels saturated with p50-over-expression and >70 uM nocodazole. These results suggest that p50 over-expression is acting on a microtubule-dependent pathway, ie, very probably on dynein. Although the additivity of p50 over-expression and low-dose nocodazole may indicate that p50 acts also on a secondary pathway, it is more likely that p50 over-expression is not completely effective in preventing dynein motor complex assembly. Thus, only when microtubules are fully depolymerized at the higher nocodazole concentrations is the residual trafficking by dynein eliminated. In parallel experiments, atrial myocytes were isolated from adult rats and treated for 6 hours with 35 uM nocodazole. 7-Frelations in Figure 2.3C and 2.3D show that sustained currents were enhanced ~2X in the nocodazole-treated myocytes but the A-type 56 currents, which underlie ITO, in these cells were unaffected (Figure 2.3E). As shown in Figure 2.3D, the increase in sustained current was across the voltage range that Kvl .5 is expected to be open. Imaging experiments confirmed an enhancement of Kv l .5 surface expression in these myocytes. Nocodazole-treated and control myocytes were stained with anti-Kvl.5 and antitubulin and visualized by confocal microscopy (Fedida et al, 2003). This treatment resulted in a loss of microtubule integrity, as evidenced by a loss of reticulate structure and a general dispersal of tubulin staining (data not shown). Nocodazole treatment dramatically increased the intensity of Kvl .5 staining at the myocyte surface compared with controls (Figure 2.4A and B). Linescan analysis on 10 randomly chosen control and nocodazole-treated myocytes showed that 26.9 + 2.1% (mean ± SEM) of Kvl.5-positive pixels were within the quartile of the cell nearest to (and including) the plasmalemma in control cells and 40.5 ± 2.6% (Figure 2.4C and D; unpaired Mest, PO.001) of positive pixels were within these boundaries in nocodazole-treated myocytes. The increased Kvl .5 staining in the outermost quartile supports the idea that enhancement of sustained currents in these cells by nocodazole was caused by block of K v l .5 retrograde trafficking. To determine the contribution of Kvl .5 to the increased sustained currents in nocodazole-treated myocytes, Kv l .5 blockers were used. 4-aminopyridine is commonly used to block K + channels by binding to the inner pore mouth (Bouchard and Fedida, 1995). Susceptibility of the current to 4-AP eliminated the possibility that anion or other currents underlie the increase in myocyte current. In nocodazole-treated myocytes, 1 m M 4-AP reduced sustained and A-type current (Figure 2.5A), confirming that nocodazole was indeed influencing potassium channel expression. However, whereas Kvl .5 reportedly 57 underlies the bulk of the sustained current in rat atrial myocytes, other channels, like Kv2.1, may also be contributors to this current at the 1 m M concentration used here (Grissmer et al, 1994; Nerbonne, 2000). To confirm that Kvl .5 underlies the enhanced sustained K + currents seen after nocodazole treatment, a Kvl.5-specific blocker, 2-(3,4-dimethylphenyl)-3-(4-methoxyphenethyl)-metathiazan-4-one (DMM), a gift from Cardiome Pharma (Vancouver, Canada), was used. The IC50 for Kvl .5 block (1.5 nM) with this drug was >2 orders of magnitude lower than for Kv2.1 or other K + channels and N a + channels present in heart (Figure 2.5B), and 100 nM D M M completely blocked Kvl .5 in HEK-293 cells (Figure 2.5C). More importantly, D M M also blocked the nocodazole-increased sustained current in rat atrial myocytes (Figure 2.5D). 2.3.4 Kvl.5 Coimmunoprecipitates With the Dynein Motor Complex To test for an association between Kvl .5 and dynein, coimmunoprecipitations of Kvl .5 and the dynein intermediate chain (DIC), or p50 (an integral part of the dynein-dynactin complex) were performed on extracts of HEK-293 cells heterologously expressing a T7-tagged Kvl .5 construct (Figure 2.6A), and rat ventricular myocyte lysates expressing the native channel (Figure 2.6B). A significant portion of Kvl .5 coimmunoprecipitated with the dynein intermediate chain in these experiments. Anti-T7 antibody brought down the DIC along with the targeted T7-tagged Kvl .5 in HEK-293 cells and anti-Kvl.5 coimmunoprecipitated DIC in myocytes (Figure 2.6A, lower panel, and B). T7-tagged Kvl .5 was also efficiently coimmunoprecipitated with p50 (Figure 2.10). Given that dynein interacts with many cellular proteins and that plasmalemma-integrated Kvl .5 is unlikely to interact with dynein, this level of coimmunoprecipitation is highly significant. Thus, like 58 C1C-2 (Dhani et al, 2003), Kv l .5 directly interacts with the motor complex that is likely trafficking it from near the cell surface in native cells to the interior. 2.3.5 A Proline Rich Region in Kvl.5 Is Essential for the Regulation of Channel Expression by Dynein Both the N and C termini of K v channels have been implicated in regulating their surface expression (Li et al, 2000; Eldstrom et al, 2003). Mutants lacking the bulk of either the N terminus (AN209) or C terminus of Kvl .5 (AC51) were tested for responsiveness to p50-over-expression (Figure 2.7A). The AC51 mutant behaved like WT, as currents were roughly doubled, but Kvl.5AN209 was wholly unresponsive to disruption of dynein motor function. A more conservative deletion (AN91) also proved unresponsive to p50 over-expression, and included in the deletion is a proline rich region (aa 65 to 93) harboring two SH3 binding domains that are consensus Src kinase binding sites (Kay et al, 2000). As tyrosine phosphorylation has been previously implicated in Kvl .5 surface expression and specifically in the regulation of Kv l .2 endocytosis (Holmes et al, 1996; Hattan et al, 2002), this region seemed a likely candidate for involvement in internalization of the channel. Deletion of aa65-93 completely eliminated the increased Kvl .5 currents seen with p50 over-expression (Figure 2.7B). The 2 consensus SH3-binding domains in this region are separated by 4 residues and both have the sequence RPLPPLP. We mutagenized the consensus sequences individually to R P L A A L P (PI and PH, Figure 2.7C). Mutation of the second consensus domain (Kvl.5 P79/80A; P-II) had no effect on Kvl .5 responsiveness to p50 over-expression (Figure 2.8B), but mutation of the first consensus SH3-binding domain (Kvl.5 59 P68/69A; P-I) eliminated the responsiveness of Kvl .5 to p50 over-expression (Figure 2.8A). Interestingly, the baseline current densities in PI mutant cells were unusually high, resembling levels seen with WT Kvl .5 cells overexpressing p50 or the dynamin inhibitory peptide (DIP, Figure 2.2). This suggested that Kvl .5 P68/69A had lost its ability to be upregulated by interference with the motor, or perhaps had been relieved of negative regulation by the dynein motor because the channel no longer interacted with it. However, given that block of endocytosis with a dynamin inhibitory peptide mimics the effect of p50 (Figure 2.2C) and that tyrosine phosphorylation has been implicated in the Kvl .5 downregulation and in endocytosis of the related K v l . 2 channel (Holmes et al, 1996; Nesti et al, 2004), we preferred the hypothesis that this loss of responsiveness might be attributable to a defect in endocytosis of the channel, which was thus no longer available for retrograde transport. We therefore repeated the PI mutant experiment with DIP to test whether that peptide could still affect surface expression of the PI-Kvl.5. As shown in Figure 2.8C, DIP had no significant effect on the expression of PI channel currents, although still affecting PII currents. Kv l .5 P68/69A expression was high whether or not the cells were pretreated with DIP. Currents were 2 to 3X those of Kv l .5 P79/80A which served as a control in these experiments. Neither DIP, nor p50 over-expression, nor a combination of the two (Figure 2.8D) had any effect on the expression of the PI mutant Kvl .5 channel. We conclude that the N-terminal-most SH3-binding domain is essential for normal endocytosis of K v l . 5 . Surface expression is regulated by endocytosis, followed by retrograde transport of the internalized channels in association with the dynein motor complex. 60 2.4 DISCUSSION 2.4.1 Dynein Motor Function Affects Kvl.5 Surface Expression This is the first report of an association of a K + channel with the dynein motor complex. In the only other report of an ion channel-dynein interaction, Dhani et al. demonstrated that a chloride channel, C1C-2, bound the heavy and intermediate dynein chains in vitro and that the channel could be coimmunoprecipitated with the dynein motor complex from murine hippocampal lysates (Dhani et al, 2003). As with K v l . 5 , interference with dynein function increased surface expression of C1C-2. Here, our data unequivocally demonstrate that inhibition of dynein motor function in HEK-293 cells with p50 over-expression, or nocodazole in myocytes increases the presence of Kvl .5 at the cell surface by about 2-fold, by electrophysiological, biochemical and imaging data (Figures 2.1, 2.3, and 2.4). p50 over-expression is well established as a powerful and specific means to inhibit dynein motor function. However, it remains possible that p50 over-expression has additional, as yet undescribed effects on the cell that are independent of its role in the dynein-dynactin complex. To exclude this possibility, nocodazole was used as a second method to inhibit dynein function, and caused significant increases in Kvl .5 currents in HEK-293 cells and in rat cardiac myocytes (Figure 2.3), that could be abolished by the K + current-selective agent 4-AP, and the more specific Kvl .5 antagonist, D M M (Figure 2.5). Although there is no requirement for a direct interaction between the complex and channel for trafficking by the motor (Kvl.5 could be dragged along in endosomes bound to the dynein motor complex independently of a specific K v l . 5-dynein association), we were also able to find a direct interaction between Kvl .5 and components of the dynein complex, p50, and DIC in HEK-293 cells and native myocytes (Figure 2.6). Also, it is interesting that, at 61 low doses of nocodazole, the effects of the drug and p50 over-expression on Kvl .5 expression in HEK-293 cells are additive (Figure 2.3). This suggests that either p50 over-expression is insufficient to fully inhibit dynein function or there is at least one additional pathway by which K v l .5 is trafficked. 2.4.2 Endocytosis and the Role of a Kvl.5 Consensus SH3 Binding Domain The most likely mechanism by which dynein block increases Kvl .5 surface expression is indirect, by affecting the balance of channel delivery and removal from the plasma membrane. Valetti et al. have shown that endocytosis is normal in HeLa cells overexpressing p50, as is the recycling of endosomes to the cell surface (Valetti et al, 1999). This does not mean, however, that Kvl .5 surface levels must remain in a steady state when retrograde trafficking is impaired. If even a fraction of the newly synthesized or internal pools of the channel continue to traffic to the cell surface, the failure of dynein to remove endocytosed channels would quickly result in an increased concentration of the channel in the endocytosing/recycling pool. Indeed, disruption of the dynein-dynactin complex by p50 over-expression has been shown to lead to a redistribution of endosomes toward the cell periphery in COS-7 cells and is probably attributable to a failure of newly formed endosomes to be further internalized (Dhani et al, 2003; Burkhardt et al, 1997). Block of endocytosis using the dynamin-inhibitory peptide mimicked the effects of p50 over-expression and nocodazole treatment (Figure 2.2) and is consistent with this model. Mutation of 2 prolines to alanines in 1 Kvl .5 consensus SH3-binding domain also eliminated the Kvl .5 current response to p50 over-expression (Figures 2.7 and 2.8). This is almost certainly because the mutant channel is not efficiently endocytosed. Current levels 62 are high (similar to those of the wild-type channel when dynein function or endocytosis are inhibited) and, more tellingly, the mutant is also insensitive to dynamin inhibition (Figure 2.8). SH3 domains are important for the recruitment of tyrosine kinases, like Src, to a protein. Tyrosine phosphorylation is well known to downregulate Kv l .5 currents, Src kinase has been shown to directly bind the proline-rich region in which this SH3-binding domain resides, and a direct role for tyrosine phosphorylation in the regulation of endocytosis has recently been demonstrated for Kv l . 2 , a related potassium channel (Holmes et al, 1996; Mason et al, 2002; Nesti et al, 2004). Thus, the simplest explanation for the role of this consensus SH3-binding domain in Kvl .5 is that it is required to promote tyrosine phosphorylation of the channel and that this in turn regulates channel internalization. Lacking this domain, the channel remains in the plasma membrane, and is inaccessible to dynein so manipulations of the motor have no effect on channel surface expression (Figure 2.8). There are 2 identical consensus SH3-binding domains in close juxtaposition in K v l . 5 . However, mutation of the second SH3-binding domain does not affect the sensitivity of the channel to p50 over-expression. Perhaps the precise context of the domain is important. Also, although deletion of the entire proline rich domain (residues 65 to 93) eliminates the responsiveness of the channel to p50 over-expression, cells expressing this mutant do not display current densities above those of cells expressing the wild-type channel in the absence of p50 over-expression. Perhaps there are additional determinants in this region that affect delivery of the channel to the plasma membrane or otherwise affect its stability at that locale. 63 2.4.3 Implications for Forward Trafficking Although pore and C-terminal motifs have been implicated in the trafficking of K v channels to the cell surface, the mechanism(s) by which these motifs affect this movement remain unclear (Manganas et al, 2001; Zhu et al, 2001; L i et al, 2000). It may relate either to interactions with trafficking molecules or to channel assembly instead. However those signals operate, the fact that inhibition of dynein function and depolymerization of the microtubule cytoskeleton increases Kvl .5 surface expression has important implications for forward trafficking. Somehow, Kvl .5 is continuing to traffic to the membrane. Kinesins are unlikely to be involved in this process. Although they are generally responsible for anterograde transport, dynein and kinesin activities are coordinated (Schliwa and Woehlke, 2003). When retrograde transport is blocked, anterograde movement also ceases and vice versa (Valetti et al, 1999; Hamm-Alvarez et al, 1993). The dynactin complex, which connects the dynein motor to its cargo, may play an essential role in this regard. p l50 g l u e d , a component of the dynactin complex, is required also for the functioning of at least 1 kinesin isoform (Deacon et al, 2003). Whatever the case, kinesins are certainly unlikely to be functional in the nocodazole-treated cells, where the microtubule cytoskeleton has been depolymerized. At least a fraction of Kvl .5 forward trafficking must therefore use a nonmicrotubule-dependent pathway. One possibility is that the channel is trafficked via the actin cytoskeleton instead. Perhaps a fraction of Kvl .5 is carried to the surface by myosin V which has been implicated in anterograde trafficking in neurons, and in complex with kinesin has been shown to be involved even in long-range anterograde vesicular trafficking (Langford, 2002). Alternatively, simple diffusion within the cell might be enough to traffic 64 Kvl .5 to the surface in the absence of a microtubule cytoskeleton. A high-affinity Kvl .5 anchoring protein in the plasma membrane or a preference of the channel for a plasma membranelike lipid environment might be sufficient to drive the density of the channel at the surface in the time frames examined here. Clearly future work needs to address these possibilities. 2.4.4 Relevance to the Heart Nocodazole-treatment increased Kvl .5 surface expression as assayed by confocal imaging and measurement of sustained K + currents in rat cardiac myocytes. The sensitivity of these increased currents to the specific Kvl .5 antagonist D M M demonstrated that they were specifically underlain by increased Kvl .5 expression in these cells. In addition, Kvl .5 was shown to directly interact with components of the dynein complex (Figure 2.6). It is highly likely, therefore, that the microtubule cytoskeleton is involved in the internal trafficking of Kvl .5 in both heterologous and physiological (cardiac) systems. It will be of great interest to determine how this system is used by the heart to modulate the expression of Kv l .5 and other ion channels as a mechanism to regulate cardiomyocyte excitability, and how other channels are affected by perturbations of retrograde trafficking. It is possible that in disease states associated with channel remodeling, like atrial fibrillation and congestive cardiac failure, the trafficking or endocytosis mechanisms described above may be of key importance in determining the final population of surface-expressed channels. For example, it has been reported that microtubule abundance is increased in the myocytes of patients with congestive heart failure (Aquila et al, 2004). 65 2.5 CONCLUSIONS The dynein motor has been strongly implicated in the retrograde trafficking of Kvl .5 in endosomes from the cell surface in HEK-293 cells and myocytes, probably via a direct interaction between the channel and the dynein motor complex. This trafficking depends on efficient endocytosis of the channel, which is, in turn, dependent on an intact SH3-binding domain in the Kvl .5 N terminus. This is a previously unexplored avenue of K v channel trafficking and regulation. 66 2.6 ACKNOWLEDGEMENTS These studies were supported by funding to D.F. from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of British Columbia and Yukon, and to WSC from a University Graduate Fellowship. We thank Richard Vallee for the gift of human p50-GFP, and Yan Liu for help with cell preparation. 67 A B CHO cells HEK 293 cells -80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) Figure 2.1 p50/dynamitin coexpression significantly increases Kvl.5 current levels and surface expression. (Legend on the following page) 68 D E pGFP Kv1.5-T7 Merged Figure 2.1 p50/dynamitin coexpression significantly increases Kvl.5 current levels and surface expression. A , Peak currents from HEK-293 cells stably expressing Kvl .5 transfected with empty vector (pGFP) or p50-pGFP. From -80 mV, cells were depolarized to between -70 and +80 mV in 10-mV steps, followed by repolarization to -40 mV. B, CHO cells transiently expressing both Kvl .5 and p50-GFP (solid line) and control cells transfected with Kvl .5 pGFP alone (dashed line). Dotted line denotes the zero current level. C, Peak current amplitudes at +80 mV from controls (filled symbols) and p50-overexpressing cells (open symbols) were normalized to cell capacitance (n=12; **P<0.0\). D, Confocal images (from a slice of a single Z axis) of HEK-293 cells stably expressing T7-tagged Kvl .5 (red) transfected with pGFP alone or p50-GFP (green). Scale bars=10 urn. E, HEK-293 cells were cotransfected with T-7 tagged Kvl .5 and either p50-GFP or pGFP and treated with externally applied Proteinase K (lanes 3 and 4) or buffer alone (lanes 1 and 2). The arrows indicate the Western blot position of surface Kvl .5 (digested form) and intracellular Kvl .5 (undigested form). The immunoblot at the bottom indicates that equal amounts of protein were loaded in each lane. 69 -80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) Figure 2.2 Kvl.5 is internalized via an endocytotic pathway. A , Kvl .5 colocalizes with the early endosome marker, EEA1. Confocal images (from a slice of a single Z axis) of HEK-293 cells stably expressing Kvl .5 immunostained for Kvl .5 (green) and EEA1 (red), with yellow indicating colocalization. Scale bar=10 urn B, Dynamin inhibition increases Kvl .5 currents. Cells incubated with 50 uM dynamin inhibitory peptide (DIP) or scrambled control peptide for 16 hours. Cells were depolarized from -70 to +80 mV in 10-mV steps and repolarized to -40 mV. Sample currents in control and a cell over-expressing DIP at +80 mV. C, Peak current amplitudes normalized to cell capacitance in control (filled symbols) and from DIP-applied cells (open symbols; **P<0.01). 70 Figure 2.3 Nocodazole pretreatment increases Kvl.5, but not transient currents HEK-293 cells and in rat atrial myocytes. (Legend on the following page) 3 o -10 J . . . . n , , , , -100-80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) Figure 2.3 Nocodazole pretreatment increases Kvl.5, but not transient currents in HEK-293 cells and in rat atrial myocytes. A , Kvl .5 current density-voltage relationships at various doses of nocodazole in HEK-293 cells over-expressing p50 or transfected with empty vector, as indicated (n=5). B, Peak current densities at +80 mV from A in nocodazole-treated cells transfected with p50 (open triangles) or empty vector. C through E, Currents from rat atrial myocytes treated with 35 uM nocodazole or solvent-(DMSO) alone. Cells were depolarized to +80 mV from -80 mV in 10-mV steps. D, Mean sustained current-voltage data ± nocodazole. **P<0.01, Student t test. E, Mean I t 0 peak current-voltage data ± nocodazole. To avoid contamination by sustained currents, the K v l . 5 -specific blocker D M M (100 nM) was included in the bath solution. 72 1 2 3 4 1 2 3 4 Distance (quarters) Distance (quarters) Figure 2.4 Nocodazole pretreatment increases Kvl.5 surface expression in rat atrial myocytes. A and B, Myocytes incubated for 6 hours without (control) or with 35 uM nocodazole, fixed and immunostained for Kvl .5 (green). Scale bar=10 um. C and D, Relative pixel intensity across cells divided into eighths and summed into quarter bins from edge to center, where 1 represents the total fluorescence intensity in the peripheral two-eighths of the cells and 4 represents the center-most quarter of the cells. **P<0.01, 1-way A N O V A . 73 Nocodazole 2 nA | +100 nM RSD1355 100 ms Figure 2.5 Nocodazole-induced increase in atrial myocyte current is caused by IRUR-A , Current traces from rat atrial myocytes (-80 to +80 mV) pretreated with 35 uM nocodazole for 6 h (above) and then exposed to 1 m M 4-AP (below). B, Concentration response curves for D M M on K v l - K v 4 subfamily channels. Numbers indicate IC50 values, n=3 to 5 for each point. C, Kvl .5 HEK-293 cell currents at +80 mV in control and 100 nM D M M . D, Sample current traces from rat atrial myocytes pretreated with 35 uM nocodazole for 6 hours (above), and the same cell then exposed to 100 nM D M M . 74 Figure 2.6 Kvl.5 coimmunoprecipitates with the dynein intermediate chain in HEK-293 cells and myocytes. A , Aliquots of cell lysates from HEK-293 cells stably expressing T7-tagged K v l .5 were mixed with either anti-T7 antibody or anti-dynein intermediate chain and precipitated with Protein A-sepharose. Immune complexes were resolved by SDS-PAGE. The immunoprecipitating antibody is indicated above each lane. Detecting antibody is indicated at right. B , As for A except that rat ventricular myocyte lysates were used, and Kvl .5 was immunoprecipitated using a C-terminal antibody. 75 A N-terminus C-terminus *PLPPLH T1 LRRST pTDLJ W T LRRST ETDU A N 2 0 9 Dynamitin (n=12) Control (n=5 Dy.iam.tm ( n ^ H T1 LRRST feTDU A N 9 1 PLPPLR T1 B A C 5 1 Dynamitin (n=7) 0.0 0.5 AN65-93 Kv1.5 1.0 1.5 LL Q. < C (A C 0) •a c 9> O 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 • pGFP (n=10) O p50 (n=8) -80 -60 -40 -20 0 20 4060 Voltage (mV) 80 100 2.0 2.5 68 79 WT: Q R D A D S G V R P L P P L P D P G V R P L P P L P E E L P R P R R P P P E D E P - I : Q R D A D S G V R P L A A L P D P G V R P L P P L P E E L P R P R R P P P E D E P - I I : Q R D A D S G V R P L P P L P D P G V R P L A A L P E E L P R P R R P P P E D E Figure 2.7 An N-terminal segment of Kvl.5 is required for sensitivity to p50 over-expression. A , HEK-293 cells stably expressing Kvl .5 constructs (wild type, AN209, AN91, and AC51, shown schematically to the left) were transfected with p50-GFP or empty vector (pGFP). Histograms compare peak current levels at +80 mV in the presence and absence of p50/dynamitin over-expression. B, Deletion of residues 65 to 93 eliminates Kvl .5 current increase in response to p50. Current densities for control and p50-overexpressing cells are plotted against voltage. A l l data analyzed by Student's t test were compared with empty vector controls (**P<0.01). C, Scheme of the proline-rich region of K v l . 5 . Residues 65 to 93 are underlined in the WT. The proline pairs mutagenized to alanine in the "P-I" (Kvl.5 P68/69A) and "P-II" (Kvl.5 P79/80A) mutants are bold. 76 B 2.5 LL Q. 2.0 1.5 & it) c 1.0 o •a > c 9> 0.5 C. i_ 3 0.0 o -0.5 • P-l Control (n=21) O P-l p50 (n=20) -80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) Q. 5 C O •a 3 o 2.0 1.5 1.0 0.5 0.0 -0.5 • P-ll Control (n=10) O P-ll p50 (n=10) -80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) 2.5 ft? 5- 2.0 < g 1.0 o T3 •g 0.5 £ 3 0.0 o -0.5 PI (n=8) PI+DIP(n=11) Pll (n=12) PII+DIP (n=6) -80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) 2.0 ft? c 10 0.5 0.0 0.5 • Pl+p50 (n=5) O PI+DIP+pSO (n=13) • Pll (n=5) -80 -60 -40 -20 0 20 40 60 80 100 Voltage (mV) Figure 2.8 The N-terminal Kvl.5 SH3-binding domain regulates Kvl.5 surface expression. A , The P68A/P69A (PI) mutation eliminates Kvl .5 current increase on p50 over-expression. B, PII mutation does not affect Kvl .5 increase in response to p50. C, The PI, but not the PII mutant, eliminates Kvl .5 increase in response to dynamin inhibitory peptide (DIP) incubation. HEK-293 cells transiently expressing PI were incubated with 50 uM DIP or scrambled control peptide for 6 hours. Data were compared with PH-expressing cells as another control. D, HEK-293 cells transiently expressing both the PI mutant and p50-GFP were incubated with 50 uM DIP or control peptide for 6 hours. Data are compared with PH-expressing cells as controls. I- V relations normalized to cell capacitance are shown in the graphs, and significant differences are shown as **P<0.01). 77 Pixel Figure 2.9 Line scan analyses of Kvl.5-EEA1 colocalization. In several cell confocal images, lines were drawn in Image J through endosomes and the intensities of Kvl .5 and EEA1 signals at each pixel were plotted on the same graph for comparison. A and B are examples of plot profiles from cells that showed a high degree of colocalization between Kvl .5 and EEA1. C illustrates a plot profile across several endosomes in a cell pretreated with Dynamin inhibitory peptide (DIP) before staining and examining for EE A 1 - K v l . 5 colocalization. To ensure a stringent test for co-localization, pixel lengths ranged from 0.1064 microns to 0.134 microns in the scans across endosomes in which Kvl .5-EEA1 were expected to be present, and were 0.242 microns in the cells where Kvl .5 and EEA1 were not obviously present together in endosomes. 78 No Ab No Ab Anti-GFP Anti-GFP Kv1.5 T7 Kv1.5-T7 + - + P50-GFP + + + + Figure 2.10 Co-immunoprecipitation of Kvl . 5 with p50. HEK-293 cells were cotransfected with T7-tagged Kvl .5 and either p50-GFP or empty vector (pGFP) as a control. The cells were harvested approximately 66 h post-transfection. Control and transfected cells were lysed and aliquots of the supernatants were employed for immunoprecipitation. 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Mcintosh (2005) A novel mechanism for the suppression of a voltage-gated potassium channel by glucose-dependent insulinotropic polypeptide: protein kinase A-dependent endocytosis. The Journal of Biological Chemistry. Vol . 280, Issue 31, 28692-28700. (*Both authors contributed equally to this work.) 83 3.1 INTRODUCTION Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 are the two major intestinal hormones (incretins) involved in the stimulation of insulin secretion during a meal (Pederson, 1994; Kieffer and Habener, 1999). Glucose-stimulated insulin secretion is mediated via closure of ATP-sensitive K + (K A TP) channels resulting in membrane depolarization, activation of voltage-dependent C a 2 + channels, and increases in intracellular Ca 2 + , followed by membrane repolarization by voltage-dependent K + (Kv) and Ca2+-sensitive K (K C a ) channels (Habener, 1993; Wahl et al, 1992; MacDonald and Wheeler, 2003). Incretins act by potentiating the events underlying membrane depolarization in addition to exerting direct effects on exocytosis. These events ultimately depend upon incretin interaction with its cognate seven-transmembrane G protein-coupled receptor and activation of proximal signal transduction pathways. In the case of GIP, these include stimulation of the adenylyl cyclase/cAMP/protein kinase A (PKA) module, and activation of phospholipase A2 (PLA 2 ) , protein kinase B (PKB), and mitogen-activated protein kinases (Ehses et al, 2001; Trumper et al, 2001; Ehses et al, 2002). There is little known regarding the effect of incretins on membrane repolarization of the P-cells (MacDonald and Wheeler, 2003). Voltage-gated potassium channels (Kv channels) belong to the six-transmembrane family of K + channels consisting of K v l to K v l l subfamilies and are involved in repolarization of excitable cells (Ottschytsch et al, 2002; Hille, 2001). They are of interest as potential therapeutic targets in diabetes, because blockade of K v channels would be expected to prolong the pancreatic P-cell action potential, sustain the opening of voltage-dependent Ca channels, and thereby potentiate glucose-induced insulin release 84 (MacDonald and Wheeler, 2003). Mammalian K v l . X s have been cloned and characterized in heterologous expression systems, and they generate at least two different types of outward potassium currents classified on the basis of their inactivation properties: delayed rectifier steady-state currents, which do not rapidly inactivate, and A-type transient currents, which inactivate rapidly (Hoshi et al, 1991; Kurata et al, 2004). Delayed rectifier current is produced by K v l . l , Kv l . 2 , Kv l . 3 , Kv l .5 , and Kvl .6 , and A-type current is produced by K v l . 4 (Brahmajothi et al, 1999; Jerng et al, 2004). Although both types of currents are present in pancreatic P-cells, most of the studies have concentrated on the delayed-rectifier K v channels. The current study focused on a potential role for GIP in the regulation of Kvl .4 , a prominent member of the K v l family and one of several channel proteins giving rise to potassium currents proposed to be involved in the regulation of insulin secretion. Using GIPR-expressing human embryonic kidney (HEK)-293 cells, we demonstrated that GIP decreases peak current amplitude of Kv l .4 via a process involving P K A activation. In parallel studies, Kv l .4 was shown to be expressed in human pancreatic P-cells, and GIP decreased A-type potassium current amplitude in these cells. Additionally, GIP was shown to induce phosphorylation and dynamin-dependent endocytosis of Kv l .4 and transient over-expression in INS-1 (832/13) p-cells of wild-type (WT) Kvl .4 , or a T601A mutant form resistant to P K A phosphorylation, resulted in reduced glucose-stimulated insulin secretion; WT over-expression potentiated GIP-induced insulin secretion, whereas the loss of PKA-dependent phosphorylation (T601A cells) ablated this effect. These results therefore provide compelling evidence for a role for GIP-induced down-regulation of Kvl .4 , via phosphorylation-dependent endocytosis of the channel protein, in the modulation of insulin secretion. 85 3.2 EXPERIMENTAL PROCEDURES 3.2.1 Generation of a GIPR-HEK-293 Cell Line HEK-293 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen), supplemented with 5% fetal bovine serum (Sigma-Aldrich), and penicillin-streptomycin (50 IU/ml, 50 ug/ml, Invitrogen) and transfected with GIPR/pcDNA3 plasmid, expressing rat pancreatic islet GIP receptor cDNA under control of the cytomegalovirus promoter. Transfections were performed using Lipofectamine 2000™ reagent (Invitrogen) for 4 h according to the manufacturer's instructions. Stably transfected cells were selected with G418 (Invitrogen), and GIPR-HEK-293 cell clones were analyzed by quantitative real-time reverse transcription-PCR to check GIPR mRNA expression levels and by Western blotting to confirm GIPR protein expression, respectively. 3.2.2 cDNA Constructions of Kvl.4 Plasmids and Transient Transfections in GIPR-HEK-293 Cells Kvl .4 cDNA was cloned into the pEGFP-N2 vector (Clontech, Palo Alto, CA) and various constructs, as detailed under "Results," were prepared by PCR with Hindlll and EcoRI insertions for directed cloning. Site-directed mutant constructs were prepared using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). A l l transfection plasmids were prepared using the Plasmid Midi kit (Qiagen, Valencia, CA). GIPR-HEK-293 cells were plated at a density of 2 x 105 cells/glass coverslip in 35-mm dishes. On the following day, transfection was performed with 1.5 of the indicated Kv l .4 plasmids using Lipofectamine 2000™ transfection reagent (Invitrogen), according to the manufacturer's instructions. 86 3.2.3 Enzyme Activity Assay of PKA P K A activity was measured using a P K A kinase activity assay kit (Stressgen, Mississauga, Ontario) according to the manufacturer's protocol. The enzyme activity was normalized to protein concentration and shown as the relative activity to control. 3.2.4 Kvl.4 protein Purification and in vitro Phosphorylation K v l . 4 cDNA was prepared by PCR and subcloned into the pGex4T3 vector (Amersham Biosciences). Glutathione S-transferase (GST)-Kvl.4 fusion protein was purified from BL21(DE3) Escherichia coli expressing pGex4T3/Kvl.4. GST fusion proteins were induced by treatment with 1 m M isopropyl-P-D-thiogalactopyranoside (Sigma) for 4 h, and the bacteria were harvested by centrifugation, resuspended in PBST-100, pH 7.4 (phosphate-buffered saline, 1% Triton X-100, 1 m M EDTA) and sonicated (Branson Ultrasonic Corp, Brandury, CT) to release the proteins. GST-Kvl .4 fusion protein was affinity-purified using a glutathione-Sepharose 4B (Amersham Biosciences) column, and bound protein was eluted with 100 mM NaCl solution containing 25 m M reduced glutathione (Sigma). After affinity purification, Kv l .4 proteins were released from GST fusion protein through thrombin digestion. In vitro phosphorylation reactions were performed at 30 °C in a volume of 40 ul of MES buffer (50 m M MES, pH 6.9, 10 mM M g C l 2 , 0.5 m M EDTA, 1 m M dithiothreitol) and initiated with the addition of 5 uCi of [y-3 2 P]ATP and unlabeled ATP to a final concentration of 100 uM. For determination of the time course of phosphorylation of target protein, 50 Lig of Kv l .4 protein was incubated with 5 |ag/ml recombinant P K A catalytic subunit (active P K A , Sigma) or GIP-treated GIPR-HEK-293 cellular extracts for 1 min to 2 h. Protein phosphorylation was determined 87 by terminating the reactions with 2 x SDS sample buffer, resolving the samples on 12.5 % SDS-PAGE gels, and drying the gels for autoradiography. 3.2.5 Western Blot Analysis Proteins (25 ug of protein/well) from each sample were separated on a 12.5 % SDS-PAGE gel and transferred onto nitrocellulose (Bio-Rad) membranes. Probing of the membranes was performed with antibodies against Kv l .4 and P-tubulin. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences) using horseradish peroxidase-conjugated IgG secondary antibodies. 3.2.6 Islet Isolation and Cell Culture Human islets were isolated from the pancreas of adult organ donors using collagenase duct perfusion, gentle dissociation, and density gradient purification at the Ike Barber Islet Transplantation Laboratory (Vancouver General Hospital, Vancouver, Canada) (Warnock et al., 2005). The Research Ethics Board of the University of British Columbia provided ethics approval. Islets were dispersed to single cells and plated on laminin-coated glass coverslips in 35-mm dishes in C M R L Medium-1066 (Mediatech, Inc.) supplemented with 10 % fetal bovine serum and penicillin-streptomycin (50 IU/ml-50 ug/ml, Invitrogen). 3.2.7 Electrophysiological Studies To record ionic current, we used a superfusion solution containing the following (in mM): NaCl, 135; KC1, 5; M g C l 2 , 1; sodium acetate, 2.8; HEPES, 10; CaCl 2 , 1; adjusted to pH 7.4 using NaOH. The patch pipettes were filled with the pipette solution containing 88 (in mM): KC1, 130; EGTA, 5; M g C l 2 , 1; HEPES, 10; Na 2 ATP, 4; GTP, 0.1; adjusted to pH 7.2 with K O H . A l l chemicals were from Sigma. Whole cell current recording and data analysis were done using an Axopatch 200B amplifier and pClamp 8 software (Axon Instruments, Foster City, CA), and pipettes with a resistance of 1-3 M Q were used. HEK-293 cells were depolarized to +60 mV for 900 ms from a holding potential of—100 mV. A l l whole cell recordings were performed at room temperature (20-23 °C). 3.2.8 Proteinase K Digestion Experiments For proteinase K digestion (Manganas and Trimmer, 2001; Campomanes et al, 2002), GIPR-HEK-293 cells were transfected with a cDNA coding for Kv l .4 bearing a green fluorescent protein (GFP) tag at the C terminus (Kvl.4-EGFP). Cells incubated with 100 nM GIP for indicated periods of time were washed three times with ice-cold PBS and incubated with 10 m M HEPES, 150. m M NaCl, and 2 m M CaCl 2 (pH 7.4) with 200 ug/ml proteinase K at 37 °C for 30 min. The cells were then harvested, and proteinase K digestion was quenched by adding ice-cold PBS containing 6 m M phenylmethylsulfonyl fluoride and 25 m M EDTA. This was followed by SDS-PAGE and immunoblotting, and probing of the membranes was performed with antibodies against GFP and P-tubulin. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences) using horseradish peroxidase-conjugated IgG secondary antibodies. 3.2.9 Confocal Microscopy GIPR-HEK-293 cells were transfected with WT or mutant forms of Kvl .4-EGFP plasmids and treated for 10 min with GIP (100 nM). Cells were then fixed and 89 immunostained successively with a-GFP antibody and a secondary Alexa fluor® 488 dye-conjugated anti-rabbit antibody. Cell nuclei were counterstained with 4',6-diamino-2-phenylindole, and transfected cells were imaged using a Zeiss laser scanning confocal microscope (Axioskop2). 3.2.10 Islet Embedding in Agar and Confocal Microscopy Human islets were fixed in 4 % paraformaldehyde in PBS for 30 min at room temperature. After centrifugation, islets were resuspended in PBS, mixed with an equal volume of 2 % agar solution, and added to a mold. Agarose-embedded sections were processed for a double immunostaining for Kv l .4 and insulin. The sections were incubated with K v l . 4 and insulin antibodies and visualized with Alexa fluor® 488-conjugated anti-mouse secondary antibody and Texas Red® dye-conjugated anti-rabbit antibody. Cell nuclei were counterstained with 4',6-diamino-2-phenylindole and imaged using a Zeiss laser scanning confocal microscope (Axioskop2). A l l imaging data were analyzed using the Northern Eclipse program (version 6). 3.2.11 Insulin Secretion P-INS-1 cells (clone 832/13) were kindly provided by Dr. C. B. Newgard (Duke University Medical Center, Durham, NC). Cells were cultured in 11 m M glucose RPMI 1640 (Sigma) supplemented with 2 m M glutamine, 50 u M P-mercaptoethanol, 10 m M HEPES, 1 m M sodium pyruvate, 10 % fetal bovine serum, 100 unit/ml penicillin G-sodium, and 100 u.g/ml streptomycin sulfate. INS-1 cells were plated at a density of 1 x 106 cells/well. On the following day, transfection was performed with 2 \ig of control vector, 90 pEGFP-N2, mutant T601A Kvl .4-EGFP, or WT Kvl .4-EGFP. Transfections were performed using Lipofectamine 2000™ reagent (Invitrogen) for 4 h according to the manufacturer's instructions. On the following day, cells were treated with GIP and incubated for 2 h at 37 °C in Krebs-Ringer buffer with HEPES (KRBH) buffer (115 m M NaCl, 4.7 m M KC1, 1.2 m M K H 2 P 0 4 , 10 m M NaHC0 3 , 1.28 m M CaCl 2 , 1.2 m M M g S 0 4 , 0.1 % bovine serum albumin, and 10 m M HEPES (pH 7.4) containing low (2.5 mM) or high glucose (25 mM). Insulin release into the medium was determined using a radioimmunoassay kit (Linco Research Inc., St. Charles, MO). 3.2.12 Statistical Analysis Data are expressed as mean ± S.E.M. with the number of individual experiments presented in the figure legend. Data were analyzed using the non-linear regression analysis program PRISM (GraphPad, San Diego, CA), and significance was tested using Student's t test or analysis of variance with a Student-Newman-Keuls post hoc test (p < 0.05) as indicated in the figure legends. 91 3.3 RESULTS 3.3.1 GIP Decreases Peak Current Amplitude of Kvl.4 in GIPR-HEK-293 Cells GIPR-expressing HEK-293 cells (GIPR-HEK-293) were used as an in vitro model system to characterize the modulation of Kv l .4 currents by GIP treatment. HEK-293 cells were stably transfected with a rat islet GIPR cDNA construct under control of the cytomegalovirus promoter. The resulting GIPR-HEK-293 cell clones transiently transfected with Kvl .4-EGFP cDNA (GIPR-HEK-293-Kv) demonstrated voltage-activated, rapidly inactivating outward currents (Figure 3.1 A) with properties similar to Kv l .4 currents previously reported in insulin-secreting cells (MacDonald and Wheeler, 2003). Administration of GIP-(l-42) to GIPR-HEK-293-Kv cells resulted in a decrease in peak current amplitude that was initiated within 1 min and decreased by 44.0 ± 5.7 % at 5 min (Figure 3.1 A and B). The decrease in peak current amplitude was not observed in GIP-(1-42)-treated HEK-293 cells, which do not express the GIPR (Control-I), or in GIPR-HEK-293-Kv cells treated with the non-insulinotropic truncated form of GIP (GIP-(19-30); Control-II). The effect of GIP on the decrease of peak current amplitude was concentration-dependent, with 1 nM GIP producing a significant reduction (Figure 3.1C). Other macroscopic properties of Kv l .4 were next studied to determine whether GIP affects the gating properties of Kvl .4 . As shown in Figure 3.1 (D-F), GIP did not alter the properties of activation (Vi / 2 control = -27.6 ± 2.5 mV versus V I / 2 G I P = —25.1 ± 1 . 1 mV), inactivation (Vi / 2 control = -54.5 ± 2.7 mV versus V i / 2 G I P = -52.9 ± 1 . 7 mV) or recovery from inactivation (ocontroi = 4.6 ± 0.6 s versus O G I P = 4.9 ± 0.3 s), indicating that the effect of GIP is not mediated by changes in the macroscopic gating properties of Kvl .4 . 92 3.3.2 GIP Activates PKA in GIPR-HEK-293-Kv Cells and GIP-stimulated PKA Activation Is Involved in the Decrease of Kvl.4 Peak Current Amplitude Mechanisms involved in the decrease of Kv l .4 peak current amplitude by GIP treatment were next studied. The possibility that changes in Kv l .4 peak current amplitude were linked to activation of adenylyl cyclase and P K A was first examined, because this is a well established GIP signalling module involved in the regulation of insulin secretion. Treatment of GIPR-HEK-293-Kv cells with 100 nM GIP resulted in increased P K A activity (Figure 3.2A), apparent 10 min after initiation of GIP treatment, thereafter quickly decreasing with time. GIP stimulation was concentration-dependent, with an EC50 value of 2.10 ± 0.22 nM (Figure 3.2B). To establish that GIP-induced activation of P K A was associated with decreases in Kvl .4 peak current amplitude, H-89 and (-ftp)-cAMP, selective inhibitors of P K A , were applied during electrophysiological recordings. The inhibitor H-89 significantly blocked GIP-stimulated P K A activation (Figure 3.2A and B) and the decreases of Kv l .4 peak current amplitude (Figure 3.2C). Similarly, the cAMP antagonist (i? p)-cAMP eliminated the effect of GIP on Kv l .4 peak current amplitude (Figure 3.2C). Taken together, these results demonstrate that GIP-stimulated P K A activation is involved in the regulation of Kvl .4 . 3.3.3 GIP-stimulated PKA Activation Resulted in the Phosphorylation of Kvl.4 and Decreases in Kvl.4 Peak Current Amplitude To determine whether P K A could phosphorylate Kvl .4 , purified recombinant Kv l .4 protein was incubated with the catalytic subunit of P K A and [ 3 2P]ATP in vitro. PKA-induced phosphorylation of Kvl .4 protein was apparent 1 min after the initiation of 93 P K A treatment, and it was sustained for 2 h (Figure 3.3A). Similar rapid onset and sustained phosphorylation of Kvl .4 was observed with GIP-treated GIPR-HEK-293-Kv cellular extracts (Figure 3.3B), indicating the involvement of GIP-stimulated P K A activation for the phosphorylation of Kvl .4 . To identify the functional region of Kvl .4 involved in GIP-mediated phosphorylation, electrophysiological responses to GIP were recorded in Kv l .4 mutants with deletions at the N or C termini. As shown in Figure 3.3C, A N 147 Kvl .4 , with 147 amino acids deleted from the N terminus, showed delayed inactivation compared with WT Kvl .4 . The delayed inactivation has been previously shown to result from deletion of an N-terminal inactivating "ball" domain, which acts as an intracellular tethered blocker of the open channel (Kurata et al, 2004). GIP was found to retain the ability to reduce peak current amplitude expressed by A N 147 Kvl .4 . On the other hand, the mutant AC55 Kvl .4 , with 55 amino acids deleted from C terminus, demonstrated no responsiveness to GIP (Figure 2.3C). These results therefore indicate that the C terminus in Kvl .4 is responsible for GIP-mediated effects. Inspection of the amino acid composition of the C terminus of Kv l .4 revealed several motifs that are potentially involved in regulating its subcellular localization and interaction with other proteins. The V X X S L motif is critical for glycosylation and surface membrane localization of the channel (Li et al, 2000), and the sequence ETDV is the binding site for PDZ domain proteins, such as PSD-95 and SAP-97 (Tiffany et al, 2000; Wong and Schlichter, 2004). Additionally, we found a potential P K A phosphorylation site, SSTSSS, in the C terminus of Kvl .4 . To determine whether phosphorylation of Kv l .4 is linked to GIP-mediated decreases in current amplitude, studies were performed on the effect of substituting alanine for each of the phosphorylatable serine and threonine residues: S599A, S600A, T601A, S602A, S603A, 94 and S604A. When in vitro phosphorylation of the purified alanine-substitution mutants was examined, all proteins, except T601A, were phosphorylated by P K A (Figure 3.3D). Electrophysiological recordings showed that GIP was able to reduce peak current amplitude with GIPR-HEK-293 cells expressing S599A, S602A, S603A, and S604A, but not T601A (Figure 3.3E). The mutant S600A was not functionally expressed in the plasma membrane (data not shown). Taken together, these results indicate that phosphorylation of Kvl .4 by GIP-stimulated P K A activation is linked to the decrease in peak current amplitude and phosphorylation of Thr-601 in the C terminus is a critical step in this process. 3.3.4 GIP-induced Decreases in Kvl.4 Peak Current Amplitude Result from Channel Endocytosis Although phosphorylation of ion channels has been reported in several systems, in most cases it is associated with changes in inactivation rate and recovery from inactivation, rather than changes in peak current amplitude. Membrane protein phosphorylation directly affects interactions with intracellular proteins involved in many cellular pathways, and it was hypothesized that GIP-mediated decreases in Kvl .4 peak current amplitude were mediated by phosphorylation-associated endocytosis. To examine this proposal, the cellular localization of Kv l .4 during treatment with GIP was studied using proteinase K digestion. The GFP tag on Kvl .4 is located intracellularly at the C terminus. Proteinase K is able to randomly digest extracellular regions of Kv l .4 channels in the plasma membrane, thus producing a digested form, whereas intracellular channel is protected. As shown in Figure 3.4A, surface membrane expression levels of Kv l .4 (S) decreased with GIP treatment, and this was evident at 5 min following initiation of treatment. Confocal microscopy also 95 revealed that Kv l .4 channels present in the plasma membrane were greatly reduced by treatment with GIP (Figure 3.4B). To determine whether GIP-mediated decreases in Kvl .4 peak current amplitude were mediated by dynamin-dependent endocytosis, electrophysiological recordings were performed on GIPR-HEK-293-Kv cells treated with GIP (100 nM) in the presence or absence of myristoylated dynamin inhibitory peptide (mry-DIP: myr-Gln-Val-Pro-Ser-Arg-Pro-Asn-Arg-Ala-Pro-NH2) (Nong et al, 2003). Dynamin is a large GTPase implicated in the budding and scission of nascent vesicles from parent membranes (Praefcke and McMahon, 2004). It has been extensively studied in the context of clathrin-coated vesicle budding from the plasma membrane, but it is also involved in the budding of clathrin-coated vesicles from other compartments and budding of caveoli, phagocytosis, and vesicle cycling at synapses (De Camilli et al, 1995; Oh et al, 1998). As shown in Figure 3.4C, mry-DIP completely abolished the effect of GIP on Kvl .4 . These results therefore strongly suggest that GIP induces the endocytosis of Kv l .4 and that these processes are responsible for the GIP-mediated decrease in Kv l .4 peak current amplitude. 3.3.5 Phosphorylation Is Involved in GIP-induced Retrograde Trafficking of Kvl.4 Next, we investigated the potential relationships between phosphorylation and endocytosis of Kv l .4 in response to treatment with GIP. The mutant channels S599A, T601A, S602A, S603A, and S604A were transiently expressed in GIPR-HEK-293 cells, and confocal microscopy was performed on fixed cells following treatment with GIP. Endocytosis was observed with S599A-, S602A-, S603A-, and S604A-transfected GIPR-HEK-293 cells with GIP treatment, but not with T601A-transfected cells (Figure 3.5). 96 These results correlate well with the electrophysiological recordings, and the combined data demonstrate that phosphorylation and endocytosis are consecutive processes responsible for the effects of GIP on K v l . 4 channel distribution. 3.3.6 Kv Channel Expression in Human Islets Kv channels have been shown to play an important role in the regulation of glucose-dependent insulin secretion in rodent islets (MacDonald and Wheeler, 2003). As shown in Figure 3.6, K v l . 4 protein is also expressed in human islets (A), mainly restricted to insulin-expressing pancreatic P-cells (B). Electrophysiological recordings from human islet cells revealed a typical A-type outward potassium current, and 100 nM GIP treatment resulted in a decrease in A-type peak current amplitude with a similar pattern to that observed in Kv l .4 currents in GIPR-HEK-293-Kv (Figure 3.6, C and D). The effect of GIP on peak current amplitude was reversible by washing-out, and by 7 min following wash-out responses had almost returned to levels achieved prior to treatment with GIP. To determine whether A-type current in human islets is mediated by Kvl .4 , 4-aminopyridine (4-AP; 1 mM), a conventional K v channel blocker, was applied (Hille, 2001). As shown in Figure 3.6E, 4-AP treatment resulted in -50 % reduction in A-type potassium current. A-type potassium channels of different types exhibit variable sensitivity to 4-AP. For example, with cloned Kvl .4 channels 73 % of peak current was found to be blocked by 1 mM 4-AP, whereas, in contrast, Kv4.2 was 7-fold less sensitive to 4-AP than Kv l .4 and Kv3.4 exhibited much greater sensitivity to 4-AP (micromolar range) (Judge et al, 1999; Roberds et al, 1993; Schroter et al, 1991). The sensitivity to 4-AP exhibited by the A-type current in human islets suggests that it is mediated by Kvl .4 , and that GIP is able to confer its 97 effect on peak current amplitude in a similar manner to that observed with GIPR-HEK-293-K v cells. 3.3.7 Phosphorylation-dependent Internalization of Kvl.4 Participates in the Effect of GIP on Insulin Secretion To establish that phosphorylation-dependent internalization of K v l .4 contributes to GIP stimulation of insulin secretion, the T601A mutant form, which is resistant to P K A phosphorylation, or WT Kvl .4 channel, were transiently expressed in insulin-secreting P-INS-1 cells and channel internalization determined by proteinase K treatment. The P-INS-1 cell line (clone 832/13) was chosen because it lacks functional A-type current (data not shown), thereby excluding the involvement of endogenous K v l . 4 current in responses to GIP. As shown in Figure 3.7A, GIP treatment did not decrease surface membrane expression of mutant T601A under either low (2.5 mM) or high (25 mM) glucose conditions, whereas WT Kvl .4 was internalized by GIP treatment in the presence of high, but not low, glucose. Expression of either WT or T601A mutant Kv l .4 channels reduced glucose-stimulated insulin secretion, compared with vector-transfected cells (Figure 3.7B), presumably because of a prolonged repolarization phase of the P-INS-1 cell action potential. As expected, GIP treatment did not significantly increase insulin secretion in the presence of low glucose in pEGFP-N2 vector-, T601A-, or WT Kvl.4-transfected groups. However, under high glucose conditions, GIP treatment resulted in increased insulin secretion in all groups. GIP responses of P-INS-1 cells overexpressing WT Kvl .4 channels were potentiated compared with pEGFP-N2-transfected cells, whereas the loss of P K A -dependent phosphorylation (T601A cells) ablated this effect. Taken together, these results 98 strongly suggest that phosphorylation-dependent internalization of Kv l .4 is an important component of GIP-potentiated insulin secretion. 99 3.4 DISCUSSION The ability of GIP to directly enhance glucose-stimulated insulin secretion in pancreatic P-cells has been attributed to GIPR activation leading to enhanced depolarization and increases in the intracellular calcium concentration as well as direct effects on insulin exocytosis (Habener, 1993; Wahl et al, 1992; Mik i et al, 2005). Ion channels are the primary determinants of membrane excitability in most cells, and they are regulated to maintain membrane potentials within specific limits. Frequently this occurs through modulation of the functional responses of the ion channel to extracellular stimuli. In pancreatic P-cells, insulin secretion is modulated by the activity of several different ionic currents. Among these are the three main potassium currents: inward rectifier potassium currents, including the ATP-sensitive (K A TP) channel and others, calcium-activated (K C a ) , and voltage-gated (Kv) currents (Seino, 1999; Ferrer et al, 1995; Nichols and Lopatin, 1997; MacDonald et al, 2002). The molecular mechanisms involved in the regulation of K A T P and K c a channels in pancreatic P-cells have been extensively studied, but considerably less is known about the Kv channels. Because these channels are considered to be potential therapeutic targets for type 2 diabetes, it is important to establish their physiological role and mechanisms involved in regulating their activity. The current study was therefore initiated with the objective of identifying potential interactions between the incretin hormone GIP and Kv channels. GIP transduces its biological effects on pancreatic P-cells by interacting with a seven-transmembrane receptor, GIPR, which is a member of the class II G protein-coupled receptor subfamily. The best characterized pathway by which GIP acts on insulin secretion in P-cells involves activation of the adenylyl cyclase/cAMP/PKA pathway. Using HEK-293 cells co-expressing the GIPR and Kv l .4 (GIPR-HEK-293-Kv 100 cells), we have now shown that GIP reduces peak current amplitude of Kv l .4 channels via a pathway inhibited by the selective inhibitors of P K A , H-89, and the cAMP antagonist, (i? p)-cAMP. In parallel experiments, it was shown that recombinant P K A catalytic subunits (Figure 3.3A) or cell extracts from GIP-stimulated GIPR-HEK-293-Kv cells (Figure 3.3B) increased phosphorylation of Kvl .4 , and active P K A phosphorylated Thr-601 in the C terminus of Kvl .4 (Figure 3.3D), thus substantiating the involvement of P K A signalling in GIP-induced effects on Kv l .4 current. This was confirmed by experiments showing that mutant T601A Kvl .4 channels could not be phosphorylated by P K A , and peak currents in this mutant were resistant to GIP (Figure 3.3D and E). The macroscopic current in cells is regulated by the following two processes: 1) biophysical and biochemical modulation of surface membrane ion channel activity and 2) biosynthesis and trafficking of channel protein (Delisle et al, 2004). Direct phosphorylation of channel proteins by serine/threonine and tyrosine kinases has been established as a mechanism by which ion channels are regulated. The delayed rectifier potassium channel Kv l .2 was the first example of a voltage-gated ion channel shown to be regulated by Ser/Thr phosphorylation and a range of voltage- and ligand-gated channels have been found to be regulated by tyrosine kinases, including JV-methyl-D-aspartic acid receptors, voltage-dependent C a 2 + channels, and a variety of potassium channels (Huang et al, 1993; Nesti et al, 2004; Davis et al, 2001; Strock and Diverse-Pierluissi, 2004). Previous studies have addressed the effects of Ser/Thr phosphorylation of the N-terminal domain of Kv l .4 on physiological responses. Calcium/calmodulin-dependent protein kinase has been shown to slow the inactivation of Kv l .4 currents by phosphorylating Ser-123 in the cytoplasmic N terminus (Roeper et al, 1997). Treatment of Kvl.4-expressing Xenopus 101 oocytes with phorbol 12-myristate 13-acetate, a protein kinase C activator, has been shown to lead to a biphasic change in the magnitude of peak current: an initial increase followed by a later reduction (Murray et al., 1994). Although in most cases the precise mechanisms underlying the effects of Ser/Thr phosphorylation on channel function are unclear, the most commonly suggested mechanism is that phosphorylation-induced changes in channel structure alter its biophysical properties (Levitan, 1994). In the present study, GIP reduced Kvl .4 peak current amplitude, without affecting macroscopic gating properties of Kvl .4 (Figure 3.1A-F), and threonine phosphorylation of the C terminus by GIP-stimulated P K A activation also resulted in a decrease in Kv l .4 peak current amplitude (Figure 3.3). These results imply that different mechanisms are involved, compared with previously reported phosphorylation of K v channels. The trafficking of ion channels is one of the processes involved in the modulation of plasma membrane macroscopic currents (Delisle et al., 2004). The regulation of expression of Kv channels in the plasma membrane begins at the level of gene transcription and biosynthesis of the channel protein (Deutsch, 2003), with further control provided during insertion of the channel into the cell surface and by its regulated retrieval and degradation. Endocytosis was initially defined as a process by which substances are taken into the cell, but it is now recognized as an essential mechanism for the regulation of a variety of membrane proteins. Endocytosis initiates the internalization of membrane-bound proteins undergoing recycling or retrograde trafficking to be degraded. Endocytosis initiated by phosphorylation of Kv channels results in decreased ionic current density (Nesti et al., 2004). In the present study, it was demonstrated that direct phosphorylation of Kv l .4 by GIP-stimulated P K A activation is involved in endocytosis of the channel protein (Figure 102 3.3 and 3.5). Retrograde trafficking of Kvl .4 resulting in decreased peak current amplitude was observed following treatment with GIP (Figure 3.4, A and B), and dynamin-dependent endocytosis was involved in this process (Figure 3.4C). In contrast, the nonphosphorylatable mutant T601A Kvl .4 was incapable of undergoing endocytosis, demonstrating the critical role played by phosphorylation in GIP-induced endocytosis of Kv l .4 (Figure 3.5). The underlying molecular mechanism by which PKA-dependent phosphorylation is linked to endocytosis of Kvl .4 is not clear at the present time. Post-translational modifications of channel proteins by signalling molecules and resulting structural changes of channel proteins may affect protein-protein interactions between channel proteins and proteins involved in the endocytotic pathway. The Kv l .4 channel was also demonstrated to be present in human pancreatic P-cells, and GIP treatment decreased A-type ionic current amplitude (Figure 3.6). There have been controversial reports regarding the expression patterns of K v l family channels in human islets. It has been reported that K v l . l , Kv l . 2 , and Kv l .4 are not found by reverse transcription-PCR in human islets, whereas Kvl .5 and Kv l .6 are present (MacDonald and Wheeler, 2003). On the other hand, Yan et al. reported that only Kvl .3 and Kvl .6 were detected by reverse transcription-PCR, with a very weak indication for Kv l . 7 (Yan et al, 2004). In the current study, we have shown that Kv l .4 protein is expressed in human pancreatic p-cells (Figure 3.6B). These discrepant results might arise from phenotypic differences in channel composition between races or individuals. What is the likely effect of A-type potassium current down-regulation on pancreatic P-cell function? A-type current has an important role in the early repolarization phase of action potentials (Hille, 2001; Sah and McLachlan, 1992). 4-AP potentiates insulin 103 secretion from rat islets and insulinoma cells stimulated by sulfonylureas, even in the absence of glucose (MacDonald and Wheeler, 2003). Therefore, it is reasonable to predict that the down-regulation of A-type current mediated by GIP-stimulated K v l . 4 endocytosis in pancreatic P-cells would enhance the duration and amplitude of action potentials, resulting in the prolongation of insulin secretion. This is supported by the demonstration that transient over-expression of WT Kvl .4 channels in INS-1 (832/13) p-cells resulted in potentiated insulin secretion in response to GIP, and that loss of a P K A phosphorylation site ablated this effect (Figure 3.7B). In summary, GIP-induced phosphorylation of K v l . 4 channel protein, resulting in endocytosis and decreases of ionic peak current amplitude, is likely to be an important pathway by which GIP acts as an insulinotropic hormone. This appears to be the first example of a physiological pathway directly linking hormone signalling to endocytosis of K v channels. The combined effects of GIP and glucagon-like peptide-1 account for ~50 % of the total insulin response to a meal, and it is therefore clear that a deeper understanding of its mechanism of action is an important issue, with strong implications for the development of therapeutic agents for type 2 diabetes. 104 3.5 ACKNOWLEDGEMENTS This work was supported by the Canadian Institutes of Health Research (CIHR) (Grant 590007), the Canadian Diabetes Association, and the Canadian Foundation for Innovation (CFI) (to C. H. S. M.); by the CIHR and the Michael Smith Foundation (to D. F.); by the CFI P. A . Woodward Foundation and the Ike Barber Diabetes Research Endowment (to G. W.); and by a University Graduate Fellowship (to W. S. C ) . We thank Dr. C. B. Newgard (Duke University Medical Center, Durham, NC) for kindly providing us with INS-1 cells (clone 832/13). 105 B1-4 1.2 1.0 _ o 0.8 ~ 0.6 0.4 0.2 0.0 OnM 1 nM 10 nM 100 nM GIP concentration * * * * * Control I Control II GIP (100 nM) 0 1 2 3 4 5 6 7 Time (min) Figure 3.1 GIP decreases peak ionic current amplitude of Kvl.4 in GIPR-HEK-293 cells. (Legend on the following page) 106 • Control O GIP 1.2 1.0 0.8 J 0.6 0.4 0.2 0.0 • Control O GIP -100-80 -60 -40 -20 0 20 40 60 80 10 -150 Voltage (mV) -100 -50 0 50 Voltage (mV) 100 1.2 1.0 I 0-8 •S 0.6 u £ 0.2 0.0 -a—2- Q • Contol O GIP 0 5 10 15 20 25 30 35 Time (s) Figure 3.1 GIP decreases peak ionic current amplitude of Kvl.4 in GIPR-HEK-293 cells. A, examples of KV1.4 currents responses to GIP measured in GIPR-HEK-293-Kv cells. Scale bar indicates 10 nA. B, time course of decreases in K v l . 4 peak current amplitude in response to GIP. GIPR-expressing HEK-293 cells were transfected with KV1.4-EGFP plasmid and treated for the indicated periods of time with 100 n M of GIP. Currents were recorded in GIP-treated GIPR-HEK-293-Kv cells (GIF), GIP-treated HEK-293 cells not expressing GIPR (Control-!), and GIPR-HEK-293-Kv cells treated with an inactive form of GIP-(19-30)-treated (Control-II). C, concentration dependence of decreases in Kv l .4 peak current amplitude in response to GIP. Cells were prepared as described above and incubated with different concentrations of GIP (in nM: 0, 1, 10, and 100). Currents were recorded at 5 min after GIP treatment. Peak current amplitudes at +60 mV were normalized to cell capacitance and represented as percentage to 0 nM GIP group (maximum=l). D-F, effect of GIP on macroscopic KV1.4 current kinetics. Activation properties (D), inactivat ion properties (E), and recovery time from inactivation (F) of KV1.4 were determined during treatment with GIP. A l l data represent the mean ± S.E.M. from three to five independent experiments, and significance was tested using Student's t test or analysis of variance with a Newman-Keuls post hoc test, where the asterisk represents p <0.05 versus basal. 107 A B WT H-89 Rp-cAMP Figure 3.2 GIP activates PKA in GIPR-HEK-293-Kv cells and PKA activation is involved in the decrease in Kvl.4 ionic currents. A , time course of P K A activation responses to GIP and effect of H-89. GIPR-HEK-293-Kv cells were stimulated for the indicated periods of time with 100 nM GIP in the presence or absence of H-89. H-89 (10 uM) was added to cells during a 30-min preincubation as well as during GIP stimulation. P K A activity assays were performed as described under "Experimental Procedures." B, concentration-response effect of GIP on P K A activation and the effect of H-89. GIPR-HEK-293-Kv cells were stimulated for 10 min with the indicated concentrations of GIP in the presence or absence of H-89. H-89 (10 uM) was added to cells during 30-min preincubation as well as during GIP stimulation. C, electrophysiological recordings. GIPR-HEK-293-Kv cells were incubated in the presence or absence of P K A inhibitor, H-89, or cAMP antagonist, (Rp)-cAMP. Current was recorded at zero time (Control) and 5 min after initiating GIP treatment (GIP). Peak current amplitudes at +60 mV were normalized to cell capacitance and represented as percentage ratio to Control (maximum=l). A l l data represent the mean ± S.E.M. from four to six independent experiments, and significance was tested using Student's t test or analysis of variance with a Newman-Keuls post hoc test, where the asterisk represents p <0.05 versus basal. 108 Time(mins): 0 1 2 5 10 30 60 120 B Time(mins): 0 1 2 5 10 30 60 120 l/l, Figure 3.3 GIP-stimulated PKA activation resulted in the phosphorylation of Kvl.4 and decreases in ionic current. (Legend on the following page) 109 D S599A S600A T601A S602A S603A S604A — WT : S S T S S S S599A: ASTSSS T601A: SSASSS S602A: SSTASS S603A: SSTSAS S604A: SSTSSA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 l/l0 Figure 3.3 GIP-stimulated PKA activation resulted in the phosphorylation of Kvl.4 and decreases in ionic current. A and B, in vitro phosphorylation of K v l . 4 by P K A . 50 ug of purified Kv l .4 protein was incubated for the indicated periods of time at 30 °C in kinase buffer containing 5 uCi of [y- 3 2P]ATP in the presence of recombinant protein kinase A catalytic subunit (A) and GIP-stimulated GIPR-HEK-293 cellular extracts (B). The reactions were terminated by boiling in SDS sample buffer, and proteins were separated by SDS-PAGE. The gels were Coomassie-stained to visualize protein, dried, and exposed to x-ray film to visualize incorporated 3 2 P . C, electrophysiological recordings. Schematic diagram of the Kvl .4 serial deletion constructs and their functional elements are presented. Peak current amplitudes are shown at the far right, for comparison between WT and N -terminally deleted (AN147) or C-terminally deleted mutants (AC55) of Kvl .4 . Currents were recorded at zero time (black bar) and 5 min after GIP treatment (white bar), and peak current amplitudes at +60 mV were normalized to cell capacitance and represented as percentage (maximum=l). In the right panel, examples of current traces of WT, A N 147, and AC55 of Kv l .4 ± GIP were shown, and the scale bar indicates 10 nA. D, in vitro phosphorylation of mutant KV1.4 by PKA. 50 ug of purified mutant Kv l .4 protein was incubated for 1 h at 30 °C in kinase buffer containing 5 uCi of [y- 3 2P]ATP in the presence of recombinant protein kinase A catalytic subunit. Reactions were performed as described in A and B. E, electrophysiological recordings. Peak current amplitudes are shown at the far right, for comparison between WT and the site-directed mutants (S599A, T601A, S602A, S603A, and S604A) of Kvl .4 . Currents were recorded at zero time (black bar) and 5 min after GIP-treatment (white bar) and peak current amplitudes at +60 mV were normalized to cell capacitance and represented as percentage ratio (maximum=l). On the right panel, examples of current traces of WT and the mutants of K v l .4 ± GIP were shown, and the scale bar indicates 10 nA. A l l data represent the mean ± S.E.M. from four to six independent experiments and significance was tested using Student's / test, where the asterisk represents p <0.05 versus basal. 110 B • GIP + GIP tubulin Time (mins): o 1 DAPI GFP V , 13 OAPI Merge JUt 10 1.2 1.0 0.8 0.6 0.4 0.2 0.0 GIP alone GIP + DIP | 0 1 2 3 4 5 6 7 Time (min) Figure 3.4 GIP treatment resulted in the endocytosis of Kvl.4 protein and decrease in ionic current. A , determination of Kvl .4 subcellular distribution by proteinase K treatment. GIPR-expressing HEK-293 cells were transfected with Kvl .4-EGFP plasmid and treated for the indicated periods of time with 100 nM GIP. Proteinase K was applied for 30 min, and cell lysates were analyzed for Kvl .4 by SDS-PAGE and immunoblotting. The arrow labeled S indicates the immunoblot of cell surface expressed Kvl .4 , and the arrow labeled I indicates the immunoblot of cytosolic Kvl .4 . B, determination of Kv l .4 subcellular distribution by confocal microscopy (from a slice of a single Z axis). GIPR-expressing HEK-293 cells were transfected with Kvl .4-EGFP and treated for 10 min ± 100 nM GIP. Control GIPR-HEK-293-Kv cells (-GIP) and GIP-treated GIPR-HEK-293-Kv cells (+GIP) are shown. Immunocytochemical staining was performed using a-GFP antibody and imaged using a Zeiss laser scanning confocal microscope (Axioskop2). A l l imaging data were analyzed using the Northern Eclipse program (version 6), and the scale bar indicates 10 um. C, electrophysiological recordings. GIPR-expressing HEK-293 cells were transfected with Kvl .4-EGFP and treated for the indicated periods of time with 100 nM GIP in the presence or absence of mry-DIP. Peak current magnitude at each time point was normalized to the peak current from zero time, and example traces from mry-DIP-treated cells are shown in the inset, and the scale bar indicates 10 nA. A l l data represent the mean ± S.E.M. from four to six independent experiments, and significance was tested using Student's t test, where the asterisk represents p <0.05 versus basal. I l l - GIP + GIP Figure 3.5 Phosphorylation is involved in GIP-induced retrograde trafficking of Kvl.4. GIPR-expressing HEK-293 cells were transfected with the indicated mutant Kvl .4-EGFP plasmids and treated for 10 min ± 100 nM GIP. Mutant Kvl .4-EGFP transfected cells without GIP treatment are shown in the left panels (-GIP), and cells with GIP treatment are shown in the right panels (+GIP), respectively. Immunocytochemical staining was performed using a-GFP antibody and imaged using a Zeiss laser scanning confocal microscope (Axioskop2). A l l imaging data (from a slice of a single Z axis) were analyzed using the Northern Eclipse program (version 6), and the scale bar indicates 10 um. 112 A B GIPR-HEK 293 GIPR-HEK 293 Human islets Figure 3.6 Kv channel expression in human islets. A , determination of Kv l .4 expression in human islets by Western blot analysis. Total cellular extracts were prepared from human islets, and Western blot analyses were performed using antibodies against Kvl .4 and 0-tubulin. Total cellular extracts from GIPR-HEK-293 cells and Kvl.4-transfected GIPR-HEK-293 cells (GIPR-HEK-293 plus Kvl.4) were used as negative and positive controls for Western blotting, respectively. B, determination of Kv l .4 expression in human pancreatic p-cells by confocal microscopy (presented as 2D-projections of image stacks). Human islets were embedded in agar, and immunohistochemical staining was performed using Kvl .4 and insulin antibody and imaging using a Zeiss laser scanning confocal microscope (Axioskop2). A l l imaging data were analyzed using the Northern Eclipse program (version 6), and the scale bar indicates 50 um. C - E , cells from human islets were briefly hyperpolarized to -100 mV followed by depolarization to +60 mV for 900 ms from a holding potential of -80 mV. A l l whole cell recordings were performed at room temperature (20-23 °C). C, example of current traces measured in human islet in response to treatment with GIP. Scale bar indicates 2 nA. D, time course of decreases in A-type peak current amplitude in response to 100 nM GIP. Peak current magnitude at each time point was normalized to the peak current from zero time. E, effect of 1 m M 4-AP on A-type current in human islet. A l l data represent the mean ± S.E.M. from three to six independent experiments, and significance was tested using Student's t test, where the asterisk represents p < 0.05 versus basal. The scale bar indicates 2 nA. 113 A tubulin: Glucose: Low Low High High Low Low High High GIP: [ ! : t J + • r DNA: T601A WTKV1.4 Figure 3.7 Phosphorylation-dependent internalization participates in the effect of GIP on insulin secretion. A , determination of subcellular Kvl .4 distribution by proteinase K treatment. p-INS-1 cells (clone 832/13) were transfected with either mutant T601A Kvl .4 -EGFP or WT Kvl .4-EGFP and incubated with 100 nM GIP for 10 min. Proteinase K was applied for 30 min, and cell lysates were analyzed for Kv l .4 by SDS-PAGE and immunoblotting. The arrow labeled S indicates the immunoblot position of surface Kvl .4 , and the arrow labeled / indicates the immunoblot position of intracellular K v l .4. 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Woo Sung Choi*, Su-Jin Kim*, Garth Warnock, Christopher H.S. Mcintosh and David Fedida. (*Both authors contributed equally to this work.) 119 4.1 INTRODUCTION Glucose-stimulated insulin secretion (GSIS) is coupled to glucose metabolism by the subsequent increase in the ATP/ADP ratio arising from glucose oxidation, which closes ATP-sensitive K + channels (K A Tp) (Bell and Polonsky, 2001; Nichols and Koster, 2002; Koster et al, 2005). The inhibition of KATP currents causes depolarization of the plasma membrane, and opening voltage-dependent calcium channels with the influx of calcium-stimulating secretion of insulin (Philipson, 1999; Fridlyand et al, 2003). Also upon membrane depolarization, voltage-gated potassium (Kv) channels open to repolarize the depolarized membrane, and subsequently limit insulin secretion. Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the two major incretin hormones that potentiate GSIS during a meal (Pederson, 1994; Kieffer and Habener, 1999; Hansotia and Drucker, 2005). GIP and the proglucagon gene products glucagon-like peptide-l(GLP-l)-(7-3 7) and GLP-1-(7-3 6)-amide are responsible for 50-70 % of postprandial insulin secretion (Fehmann et al, 1995; Vilsboll and Hoist, 2004). The insulinotropic effects of these hormones are mediated through the activation of their 7-transmembrane G protein-coupled receptors (GPCR) present in pancreatic P-cells (Hoist, 2004). GIP and GLP-1 have been shown to stimulate adenylyl cyclase, leading to increased intracellular cyclic A M P (cAMP) and potentiation of insulin secretion by activation of protein kinase A (PKA)-anaVor cAMP-guanine nucleotide exchange factor-2 (GEF-2)-mediated signalling in pancreatic P-cells (Fehmann et al, 1995; Drucker et al, 1987; Kashima et al, 2001). Although GIP and GLP-1 share this common signalling pathway, distinct effects of the two peptides on insulin secretion are also likely, and it is possible that selective changes in signal transduction pathways are involved in the 120 markedly reduced or blunted responses to GIP observed in type 2 diabetes (Nauck et al., 1993; Hoist and Gromada, 2004). K v channels are involved in the repolarization of excitable cells and electrophysiological studies in human and rodent pancreatic P-cells have suggested the existence of K + currents (Smith et al., 1990; Herrington et al., 2005; Kim et al., 2005; MacDonald and Wheeler, 2003). Kv channels are classified from K v l . X to K v l 2 . X , based on the homology of amino acid sequences, and K v l , Kv2 and Kv3 families are considered to be involved in insulin secretion by modulating the amplitude and duration of action potentials (Roe et al., 1996; MacDonald et al., 2001). While Kv l .4 is a main component of transient outward current (I t0), more than one subtype of Kv channels, including Kv2.1, have been suggested to be involved in the overlapping functions of delayed rectifier currents (IDR) in human pancreatic P-cells (Philipson, 1999; Yan et al., 2004; Herrington et al., 2005). Recently, GIP and GLP-1 have been reported to reduce the Kv channel currents in pancreatic p-cells, suggesting the potential involvement of K v channels in the insulinotropic actions of these hormones (Kim et al., 2005; MacDonald et al, 2002a; MacDonald et al., 2002b; MacDonald et al., 2003). Given the number of different Kv channels that make up islet cell IDR, there is great potential for GIP and GLP-1 to use different subtypes of Kv channels to confer their distinct actions on pancreatic p-cells. The present study was therefore initiated in order to determine which subtypes of delayed rectifier K v channels are involved in the insulinotropic actions of GIP and GLP-1, and to understand its underlying molecular mechanisms. Using human and mouse islets, we have shown that GIP and GLP-1 decrease macroscopic Kv channel currents involved in the generation of IDR. Using GIP receptor (GIPR) or GLP-1 receptor (GLP-1 R) expressing 121 HEK-293 cell systems, we have demonstrated that they use different subtypes of Kv channels to exert their insulinotropic effects on pancreatic P-cells. Additionally, we have shown that GIP and GLP-1-stimulated P K A activation and endocytosis of the channels are involved in the regulation of islet cell IDR. These results therefore strongly suggest important but distinct roles for the different molecular components of IDR in the enteroinsular axis. 122 4.2 RESEARCH DESIGN AND METHODS 4.2.1 Islet isolation and cell culture Human islets were isolated from the pancreas of adult organ donors (n = 5) using collagenase duct perfusion, gentle dissociation and density gradient purification at the Ike Barber Islet Transplantation Laboratory (Vancouver General Hospital, Vancouver, Canada) (Warnock et al, 2005). The Research Ethics Board of the University of British Columbia provided ethics approval. Mouse islets were isolated from male C57/BL6 mice (Charles River, Wilmington, M A ) by collagenase digestion, and cultured in RPMI 1640 medium. For electrophysiological recording, islets were dispersed to single cells. 4.2.2 Western blot analysis Protein samples were separated on a 12.5 % SDS-PAGE gel and transferred onto nitrocellulose (Bio-Rad Laboratories, Mississauga, ON) membranes. Probing of the membranes was performed with antibodies against Kvl .5 (Fedida et al, 2003), Kv2.1, Kv3.2 (Almone Labs, Jerusalem, Israel) and P-tubulin (Santacruz, CA). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia) using horseradish peroxidase-conjugated IgG secondary antibodies. 4.2.3 Immunocytochemistry Human islets were subjected to a double immunostaining with primary antibodies reactive against Kv channels and insulin, followed by Alexa fluor® 488 or Texas Red® conjugated secondary antibodies. Cell nuclei were counterstained with DAPI and imaged using a Zeiss laser scanning confocal microscope (Axioskop2). A l l imaging data were 123 presented as 2D-projections of image stacks and analyzed using the Northern Eclipse program (ver.6). 4.2.4 Electrophysiological recordings To record ionic current, we used a superfusion solution containing the following (in mM): NaCl, 135; KC1, 5; M g C l 2 , 1; sodium acetate, 2.8; HEPES, 10; CaCl 2 , 1; adjusted to pH 7.4 using NaOH. The patch pipettes were filled with pipette solution containing (in mM): KC1, 130; EGTA, 5; M g C l 2 , 1; HEPES, 10; Na 2 ATP, 4; GTP, 0.1; adjusted to pH 7.2 with K O H . A l l chemicals were from Sigma. Whole cell current recordings were done using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) at room temperature. 4.2.5 Measurement of GSIS P-INS-1 cells (clone 832/13) were transfected with 2 \ig of the indicated K v plasmids using Lipofectamine 2000™ reagent (Invitrogen Inc, Burlington, ON). On the following day, transfected P-INS-1 cells or islets were incubated with 100 nM GIP or GLP-1 for 2 h at 37 °C in KRBH-buffer containing low (2.5 mM) or high glucose (25 mM). TEA (5 mM) was added to islets during 1 h preincubation as well as during GIP or GLP-1 stimulation. Insulin release was determined using a radioimmunoassay kit (Linco Research Inc, St. Charles, MO). 4.2.6 Proteinase K digestion experiments For proteinase K digestion (Campomanes et al., 2002), human islets incubated with 100 nM of each peptide for indicated periods of time were washed three times with ice-cold 124 PBS and incubated with 10 mM HEPES, 150 m M NaCl, and 2 m M CaCl 2 (pH 7.4) with 200 ug/ml proteinase K at 37 °C for 30 min. The cells were then harvested, and proteinase K digestion was quenched by adding ice-cold PBS containing 6 m M phenylmethylsulfonyl fluoride and 25 m M EDTA. This was followed by SDS-PAGE and immunoblotting, and probing of the membranes was performed with antibodies against Kvs and P-tubulin. 4.2.7 Generation of GIPR-HEK-293 and GLP-1 R-HEK-293 cell lines and transient transfections of Kv plasmids HEK-293 cells were transfected with GIPR/pcDNA3 or GLP-1 R/pcDNA3 plasmids, allowing expression of pancreatic islet GIPR or GLP-1 R cDNAs under control of the cytomegalovirus (CMV) promoter. Transfections were performed using Lipofectamine 2000™ reagent, according to the manufacturer's instructions. Stably transfected cells were selected with G418 (Invitrogen) and the resulting GIPR-HEK-293 and GLP-1 R-HEK-293 cell clones were transfected with 1.5 ug of the indicated K v plasmids using Lipofectamine 2000™ reagent. 4.2.8 Data analysis Data were analyzed using p C L A M P 8.1 (Axon Instruments, Foster City, CA) and PRISM 4.0 (GraphPad, San Diego, CA). The steady-state activation and inactivation curves were fitted with the Boltzmann functions: y - 1/(1 + exp[ (V\a-V)/k] (1) and y = 1/(1 + exp[(V- V\i2)lk\ + C (2), respectively, where Vis the test potential, Via'is the potential at which the conductance was half maximal, k represents the slope factor, and C represents the fraction of non-inactivating channels at potentials where inactivation was most complete. 125 A l l data are presented as the mean ± S.E.M., and significance was tested using A N O V A with a Newman-Keulspost hoc test or Student's / test. 126 4.3 RESULTS 4.3.1 Expression of delayed rectifier Kv channels in rodent and human islets To determine which subtypes of Kv channels are expressed in pancreatic P-cells, Western blot hybridization were performed in rat and human islets. As shown in Figure 4.1 A , Kv2.1 was found in a Vancouver strain of the diabetic fatty (fa/fa;VDF) Zucker rat, an animal model of Type 2 diabetes (Hinke et al, 2002; Pospisilik et al, 2002), and insulin-positive pancreatic P-cells of human islets (Figure. 4.1 A and B). Kv l .5 expression was also detected in VDF rats and human pancreatic P-cells, however, it was undetectable in lean rats (Figure. 4.1C and D). Kv3.2 was expressed in lean and diabetic VDF rats, as well as human pancreatic p-cells (Figure. 4.IE and F). Altogether, these results demonstrate the expression of K v channel subtypes in both rodent and human islets, but with changes in expression levels depending on the metabolic status of the animal (i.e. non-diabetic lean vs. diabetic fatty). 4.3.2 Multiple delayed rectifier Kv channels contribute to the generation of IDR in human islets. A previous report has shown that Kv2.1 is functional in human pancreatic P-cells (Harrington et al, 2005). To determine whether Kvl .5 and Kv3.2 are functionally expressed in human islets, we applied 4-aminopyridine (4-AP) and BDS II (blood depressing substance) with the combination of an inactivating prepulse (which inactivates I t 0). BDS II binds to voltage sensing regions of Kv3 family, which dramatically changes the channel gating and inhibits the ionic current voltage-dependently (IC50, 0.75 uM at +40 mV) (Yeung et al, 2005). Similar to the results observed in cloned Kv3 channels, 1 uM 127 BDS II reduced peak currents of human islet cells with slowing of activation (Figure. 4.2 A), and moved V i / 2 rightward significantly (Vi / 2 c o n t r o l , -9.591 ± 0.81 mV; V I / 2 B D S I I , 3.102 ± 0.98 mV) (Figure. 4.2B). These results suggest that Kv3.2 is functionally expressed in human islet cells. On the other hand, 4-AP blocks Kvl .5 and Kv3.2 channels with IC5o of 50 uM and ~1 mM, respectively, whereas much higher concentration (IC50, 4.5 mM) is required to block Kv2.1 channels (Bouchard and Fedida, 1995; Mathie et al, 1998; Patel et al, 1997). As shown in Figure 4.2C, the K + current was partially blocked by 0.1 m M 4-AP, and the existence of 4-AP sensitive current suggest the functional expression of Kvl .5 in human islet cells. When 1 m M 4-AP was applied to block most of Kvl .5 and half of Kv3.2 currents, approximately 34 % of K + current was blocked. Therefore, most of the remaining K + current is potentially mediated by Kv2.1 (Figure. 4.2A and D). Taken together, these results suggest that Kv2.1, Kv l .5 and Kv3.2 are functionally involved in the generation of human islet cells IDR. 4.3.3 G I P and G L P - 1 inhibit human islet cell I D R The effects of GIP and GLP-1 on human islet cell IDR were next investigated to determine whether incretins modulate electrical properties of human islet. Human islets were dispersed to single cells, and the current density of IDR was determined with voltage step protocol (data not shown). The current-voltage (I-V) and conductance-voltage (G-V) relations in our batch of human islets were very similar to previously reported electrophysiological properties of human pancreatic p-cells (Herrington et al, 2005). To examine the effects of GIP and GLP-1 on I D R, dispersed human islet cells were depolarized briefly from the holding potential of-80 mV to -40 mV (to prevent contamination with I t 0) 128 and pulsed to +60 mV to observe IDR. N O significant reduction of the current was observed during 20-min recordings in control cells, not treated with GIP or GLP-1 (Figure. 4.3A and D). GLP-1 (7-36, 100 nM)-treated cells tended to show a steeper pattern of decrease, compared to GIP (1-42, 100 nM)-treated cells (Figure. 4.3D). However, GIP and GLP-1 decreased the IDR by almost the same level at 20 min of incubation. Human islet cell I D R was decreased by 40.4 % and 40.5 % after 20 min-incubation with GIP and GLP-1, respectively (Figure. 4.3B-D). The effects of incretins on I D R were reversible with the elimination of GIP and GLP-1, and K + current was almost returned to the basal following 10 min-washing out (Figure. 4.3B and C). The decrease of human islet cell IDR demonstrated concentration-dependent responses to incretins, with 1 nM GIP or GLP-1 producing a significant reduction (Figure. 4.3E). Activation and inactivation properties of human islet cell IDR were next studied to determine whether incretins affect the gating properties of IDR. As shown in Figure 4.3F, GIP and GLP-1 did not significantly alter the activation (VMControl = -4.68 ± 0.93 mV; VV2GIP = -7.60 ± 1.39 mV; VV2GLP-\ = -9.54 ± 1.73 mV). On the other hand, a hyperpolarizing shift in the voltage dependence of steady-state inactivation was observed in GIP or GLP-1 treated cells (Figure. 4.3G, Vm c o n t r o l = -25.22 ± 0.83 mV; VmGlP = -40.79 ± 1.38 mV; VmGLp-\ = -38.71 ± 1.89 mV). In addition, the properties of inactivation in GIP or GLP-1 treated cells were converted to more Kv2.1-like type. This may represent that the remained current is mainly mediated by Kv2.1. Taken together, these results suggest that incretins modulate the peak current amplitude and the steady-state inactivation of the K v channels in human islet cells. 129 4.3.4 Suppression of Kv channels resulted in the decreased insulinotropic effects of incretins in human islets To determine whether delayed rectifier Kv channels participate in the insulinotropic activity of GIP and GIP-1 in human islets, Kv channels were blocked with tetraethylammonium (TEA), a pore blocker of Kv channels, and GSIS was determined during treatment with GIP (1-42, 100 nM) or GLP-1 (7-36, 100 nM). As shown in Figure 4.4, TEA pre-treatment (5 mM) resulted in significantly enhanced secretion of insulin under the high glucose condition, strongly suggesting the involvement of K v channels for the regulation of insulin secretion. Strong effects of GIP and GLP-1 on insulin secretion were observed in human islets, however, this stimulation was significantly reduced when the Kv channels were blocked by TEA pre-treatment. Complete inhibition of incretin effects was not observed by TEA pre-treatment, suggesting pleiotropic effects of GIP and GLP-1 on human pancreatic P-cells. Taken together, these results strongly suggest that GIP and GLP-1 confer insulinotropic effects on human pancreatic p-cells by modulating delayed rectifier K v channels. 4.3.5 G I P and GLP-1 inhibit mouse islet cell IDR and Kv channels participate in the insulinotropic activity of G I P and GIP-1 To complement and compare the effects of incretins on human islet cells, the electrical properties of mouse islets were next investigated. As shown in Figure 4.5A-D, there was no significant decrease in IDR during 20-min recordings in control cells, not treated with GIP or GLP-1 (Figure. 4.5A and D). On the other hand, GIP (1-42, 100 nM) or GLP-1 (7-36, 100 nM) treatment resulted in the 35.7 % and 31.9 % reduction of mouse islet 130 cell IDR, respectively (Figure. 4.5B-D). Activation and steady-state inactivation properties of mouse islet cell IDR were next studied. As we shown in Figure 4.5E, GIP and GLP-1 did not alter activation (Figure. 4.5E, Vmcontrol = -5.46 ± 0.65 mV; VmGl? = -7.91 ± 0.29 mV; V\a G L P - i = -11.57 ± 0.38 mV), however, a significantly leftward shifted V\a of the steady-state inactivation was observed with the treatment of GIP and GLP-1 (Figure. 4.5F, V\a c o n t r o l = -26.82 ± 0.76 mV; VmGve = -38.32 ± 1.84 mV; VmGL?-i = " 3 2 . 9 ± 1.17 mV). These results are consistent with the results observed in human islet cell IDR with the treatment of GIP and GLP-1 (Figure. 4.3F and G). To determine whether delayed rectifier K v channels are involved in the insulinotropic activity of GIP and GIP-1 in mouse islets, Kv channels were blocked with 5 mM TEA, and GSIS was determined in mouse islets with the treatment of GIP (1-42, 100 nM) or GLP-1 (7-36, 100 nM). Similar results found in human islets were observed with mouse islets. GIP and GLP-1 significantly enhanced insulin secretion under the high glucose condition, however, this stimulation was significantly reduced when the K + currents were blocked, by TEA (Figure. 4.6A and B). To further identify each subtypes of Kv channels involved in the insulinotropic activity of GIP and GIP-1, Kv2.1, Kv l .5 and Kv3.2 were transiently overexpressed in P-INS-1 cell lines (clone 832/13), and GSIS was determined during treatment with GIP (1-42) or GLP-1 (7-36). As shown in Figure 4.6C and D, GIP or GLP-1 treatment did not significantly increase insulin secretion in the presence of low glucose in the pcDNA3 vector control group, or the Kv2.1-, K v l . 5 - or Kv3.2-transfected groups. However, under high glucose conditions, GIP treatment resulted in increased insulin secretion in all groups (Figure. 4.6C). This difference was greater in the Kv-transfected groups than in those transfected with vector. On the other hand, GLP-1 treatment resulted in increased insulin secretion in the Kv2.1 and 131 Kvl .5 groups, but not in the Kv3.2-transfected group (compared with control, p > 0.05, Figure. 4.6D). These results therefore strongly suggest that GIP and GLP-1 confer insulinotropic effects on rodent pancreatic P-cells by modulating delayed rectifier Kv2.1, Kv l .5 and Kv3.2 channels. 4.3.6 GIP and GLP-1 regulate Kv2.1, Kvl.5 and Kv3.2 macroscopic current in HEK-293 cells expressing incretin hormone receptors To further characterize the macroscopic currents of Kv channels modulated by GIP and GLP-1, HEK-293 cells were used as in vitro model systems. The effects of GIP and GLP-1 on Kv2.1, Kv l .5 and Kv3.2 currents were examined by transient transfection of Kv2.1, Kv l .5 and Kv3.2 cDNAs into incretin hormone receptor expressing HEK-293 cells. In GIP receptor expressing HEK-293 cells, GIP treatment resulted in 42 %, 52 % and 22 % reductions in Kv2.1, Kv l .5 and Kv3.2 macroscopic currents respectively (Figure. 4.9). Conversely, GLP-1 treatment resulted in 50 % and 59 % decreases in Kv2.1 and K v l . 5 , but not in Kv3.2 currents in GLP-1 receptor expressing HEK-293 cells (Figure. 4.10). These findings are consistent with GSIS results in Kv channel overexpressing p-INS-1 cells showing increased insulin secretion in Kv2.1, K v l . 5 , K v 3.2 groups by GIP treatment, but only increased insulin secretion in Kv2.1 and Kvl .5 by GLP-1 treatment (Figure. 4.6C and D). For this experiment, we used three groups of control, 1) no incretin treated, 2) inactive form of incretin treated, 3) incretin treated in HEK-293 cells, which do not express incretin receptors, and any significant decrease in Kv currents was not observed in each control group, suggesting the specificity of this reaction. Taken together, both GIP and GLP-1 decrease macroscopic K v channel currents involved in the generation of IDR via interaction 132 with their specific receptors, and they use different subtypes of K v channels with different kinetics to exert their insulinotropic effects on pancreatic P-cells. 4.3.7 Suppression of PKA resulted in the blocking of incretins effects on delayed rectifier Kv channels Underlying molecular mechanisms involved in the decrease of IDR by GIP and GLP-1 were next studied. The possibility that the decrease of IDR were related with the activation of adenylyl cyclase and P K A was first examined, because this is a well characterized incretins signalling module involved in the regulation of insulin secretion. To determine whether adenylyl cyclase and P K A are involved in the decrease of delayed rectifier K v currents, selective inhibitor of P K A , H-89 and 8-Br-Rp-cAMP were applied during electrophysiological recordings. As shown in Figure 4.7A, H-89 and 8-Br-Rp-cAMP significantly blocked GIP and GLP-1 mediated inhibition of human islet cell IDR. Similar effects of P K A inhibitors were also observed in mouse islet cell IDR (Figure. 4.7B). These results therefore indicate that GIP and GLP-1-stimulated P K A activation is involved in the regulation of islet cell IDR inhibition. 4.3.8 GIP and GLP-1 reduce Kv channel surface expression To investigate whether GIP and GLP-1 reduced human islet cell IDR by internalization of delayed rectifier Kv channels protein from the plasma membrane, we performed proteinase K sensitivity experiments. Proteinase K randomly digests extracellular regions of K v channels, thus producing a digested form, whereas intracellular channel is protected. As shown in Figure 4.8A, C and E, surface membrane expression 133 levels of Kv2.1, Kv l .5 and Kv3.2 decreased with the treatment of G I P (1-42, 100 nM). On the other hand, G L P - 1 (7-36, 100 nM) treatment resulted in decreased membrane expression of Kv2.1 and K v l . 5 , but not of Kv3.2 (Figure. 4.8A, C and E). These findings are correlated well with the results showing the effect of over-expression of K v channels on G S I S in P-INS-1 cells (Figure 4.6C and D) as well as the electrophysiological studies on the HEK293 cells showing reductions in Kv2.1, Kv l .5 and Kv3.2 currents by G I P treatment, but only reductions of Kv2.1 and Kvl .5 by G L P - 1 treatment (Figures 4.8B, D, F, 4.9 and 4.10). On the other hand, there was no significant decrease of surface expression of K v channels with the treatment of inactive form of incretin, G I P (19-30) and G L P - 1 (9-36). Taken together, these results strongly suggest that endocytosis of the channel protein is involved in the incretin-mediated inhibition of human islet cell IDR. 134 4.4 DISCUSSION Delayed rectifier Kv channels are important in the regulation of membrane potential of electrically excitable cells, and each Kv channel subtype possesses different kinetics, different functional and signalling domains, and sensitivity to different specific blockers (Yeung et al, 2005; Bouchard and Fedida, 1995; Mathie et al, 1998; Patel et al, 1997; Tamarina et al, 2005). K v cc-subunits can co-assemble as hetero-tetramers in a family specific manner, and some of these associate with cytosolic or transmembrane accessory subunits. Kv channels are potential mediators of membrane repolarization and termination of insulin secretion in pancreatic P-cells, but the exact contribution of different K v channel subtypes to IDR and the subsequent regulation of insulin secretion are still unclear. These divergent properties of K v channels may allow them unique roles in P-cell function, potentially through coupling with different signalling pathways activated by extracellular stimuli. In the diabetic state, the Kv channel activity of vascular smooth muscle cells in streptozotocin-induced diabetic rat is suppressed, and hyperglycemia-induced superoxide production also reduces Kv channel activity, strongly suggesting a functional involvement of K v channels in the pathophysiology of the disease (Chai et al, 2005; Liu et al, 2002). Using a dominant-negative approach, it has been demonstrated that K v l and Kv2 channels mediate 25 % and 60 % of rat P-cell IDR, respectively, and knockout of these channels enhances GSIS (MacDonald et al, 2001). Ultimately though, transient specific knockouts of each K v channel subtype will be required to determine the distinct function of each Kv channel subtype. The current study was initiated with the hypothesis that GIP and GLP-1 may utilise different Kv channel subtypes to regulate insulin secretion. We have shown that both rodent 135 and human islets express multiple subtypes of delayed rectifier K v channels (Figures 4.1 and 4.2) and that their expression levels are also altered in disease states. In the VDF rat, an animal model of Type 2 diabetes (Hinke et al, 2002; Pospisilik et al, 2002), protein expression levels of Kv2.1 and Kvl .5 were increased, compared to non-diabetic control lean rats (Figure 4.1 A and C). On the other hand, there was no significant difference in Kv3.2 protein expression levels between lean and obese animals (Figure 4.IE). This increased expression of Kv2.1 and Kvl .5 may reflect compensatory mechanisms activated in Type 2 diabetes to overcome the relative insulin deficiency resulting from p-cell dysfunction and insulin resistance. Indeed, blocking K v currents resulted in the decreased insulinotropic effects of incretins in human and mouse islets (Figures 4.4A-B and 4.6A-B), and over-expression of K v channels improved insulin secretory responses to incretins in |3-INS-1 cells (Figure 4.6C and D). Although the underlying basis of this potentiation is not known, one possibility is that the level of K v channel expression in P-INS-1 cells is a limiting factor in the regulation of action potential frequency. Channel over-expression may allow the incretins to repolarize the plasmalemma more efficiently via regulation of K v channel, resulting in increased frequency of action potential generation and potentiated insulin secretion. Heterologous interaction of the overexpressed channels with endogenous Kv channel subunits may also play a role. Using HEK-293 cells expressing GIPRs or GLP-1 Rs, we have demonstrated responses of three subtypes of K v channels to treatment with GIP and GLP-1. Administration of GIP resulted in 42 % and 52 % decreases in Kv2.1 and Kvl .5 ionic currents, respectively. GIP also reduced Kv3.2 current but to a lesser extent, 22 %, compared with Kv2.1 and Kv l .5 , (Figure 4.9). On the other hand, GLP-1 treatment resulted 136 in 50 % and 59 % decreases in peak Kv2.1 and Kvl .5 current amplitudes, respectively. However, there was no significant change in Kv3.2 current with a 20-min incubation of GLP-1 (Figure 4.2). Similar effects of GIP and GLP-1 were also observed on human islet cell IDR: GLP-1-treated cells tended to show a steeper pattern of decrease, compared to GIP-treated cells (Figure 4.2). However, GIP and GLP-1 decreased the I D R by almost the same level at 20 min of incubation. These results strongly suggest that multiple Kv channels are involved in the generation and regulation of human islet cell I D R, and GIP and GLP-1 utilise different subtype of Kv channels to exert their effects on P-cells. In human islets, it has been reported that K v l . 3 , Kv l .6 , Kv2.1, Kv3.2, Kv6.2, and Kv9.3 are present in p-cells (Yan et al, 2004), while Kvl .5 was not found. In other studies of human islets, Kv l .5 , Kv l . 6 and Kv2.1 have been shown to be expressed in P-cells (MacDonald et al, 2001). In the present study, we have shown that Kv2.1, Kv l .5 and Kv3.2 are functionally expressed in human islets (Figures 4.1 and 4.2). These data suggest the variances in channel composition between the species or individuals, and highlights the importance of the identification of the relevant subunits in human pancreatic P-cells. Recently, it has been reported that ShK, a potent blocker of Kv3.2 in heterologous systems, has no effect on human p-cell IDR, suggesting that Kv3.2 is electrically silent in these cells (Herrington et al, 2005; Yan et al, 2005). However, in the current study, we have demonstrated the presence of BDS II (1 uM) sensitive I D R in human islet cells (Figure 4.2A and B). As BDS II inhibits Kv3 family channels, and Kv3.2 is the only subtype of Kv3 family has been reported to be present in pancreatic P-cells (Yan et al, 2004), the presence of BDS Il-sensitive current indicates the functional expression of Kv3.2 in human islet cells. Furthermore, 1 uM BDS II treatment resulted in the rightward shift of G-Vcurve, a typical phenotype observed with 137 the blocking of Kv3 channels. Interestingly, human islets showed a smaller rightward shift of G-Vcurve (+12.69 mV) by 1 uM BDS II, compared to the shift in cloned Kv3.2 (~+20 mV) by 0.5 uM BDS II. These results suggest the potential heterodimer formation of Kv3.2 with other accompanying molecular components in human islet cells. Back to the ShK blocker, it also cannot be completely ruled out that conformational changes in crystal structure, with the formation of a heteromultimer complex between Kv3.2 and other K v <x-subunits, may result in a different responsiveness or sensitivity to ShK in human pancreatic P-cells. There is emerging evidence for the involvement of signalling molecules and trafficking of ion channels for the regulation of ionic currents. It has been previously reported that GLP-1 regulates Kv current by modulating c A M P / P K A and phosphatidylinositol 3-kinase (PI-3K)/protein kinase CC, (PKCQ signalling molecules (MacDonald et al, 2003). GIP has been shown to regulate Kv l .4 A-type current through PKA-mediated phosphorylation of Kv l .4 and resulting in the channel endocytosis (Kim et al., 2005). In the present study, we have shown that P K A inhibitors significantly abolished GIP and GLP-1-mediated inhibition of human islet cell IDR, suggesting the involvement of P K A activation for the regulation of human and mouse islet cell IDR (Figure 4.7). Furthermore, it has been demonstrated that endocytosis of the channel protein is involved in the GIP and GLP-1-mediated inhibition of human islet cell IDR (Figure 4.8), implying retrograde trafficking of K v channels is one of the underlying molecular mechanisms that are responsive to incretin action. Interestingly, GIP decreased surface expression of Kv2.1, Kv l .5 and Kv3.2, whereas GLP-1 only decreased Kv2.1 and Kvl .5 (Figure 4.8A, C, E). GIP treatment resulted in enhanced insulin secretion in Kv2.1, Kv l .5 and Kv3.2-transfected 138 P-INS-1 cells, whereas GLP-1 treatment resulted in increased insulin secretion in the Kv2.1 and Kvl .5 groups, but not in the Kv3.2-transfected group (compared with control, p > 0.05, Figure 4.6C and D). Also, the electrophysiological recordings on HEK293 cells showed reduction in Kv2.1, Kv l .5 and Kv3.2 currents by GIP treatment, but only reductions of Kv2.1 and Kvl .5 by GLP-1 treatment (Figures 4.8B, D, F, 4.9 and 4.10). Taken together, these results strongly suggest the distinct effects of GIP and GLP-1 on each subtype of Kv channels expressed in pancreatic P-cells, and they utilise different subtype of K v channels with different kinetics to exert their insulinotropic effects on pancreatic P-cells. Further study will be required to determine which specific signalling modules are selectively involved in the regulation of each subtype of Kv channels. In summary, we have shown that human islets express Kv2.1, Kv l .5 and Kv3.2 delayed rectifier K v channels, and GIP and GLP-1 can inhibit K v currents to exert insulinotropic effects on pancreatic P-cells. They can regulate Kv channel subtypes to different degrees and with different pattern. Different control mechanisms and preferences of these incretins in using Kv channels may contribute to the fine tuning of insulin secretion. As incretins receptor agonists and DPP-IV inhibitor enter the clinical arena, determining molecular components of IDR and underlying mechanisms that are responsive to incretin action will be important for understanding their role in P-cell physiology and evaluating their potential as drug target for Type 2 diabetes. 139 4.5 ACKNOWLEDGEMENTS These studies were generously supported by funding to DF from the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundations of British Columbia and Yukon, to CHSMc from CIHR, the Canadian Diabetes Association and the Canadian Foundation for Innovation (CFI), to GW from CFI, PA Woodward Foundation and the Ike Barber Diabetes Research Endowment, and to WSC from a University Graduate Fellowship. We would like to thank Dr. C. B.Newgard (Duke University Medical Centre, NC) for INS-1 cells (clone 832/13), and Dr. Z. Ao (Ike Barber Islet Transplantation Laboratory, UBC) for the gifts of human islets. We also would like to thank Dr. S. Kehl (Cellular and Physiological Sciences, UBC) for his comments and helpful discussion. 140 Figure 4.1 Expression of delayed rectifier Kv channel in rat and human islets. A, C, E: Determination of Kv2.1 (A), Kvl .5 (C) and Kv3.2 (E) expression in rat and human islets by Western blot analysis. B, D, F: Determination of Kv2.1 (B), Kvl .5 (D) and Kv3.2 (F) expression in human islets by immunostaining (n=5). Scale bars indicate 25 um. See Materials and methods. 141 A. 3 200 ms -80 60 mV-rrTV Control Washout 1 pM 0 mV -80 -60 -40 -20 0 20 40 60 80 100 120 Voltage (mV) c. 1.00-Control 0.1 mM _0.50-4-AP 0.25-0.00-200 ms Control 1 uM 0.1 mM 1 mM BDS II 4-AP 4-AP Figure 4.2 Functional expression of Kv2.1, Kvl.5 and Kv3.2 in human islets. A , C : Current recordings at +60mV for 400 ms after a prepulse at -40 mV from -80 mV in human islet cells. A : Current trace before and during stable inhibition by 1 uM BDS II. After washing out, 1 m M 4-AP was applied. B: Normalized G-V curves of control human islet cell IDR and in the presence of 1 uM BDS II (same cells). Curves were fitted with the Boltzmann function described in the Methods. C : Current trace before and during steady-state inhibition by 0.1 m M 4-AP. D: Normalized peak current in the IDR- A l l data represent the mean ± S.E.M. from 3-5 independent recordings. */J<0.05 vs. Control. 142 B . to d -0 min Control 20 min 200 ms -80 mV 60 mVi , _ f J L^-40 mV 0 5 10 15 20 25 Time (min) -60 -40 -20 0 20 40 60 80 100 Voltage (mV) Control GIP (1-42) GLP-1 (7-36) -60 -40 -20 0 Voltage (mV) 20 40 Figure 4.3 Effects of incretins on IDR of human islet cells. A - C : Voltage clamp current traces from human islet cells during 400 ms pulses from -40 to +60 mV before and after 20 min exposure, in Control (A), GIP (1-42, 100 nM, B) and GLP-1 (7-36, 100 nM, C). D : Plot of the normalized peak current vs. time for the cells in A , B and C. E: Effect of different concentration of incretins on human islet IDR. Human islet cells were incubated with the indicated concentration of GIP or GLP-1, and currents were recorded for 20 min with GIP or GLP-1 treatment. F-G: Effect of incretins on activation (F) and inactivation (G) of human islet IDR. Curves were fitted with the Boltzmann function described in the Methods. A l l data represent the mean ± S.E.M. from 5-7 independent recordings. *P<0.05; #P<0.05 vs. Control. 143 & Glucose: Low Low High High Low Low High High GIP: - + - + - + - + T E A : . - - - + + + + & Glucose: Low Low High High Low Low High High GLP-1 : - + - + - + - + T E A : - - - ' - + + + + Figure 4.4 Effects of TEA on GSIS of human islet cells. Human islets were incubated with 100 nM GIP (1-42, A) or GLP-1 (7-36, B) for 2 h at 37 °C in low (2.5 mM) or high glucose (25 mM), in the presence or absence of TEA (5 mM). TEA was added to islets during 1 hr preincubation as well as during GIP or GLP-1 stimulation. Insulin release into the medium was determined by radioimmunoassay. A l l data represent the mean ± S.E.M. from two independent experiments, each carried out in triplicate. &P<0.05 vs. High glucose and *P<0.05 vs. High glucose, +GIP. 144 Figure 4.5 Effects of incretins on IDR of mouse islet cells. A - C : Current traces from mouse islet cells during 400 ms pulses from -40 to +60 mV before and after 20 min exposure, in Control (A), GIP (1-42, 100 nM, B) and GLP-1 (7-36, 100 nM, C). D: Plot of the normalized peak current for the cells in A , B and C. E-F: Effect of incretins on activation (E) and inactivation (F) of mouse islet IDR. *P<0.05 VS. Control. 145 Figure 4.6 Kv channels participate in the insulinotropic activity of GIP and GIP-1. A-B. Effects of TEA on GSIS of mouse islet cells. (Legend on the following page) 146 Figure 4.6 Kv channels participate in the insulinotropic activity of GIF and G1F-I. A-B. Effects of TEA on GSIS of mouse islet cells. Islets were isolated from C57/BL6 mice and incubated with 100 nM GIP (1-42, A) or GLP-1 (7-36, B) for 2 h at 37 °C in low (2.5 mM) or high glucose (25 mM), in the presence or absence of TEA. TEA (5 mM) was added to islets during 1 hr preincubation as well as during GIP or GLP-1 stimulation. Insulin release into the medium was determined by radioimmunoassay. C-D. Ef feet of over-expression of delayed rectifier Kv channels on GSIS of P-INS-1 cells. p-INS-1 cells (clone 832/13) were transfected with pCDNA3 vector, Kv2.1, Kv l .5 or Kv3.2 and incubated with 100 nM GIP (1-42, C) or GLP-1 (7-36, D) for 2 h at 37 °C in low (2.5 mM) or high glucose (25 mM). Insulin release into the medium was determined by radioimmunoassay. A l l data represent the mean ± S.E.M. from two independent experiments, each carried out in triplicate. &PO.05 vs. High glucose; *P<0.05 vs. High glucose, +GIP and #P<0.05 vs. High glucose, +GLP-1; AP<0.05 vs. High glucose, +GIP, pCDNA3 and vs. High glucose, +GLP-1, pCDNA3. 147 Figure 4.7 Effect of PKA inhibitor on incretin-mediated inhibition of human and mice islet IDR. Human islets (A) and mouse islets (B) were incubated in the presence or absence of P K A inhibitor, H-89, or cAMP antagonist, (Rp)-cAMP. Current was recorded at zero time (Control) and 20 min after GIP or GLP-1 treatment from same cells. Normalized peak current values were presented in the plots. A l l data represent the mean ± S.E.M. from 4-6 independent experiments. *P<0.05 vs. Respective control. 148 A. Kv2.1 0) -» Kv2.1 (S) P-Tubulin ->£ Peptide: c. Kv1.5(7)-> «vi.5(sj-> p-Tubulin -»£ Peptide: E. Kv3.2 (I) -> Kv3.2 (S) -> P-Tubulin ->£ Peptide: GIP (19-30) GIP (1-42) GLP-1 (9-36) GLP-1 (7-36) GIP (19-30) GIP (1-42) GLP-1 (9-36) GLP-1 (7-36) GIP (19-30) GIP (1-42) GLP-1 (9-36) GLP-1 (7-36) 1.00 B. _ 0.50 0.25 0.00 nil in. GIP GIP GLP-1 GLP-1 Peptide: - ( 1 9 . 3 0 ) - ( 1 j , 2 ) - ( 9 . 3 6 > • ( 7 . 3 6 ) D. 1 0 0 0.75 _ 0.50 0.25 III. Ill, GIP GIP GLP-1 GLP-1 Peptide: - ( 1 9 . 3 0 ) " ( 1 ^ 2 ) ' ( 9 . 3 6 ) " ( 7 . 3 6 ) O.OOi-l „ GIP GIP Peptide: - ( 1 9 . J 0 ) - ( 1 - 4 2 ) GLP-1 GLP-1 (9-36) " (7-36) Figure 4.8 GIP and GLP-1 reduce Kv channel surface expression. A, C, E: Determination of subcellular distribution of Kv2.1 (A), Kv l .5 (C) and Kv3.2 (E) by proteinase K treatment. Human islet cells were treated with 100 nM of indicated peptides, GIP (1-42), GIP analog (19-30), GLP-1 (7-36) and GLP-1 analog (9-36). Proteinase K was applied for 30 min, and cell lysates were analyzed for each subtype of Kvs by SDS-PAGE and immunoblotting. The arrow labeled S indicates the immunoblot position of surface Kvs, and the arrow labeled I indicates the immunoblot position of intracellular Kvs. B, D, F: Effect of indicated peptide on the peak current of Kv2.1 (B), Kv l .5 (D) and Kv3.2 (F). Currents were recorded for 20 min with treatment of indicated peptide. Normalized peak current values were presented in the plots. A l l data represent the mean ± S.E.M. from 3-5 independent experiments. *P<0.05 vs. respective controls. 149 < 5'-i r — 0 min GIP 20 min 50 ms -80 mV 60 mV L^40mV c. < c "0 min GIP 20 min 50 ms 0 min 50 ms B. 1 2 1.0 0.8 _ E 0.6 0.4 0.2 0.0 1.0 0.8 x - ? 0.6 0.4 0.2 0.0 F. 1 2 1.0 0.8 -I 0.6 0.4 0.2 0.0 5 10 15 Time (min) 20 25 5 10 15 Time (min) 5 10 15 Time (min) 20 25 * * * * * * - • - GIP-KV3.2 —v- Control 1 —•— Control 2 - O - Control 3 20 25 Figure 4.9 Effects of GIP on Kv2.1, Kvl.5 and Kv3.2 in HEK-293 cells. Electrophysiological recordings from Kv2.1-GIPR-HEK-293 cells (A, B), Kvl.5-GIPR-HEK-293 cells (C, D) and Kv3.2-GIPR-HEK-293 cells (E, F). A , C, E: Original data at +60 mV, and protocol. B, D, F: Plot of the normalized peak current vs. time. Control 1, no GIP; Control 2, GIP (19-30); and Control 3, GIP (1-42) without GIPR. A l l data represent the mean ± S.E.M. from three to seven independent recordings. *P<0.05. 150 < c r -0 min GLP-1 20 min 50 ms -80 mV 60 mV c. < c - 0 min GLP-1 20 min 50 ms B. 12 1.0 0.8 _= 0.6 0.4 0.2 0.0 D . 1 2 1.0 0.8 _= 0.6 0.4 0.2 0.0 F . 12 1.0 0.8 x < - s 0.6 0.4 0.2 0.0 5 10 15 Time (min) 10 GLP-1-KV3.2 Control A Control B Control C 15 5 10 15 Time (min) 20 25 T* - • - GLP-1-KV1.5 * * * * * * * * —v— Control A —•— Control B —©— Control C 20 25 20 25 Figure 4.10 Effects of GLP-1 on Kv2.1, Kvl.5 and Kv3.2 in HEK-293 cells. Electrophysiological recordings from Kv2.1-GLP-1 R-HEK-293 cells (A, B), Kvl .5 -GLP-1R-HEK-293 cells (C, D) and Kv3.2-GLP-1 R-HEK-293 cells (E, F). A , C, E: O riginal data at +60 mV, and protocol. B, D, F: Plot of the normalized peak current vs. time. Control A , no GLP-1; Control B, GLP-1 (9-36); and Control C, GLP-1 (7-36) without GLP-1 R. A l l data represent the mean ± S.E.M. from three to seven independent recordings. *Z5<0.05. 151 4.6 REFERENCES Bell GI, Polonsky KS. Diabetes mellitus and genetically programmed defects in (3-cell function. Nature. 414:788-91, 2001 Bouchard R, Fedida D. Closed- and open-state binding of 4-aminopyridine to the cloned human potassium channel K v l . 5 . J Pharmacol Exp Ther. 275:864-76, 1995 Campomanes CR, Carroll KI , Manganas L N , Hershberger M E , Gong B, Antonucci DE, Rhodes KJ , Trimmer JS. Kv p-subunit oxidoreductase activity and K v l potassium channel trafficking. J Biol Chem. 277:8298-305, 2002 Chai Q, Liu Z, Chen L. 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Diabetes. 53:597-607, 2004 Yan L, Herrington J, Goldberg E, Dulski P M , Bugianesi R M , Slaughter RS, Banerjee P, Brochu R M , Priest BT, Kaczorowski GJ, Rudy B, Garcia M L . Stichodactyla helianthus peptide, a pharmacological tool for studying Kv3.2 channels. Mol Pharmacol. 67:1513-21,2005 Yeung SY, Thompson D, Wang Z, Fedida D, Robertson B. Modulation of Kv3 subfamily potassium currents by the sea anemone toxin BDS: significance for CNS and biophysical studies. J Neurosci. 25:8735-45, 2005. 155 CHAPTER 5. GENERAL DISCUSSION AND FUTURE DIRECTIONS 156 Every cell must communicate with its environment and maintain homeostasis. Integral membrane proteins expressed on the cell surface transmit signals from outside to inside of the cell as receptors, or distribute substances across the membrane as transporters. Ion channels respond to physiological or environmental stimuli. Net flux through channels, however, is entirely passive and thus is always downhill with respect to the ion's electrochemical gradient across the membrane. Therefore, cells may require additional approaches to regulate net fluxes of the ions. Cells control voltage-gated ion channel activity in two ways. In the short term, the channels open and close in response to changes in membrane potential. When the cell membrane is polarized, such that the interior of the cell is quite negative relative to the exterior, K v channels remain closed. When the membrane depolarizes, these channels open rapidly, allowing K + ions to flow passively down their electrochemical gradients, at rates close to diffusion in water. Kv channels function in membrane repolarization at the end of an action potential. Over the longer term, cells regulate the numbers and types of channels expressed at their surfaces. Each cell type expresses a unique repertoire of channels from among hundreds of channel genes. As a further level of control, an elaborate internal transport system functions to take up the channels from the cell surface for recycling or degradation. For this process, cells are likely to exploit endocytosis mechanisms that include internalization and retrograde trafficking pathways. The principal purpose of this thesis is to gain some understanding of the endocytosis mechanisms involved in the regulation of Kv channel expression in plasma membrane. 157 5.1 Endocytosis mechanism of Kvl.4 and Kvl.5 As shown in Chapters 2 and 3, endocytosis of channel proteins modulates macroscopic K + currents from Kvl .4 and Kvl .5 without any significant change in channel biophysical properties: disruption of Kvl .5 retrograde trafficking magnifies channel current density, and Kvl .4 endocytosis induced by an external stimulus results in a reduction of the channel current. In both cases, changes of the channel protein expression in cell surface were confirmed by biochemical and confocal imaging experiments. From the data, phosphorylation of the channel protein is likely to be important in initiating endocytosis. It is clear in the study of Kv l .4 modulation by GIP that Kv l .4 endocytosis is induced by indirect stimulation of Kv l .4 phosphorylation by the hormone (Figure 5.1). Activated GIP receptors, GPCRs, promote enzymatic activity of P K A , resulting in phosphorylation at threonine 601 in the C-terminus of the channel protein (Figure 3.3). In addition several P K A inhibitors, for instance, H-89 and Rp-cAMP, inhibit the enzymatic activity as well as the effect of GIP on Kv l .4 macroscopic current (Figure 3-2). Recently, MacDonald and his colleagues indicated that PKA- , P K C - or PI3K-dependent phosphorylation of Kv2.1 is involved in the down-regulation of the channel macroscopic current without any significant changes in biophysical properties in GLP-1 treated pancreatic P-cells (MacDonald et al, 2003). Kv l .5 endocytosis, too, may be regulated by phosphorylation. Kvl .5 amino acid residues 65 to 93 comprise a proline-rich region containing two consensus Src kinase binding sites (Figure 2.7); Src may well bind to this region and phosphorylate one or more tyrosine residues. Since tyrosine phosphorylation has been reported to affect Kvl .5 surface expression, as well as Kv l .2 endocytosis, this proline-rich domain seemed a likely candidate for an important role in channel 158 internalization (Holmes et al, 1996; Nesti et al, 2004). Mutation of the first proline-rich domain, the core of an SH3 domain binding site, in the Kvl .5 N-terminus abolished the effect of p50 over-expression and substantially increased baseline current densities such that they were similar to those of the wild-type channel in p50 over-expressing cells (Figure 2.8) or dynamin inhibitory peptide (DlP)-treated cells. Interestingly, this deletion mutant also did not respond to incubation with DIP (Figure 2.8). These results suggest that phosphorylation of the cytosolic residues of the channel may trigger internalization of the channel protein. Both Kv l .4 and Kvl .5 channel proteins are internalized through a dynamin-dependent endocytosis mechanism. Data in figures 2.2 and 3.4 show that DIP mimics the effect of p50 over-expression on redistribution of Kvl .5 channel proteins, and that DIP inhibits the endocytosis normally induced by activation of GIP receptor on Kvl .4 . Data in figures 2.1 and 2.6 demonstrates that disruption of dynein function carrying a vesicle cargo via p50 over-expression increases Kvl .5 channel protein expression at the cell surface and the dynein motor interacts, directly or through a linker protein complex, with the channel protein. This clearly indicates that dynein motors are involved in Kvl .5 retrograde trafficking. This also suggests the coordination of different motors, for example, non-conventional myosin and minus-directed microtubule-based motors in the endocytosis mechanism. This will be discussed below. 5.2 Recycle or degradation? What is the destination of the endocytosed channel protein? Two possible fates exist for the endocytosed protein: it may be recycled to the plasma membrane or it may be degraded. Sorting in early endosomes is required in making this determination. 159 Lakadamyali and his colleagues reported that endocytosed protein are sorted in the plasma membrane depending on binding specific adaptor proteins, and transported into recycling endosome or degradation endosome (Lakadamyali et al, 2006). This study suggests that multiple internalization mechanisms may be involved in the sorting of a single protein, regulating the destination of that protein. For example, a certain channel protein could be sorted into a static early endosome to be recycled, and then, after a certain time, it could be directed to a dynamic early endosome for degradation. One possible candidate process to regulate this sorting might be phosphorylation. For further study, investigations using Rab proteins and their binding partners may be very important. Rab proteins can be used as specific markers that indicate subcellular organelles (Zerial and McBride, 2001). In the case of K v l . 5 , as presented in Chapter 2, the internalized channels colocalize with EEA1 in the peripheral region of the cell, indicating the presence of endocytosed Kvl .5 in the early endosome (Figure 2-x). EEA1, another integral protein in early endosomal membrane, is a binding partner of GTP-bound Rab5, functioning as a Rab5 effector, directing transport vesicles from the plasma membrane to early endosome (Zerial and McBride, 2001). 5.3 Complexity of vesicle transport: Motor coordination Vesicle transport operated by molecular motors is not simple. As mentioned above, different cytoplasmic motors must be coordinated to orchestrate vesicle transport for at least three reasons. Firstly, actin filaments do not reach all organelles in the vesicle transport pathway, from the ER to the cell surface and from the plasma membrane to late endosomes. Actin filaments are cortical and do not extend far into the cell. Secondly, 160 microtubules do not cover the whole surface of the cytoplasmic side of the plasma membrane, so that coordination between actin-based motors and microtubule-based motors is required. Finally, both anterograde and retrograde trafficking are required for each protein because the protein must be transported from the cytosol to the cell surface after synthesis and be retrieved from the membrane to cytosol for recycling or degradation. For coordination of molecular motors, direct or indirect linkage between two different motors may be required. Evidence of such a connection has been reported recently. For example, myosin V binds to the microtubule plus-end via the order like myosin V -melanophilin-EB 1 -microtubule, and myosin VI also interacts with the microtubule plus-end in the sequence of myosin Vl-cytoplasmic linker protein-170 (CLIP-170), another plus-end protein involved in dynein targeting, binding to microtubule (Wu et al, 2005; Lantz et al, 1998; Sheeman et al, 2003). Myosin V not only interacts directly with conventional kinesin via its tail domain but myosin V also shares a subunit, DLC8, with the dynein motor (Huang et al, 1999; Espindola et al, 2000). Furthermore, recent reports suggest an interaction of F-actin with microtubule-based motors such as KIF9 kinesin and CHOI, a kinesin of the KLP1 subfamily (a minus-directed kinesin motor) (Piddini et al, 2001; Kuriyama et al., 2002). Interestingly, Deacon and his colleagues demonstrated that p l50 g l u e d , a subunit of dynactin, directly interacts with K A P , a subunit of the kinesin II (KIF3) heterotrimer, and they showed that dynamitin (p50) over-expression inhibits kinesin II activity, suggesting the role of dynactin in bidirectional transport, in terms of both forward and retrograde trafficking (Deacon et al., 2003). The preliminary data presented below from my additional experimental work seem to provide evidence for the motor coordination on Kv channels. 161 5.4 Effect of dynamitin over-expression in Kvl.4, Kv4.2 and hERG channels As shown in Figure 5-2, the effects of p50 over-expression on Kvl .4 , Kv4.2 and hERG channels in HEK-293 cells were opposite to those observed in Kvl .5 currents. Over-expressed p50 reduced Kvl .4 , Kv4.2 and hERG current densities: from 1.8 ± 0.15 nA/pF, «=12 to 0.9 ± 0.12 nA/pF, n=12, p<0.01(Figure 5-2 A , B); from 0.6 ± 0.08 nA/pF, w=12 to 0.3 ± 0.03 nA/pF, n=7, p<0.05 (Figure 5-2 C, D); and from 49.7 ± 2.45 pA/pF, n=5 to 6.73 ± 1 . 4 pA/pF, n=6; p O . O l , respectively (Figure 5-2 E, F). Furthermore, Kv l .4 current density was decreased by treatment with nocodazole, a strong microtubule depolymerising drug, suggesting that the effect of p50 over-expression on Kv l .4 current is indeed likely to be mediated by changes in the functioning of a microtubule-based molecular motor (Figure 5-3). To determine whether p50 over-expression affects the macroscopic kinetics of Kvl .4 , activation and inactivation properties were tested in HEK-293 cells stably expressing Kvl .4 . The half-activation value in p50 over-expressed cells (-26.4 ± 3.26 mV) was very close to that of cells expressing only empty vector (pGFP) (-27.7 ± 2.76 mV) (Figure 5-4 A); and inactivation properties were not significantly different between the two groups (-57.5 ± 1.6 mV and -57.9 ± 2.6 mV in p50 over-expressed and empty vector, respectively) (Figure 5-4 B). These results suggest that reduction of Kv l .4 macroscopic currents was not caused by changes in kinetic properties but instead by a reduction of channel expression at the plasma membrane. Next, I exploited deletion analysis of the Kv l .4 N-terminus and an analysis of chimeric substitution of residues from the Kvl .5 channel to identify specific domains of Kv l .4 important for cell surface expression that is p50 over-expression-dependent. The effect of p50 over-expression was tested on two N-terminally deleted mutants: in AN19, 162 Kvl .4 currents were still decreased by p50 over-expression (Figure 5-5 A), but the AN147 mutant was unresponsive (Figure 5-5 B). Interestingly, a chimera in which the Kvl .4 N -terminus replaces that of Kvl .5 in the Kvl .5 channel behaved as the Kv l .4 wild type did in p50 over-expressed cells (Figure 5-5 C, D). These results strongly indicate that the N -terminus of K v l .4 is important for the effect of p50 over-expression. Taken together, p50 over-expression effects on the channel expression may be positive or negative depending on the specific channel, suggesting that p50 over-expression disrupts both anterograde and retrograde transport pathways and specificity seems to be determined by residues in the N-termini residue of both Kvl .5 and Kvl .4 . Therefore, a key to understanding these phenomena may be whether a kinesin involved in a specific channel trafficking recruits dynactin to associate with its cargo. 5.5 How to overcome a dilemma of motor coordination? An important issue arising from studies on coordination among microtubule-based motors is the question of a possible 'traffic jams' because plus- and minus-directed microtubule-based motors use the same track. To overcome this problematic issue, molecular motors use several strategies to get past each other. In principle, motor activity can be regulated at two levels: by turning the motor on or off, and by inhibiting or promoting its association with the track. When kinesin does not bind its cargo, the motor protein enters into an inhibited state exploiting an intramolecular folding mechanism. The inhibition is mediated by interaction of the motor tail domain with its motor domain, leading to low ATPase activity which minimizes the stepping rate of the motor (Coy et al, 1999; Friedman and Vale, 163 1999) . In the case of dynein, association of the motor with dynactin increase processivity, i.e., the run-length, as well as the ATPase activity (King and Schroer, 2000; Kumar et al, 2000) . This inhibition, at least in the case of dynein, is regulated by phosphorylation, because p l50 g l u e d , a subunit of dynactin, can bind dephosphorylated intermediate chains (IC) of the dynein motor, but not phosphorylted IC, a property confirmed with a site-directed mutation analysis at Serine 84 (Vaughan et al, 2001). In addition, kinesin moves along a single microtubular protofilament with high processivity, while dynein is able to jump from a bound protofilament to an adjacent one (Wang et al, 1995; Ray et al, 1993). Therefore, as a stronger processive motor, a kinesin with cargo could 'bulldoze' through and pass an inhibited dynein, in other words, dynein adopts a 'yield when necessary' strategy (Mallik and Gross, 2004). 5.6 Summary The work described in this thesis examined some of the mechanisms involved in Kv channel trafficking, with a particular focus on endocytotic processes. It demonstrates that Kv channel endocytosis contributes to channel surface expression, and suggests that dynein, a minus-directed microtubule-based motor, plays a critical role in the retrograde trafficking of internalized Kv channel proteins. It also demonstrates that activation of G-protein coupled receptor signalling initiates K v channel endocytosis through a phosphorylation-dependent mechanism, which results in the down-regulation of Kv channel currents. Overall, these results suggest that extracellular stimuli can control cell excitability over the long-term via the regulation of functional Kv channel expression in the plasma membrane. 164 Figure 5.1 Endocytosis of Kvl.4 protein resulted from PKA-dependent phosphorylation. 1. GIP receptor is activated by GIP binding. 2. G-protein heterotrimer a-subunit is dissocated from Py-subcomplex and stimulates adenylate cyclase. 3. PKA-enzymatic activity is increased and P K A phosphorylates T601 in the Kvl .4 C-terminus. 4. K v l .4 protein endocytosis occurs. 165 Kv1.4 A B Dynamitin Kv4.2 C D Dynamitin Figure 5.2 p50 over-expression significantly decreases Kvl.4, Kv4.2, and hERG current levels. (Legend on the following page) 166 Figure 5.2 p50 over-expression significantly decreases Kvl.4, Kv4.2, and hERG current levels. (A, B) Effect of p50 over-expression on Kv l .4 . Peak currents from HEK-293 cells stably expressing Kv l .4 transfected with empty vector (pGFP), or p50-pGFP. From -100 mV, cells were depolarized to between -70 and +80 mV in 10-mV steps. (C, D) Effect of p50 over-expression on Kv4.2. Peak currents were collected in Kv4.2 stably expressing HEK-293 cells. Holding potential was -80 mV and cells were depolarized from -70 to +80 mV. (E, F) Effect of p50 over-expression on hERG. HEK-293 cells stably expressing hERG were used to detect peak currents. From -80 mV, cells were depolarized to between -50 and +60 mV in 10-mV steps. p50-GFP and empty vector were transfected for p50 over-expression and control, respectively. Dotted line denotes the zero current level. 167 A B Figure 5.3 Over-expression of p50 does not alter the macroscopic kinetics of Kvl.4. (A) Activation properties and (B) inactivation properties of Kv l .4 were determined in p50 over-expressed HEK-293 cells. A l l data represent the mean ± S.E.M. from three to five independent experiments and significance determined using Student's /-test. 168 Figure 5.4 Nocodazole pretreatment decreases Kvl.4 currents in HEK-293 cells. (A) HEK-293 cells stably expressing Kvl .4 were pretreated with 35 u M nocodazole. Only DMSO was added to control cells. Dotted and solid lines represent traces in control and Nocodazole-pretreated cells, respectively. (B) Peak current densities from nocodazole-treated cells (open-circles) and control cells (closed-circles) (*: p< 0.05). 169 AN19 Kv1.4 AN 147 Kv1.4 Figure 5.5 The N-terminus of Kvl.4 is essential for the response to p50 over-expression. (A, B) Peak currents were measured in A N 19 (A) and AN 147 Kv l .4 (B) H E K -293 cells expressing the Kv l .4 N-terminus truncated mutants with depolarization between -70 and +80 mV in 10-mV step from -100 mV. A l l data represent the mean ± S.E.M. and significance was tested using Student's Mest (**: p< 0.01). 170 Voltage (mV) Figure 5.6 Kvl.4 N-terminus endows Kvl.5 with the opposite result of p50 over-expression. (A) Schematic diagram of a chimeric substitution of residues from the Kvl .5 channel with the Kvl .4 N-terminus. (B, C) Peak currents were measured in Kvl .4N/Kvl .5 chimera-expressing HEK-293 cells during depolarization between -70 and +80 mV in 10-mV steps from -80 mV. (B) Example traces of the chimera with empty vector (pGFP) (top) and p50 over-expression (bottom). The chimera showed N-type inactivation and responded as wild type Kvl .4 did. (C) Peak current amplitudes at +80 mV from controls (filled symbols) and p50-overexpressing cells (open symbols) were normalized to cell capacitance. A l l data represent the mean ± S.E.M. and significance was tested using Student's t-test (**: p< 0.01). 171 Figure 5.7 Dynamitin (p50) over-expression affects bidirectional trafficking. Over-expressed p50 may not only inhibit motor activity that is carrying cargo but it also may reduce the rate of binding to microtubules in both kinesin and dynein motors. Blue beads, a microtubular protofilament used by both kinesins and dyneins; Yellow tuning forks, kinesins; Red tuning forks, dyneins. 172 5.7 REFERENCES Coy DL, Hancock WO, Wagenbach M , Howard J. Kinesin's tail domain is an inhibitory regulator of the motor domain. Nat Cell Biol. 1:288-92, 1999 Culver-Hanlon TL, Lex SA, Stephens A D , Quintyne NJ, King SJ. A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules. Nat Cell Biol. 8:264-70, 2006 Deacon SW, Serpinskaya AS, Vaughan PS, Lopez Fanarraga M , Vernos I, Vaughan KT, Gelfand VI. Dynactin is required for bidirectional organelle transport. J Cell Biol. 160:297-301,2003 Espindola FS, Suter D M , Partata L B , Cao T, Wolenski JS, Cheney RE, King SM, Mooseker MS. The light chain composition of chicken brain myosin-Va: calmodulin, myosin-II essential light chains, and 8-kDa dynein light chain/PIN. Cell Motil Cytoskeleton. 47:269-81, 2000 Friedman DS, Vale RD. Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain. Nat Cell Biol. 1:293-7, 1999 Holmes TC, Fadool DA, Ren R, Levitan IB. Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain. Science. 274:2089-91, 1996 Huang JD, Brady ST, Richards BW, Stenolen D, Resau JH, Copeland N G , Jenkins N A . Direct interaction of microtubule- and actin-based transport motors. Nature. 397:267-70, 1999 King SJ, Schroer TA. Dynactin increases the processivity of the cytoplasmic dynein motor. Nat Cell Biol. 2:20-4, 2000 Kumar S, Lee IH, Plamann M . Cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation. J Biol Chem. 275:31798-804, 2000 Kuriyama R, Gustus C, Terada Y , Uetake Y , Matuliene J. CHOI , a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis. J Cell Biol. 156:783-90, 2002 Lakadamyali M , Rust MJ , Zhuang X . Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell. 124:997-1009, 2006 Lantz V A , Miller K G . A class VI unconventional myosin is associated with a homologue of a microtubule-binding protein, cytoplasmic linker protein-170, in neurons and at the posterior pole of Drosophila embryos. J Cell Biol. 140:897-910, 1998 173 MacDonald PE, Wang X , Xia F, El-kholy W, Targonsky ED, Tsushima RG, Wheeler M B : Antagonism of rat P-cell voltage-dependent K + currents by exendin-4 requires dual activation of the cAMP/protein kinase A and phosphatidylinositol 3-kinase signalling pathways. J Biol Chem. 278:52446-53, 2003 Mallik R, Gross SP. Molecular motors: strategies to get along. Curr Biol. 14:R971-82, 2004 Nesti E, Everill B, Morielli A D . Endocytosis as a mechanism for tyrosine kinase-dependent suppression of a voltage-gated potassium channel. Mol Biol Cell. 15:4073-88, 2004 Piddini E, Schmid JA, de Martin R, Dotti CG. The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9. EMBO J. 20:4076-87, 2001 Ray S, Meyhofer E, Milligan RA, Howard J. Kinesin follows the microtubule's protofilament axis. J Cell Biol. 121:1083-93, 1993 Sheeman B, Carvalho P, Sagot I, Geiser J, Kho D, Hoyt M A , Pellman D. Determinants of S. cerevisiae dynein localization and activation: implications for the mechanism of spindle positioning. Curr Biol. 13:364-72, 2003 Valetti C, Wetzel D M , Schrader M , Hasbani MJ , Gil l SR, Kreis TE, Schroer TA. Role of dynactin in endocytic traffic: effects of dynamitin over-expression and colocalization with CLIP-170. Mol Biol Cell. 10:4107-20, 1999 Vaughan PS, Leszyk JD, Vaughan KT. Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin. J Biol Chem. 276:26171-9, 2001 Wang Z, Khan S, Sheetz MP. Single cytoplasmic dynein molecule movements: characterization and comparison with kinesin. Biophys J. 69:2011-23, 1995 Wu X S , Tsan GL, Hammer JA 3rd. Melanophilin and myosin Va track the microtubule plus end on EB1. J Cell Biol. 171:201-7, 2005 Zerial M , McBride H. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol. 2:107-17, 2001 174 APPENDIX I Animal care certificates The University of British Columbia ANIMAL CARE CERTIFICATE ••'ROior.oi NUMHfcR A00-0049 I N V E S T I G A T O R O R C O U R S E D I R E C T O R : Fedida, 1). D r P A R r n i : N r - Physiology P R O J E C T O R C O U R S E T I T L E : Cytoskeletal interactions with voltage-gated cardiac potassium channels A N I U A L S : Rabbits 20 rats - pups 100 Mice 30 S T A R T D A T E : 98-07-01 A P P R O V A L D A T E : June 24, 2002 F U N D I N G A G E N C Y : Heart and Stroke Foundation of B.C. & Yukon T h e A n i m a l C a r e C o m m i t t e e h a s e x a m i n e d a n d a p p r o v e d t h e u s e of a n i m a l s for t h e a b o v e ; e x p e r i m e n t a l p r o j e c t ; o r ; : t e a c h i n g ; c o u r s e ? a n d - i h a v e i b e e n ^ g i v e n i a n i a s s u r a n c e i t h a t i t h e i a n i m a l s ' i h v o l v e d i w i i l b e c a r e d i i f o r i n a c c o r d a n c e i . w i t h i t h e i i p r i n c i p A n i m a l s - A G u i d e f o r C a n a d a , p u b l i s h e d by 1 t h e C a n a d i a n C o u n c i l o n A n i m a l C a r e . A p p r o v a l of t h e U B C C o m m i t t e e o n A n i m a l C a r e b y o n e of: D r . W . K . M i l s o m , C h a i r O r . J . L o v e , D i r e c t o r , A n i m a l C a r e C e n t r e • M s . L . M a c d o n a l d , M a n a g e r . A n i m a l C a r e C o m m i t t e e T h i s c e r t i f i c a t e is v a l i d for o n e y e a r f r o m t h e a b o v e s t a r t o r a p p r o v a l d a t e ( w h i c h e v e r i s l a t e r ) ^ p r o v i d e d t h e r e i s i n o e h a n g e i m t h e e ^ C C A C a n d s o m e g r a n t i n g a g e n c i e s . A copy of this certificatemust be displayed in your animal facility. ; Office of Research :Services:and Administration 323-2194 Health Sciences Mall, Vancouver, V6T 1Z3 Phone: 604-822-8155 FAX: 604-822-5093 

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