REGULATION OF ATP-SENSITIVE POTASSIUM CHANNELS IN THE HEART DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Pharmacy in the Graduate School of The Ohio State University By Vivek Garg, M.Pharm ********* The Ohio State University 2009 Dissertation Committee: Approved by Professor Keli Hu, Advisor Professor Terry S. Elton Professor Lane J. Wallace ------------------------------- Advisor Professor Dale G. Hoyt Graduate Program in Parmacy Copyright by Vivek Garg 2009 ABSTRACT ATP-sensitive potassium channels (KATP channels) link the cellular energy levels to membrane potential and excitability in various cell types. They control many important functions like insulin secretion in pancreatic β- cells; vascular tone in vascular smooth muscle cells; and action potential duration in cardiac myocytes and neurons under ischemic conditions. In cardiac myocytes, it has been shown that KATP channels on plasma membrane are composed of Kir6.2 and SUR2A subunits in 4:4 stoichiometry. Though many regulators of KATP channels and signal transduction mechanisms regulating opening or closing of KATP channels have been identified, much less is known about the subcellular localization of KATP channels which can have a profound impact on the temporal and spatial regulation by its signaling modulators. Many studies have localized KATP channels to nucleus and mitochondria, besides cellular plasma membrane. However, it not known if these are the same cell surface KATP channels, which are also targeted to mitochondria, or some other isoform of KATP channel. The plasma membrane itself is not homogenous through out. It is interspersed by sub-domains rich in sterols and glycosphingolipids. In majority of cases, this sub-domain organization is orchestrated by special proteins called as ii caveolins, which are the main structural components of caveolae. Caveolae are small (50 to 100 nm), cholesterol and sphingolipid enriched “cave”-like invaginations of the surface membrane. These specialized lipid microdomains have the ability to selectively compartmentalize many signaling molecules, including many modulators of KATP channels. Since little is known about the subcellular locations of KATP channel protein, we designed our studies to characterize the localization of KATP channel protein in cardiac myocytes. We first endeavored to look at KATP channel localization along the plasma membrane in rat cardiac myocytes. Using a variety of different techniques on isolated murine cardiomyocytes, we found that majority of cardiac KATP channels are localized to caveolin-enriched membrane microdomains. Further, whole-cell voltage-clamp recording in both adult and neonatal cardiac myocytes confirmed our hypothesis that caveolae integrity is essential for activation of KATP channel by its modulator adenosine (Chapter 1). Adenosine released from ischemic myocardium is a very important modulator of KATP channels. These findings have significant implications for cardioprotective role of KATP channels during ischemic conditions. A signaling function for caveolins either on their own (direct) or by acting as scaffolding proteins (indirect) has also been described. To test its relevance for KATP channels, we employed HEK293T cells transfected with recombinant cardiac KATP channels (Kir6.2/SUR2A) with or without caveolin-3 (Cav-3, a muscle-specific caveolin isoform). We found that Cav-3 has significant inhibitory effect on KATP channel current density which can be iii reversed by a scaffolding domain peptide from caveolin-3 protein sequence (CSD) (Chapter 2). This crucial experiment indicates a very interesting, dual regulation of KATP channels by caveolins and caveolae. Though caveolae structure ensures that KATP channel modulators are close to the channel proteins for efficient signal transduction, caveolin-3 protein through direct or indirect interaction with channel proteins makes sure that they remain inhibited until required. Whether Kir6.2 containing KATP channel are present in mitochondria or not is controversial; nevertheless no one has ever studied the trafficking aspect of KATP channel to mitochondria. In our effort to elucidate the sub- cellular localization of cardiac KATP channels, we hypothesized that localization of Kir6.2-containing KATP in mitochondria can be increased by activation of protein kinase C (PKC). Utilizing KATP-deficient COS-7 cells, we reported a novel finding that a specific protein kinase C (PKC) isoform, PKCε, promotes mitochondrial import of Kir6.2-containing KATP channels from cytosol. These findings were further corroborated with functional data using mitochondrial potential measurement studies. Collectively, our data demonstrated that besides bulk plasma membrane, Kir6.2 containing cardiac KATP channels are localized to two distinct sub-cellular locations, namely caveolae and mitochondria. Their localization can be modulated by specific regulatory pathways, and furthermore iv furthermore their regulation can be affected by their sub-cellular localization. This provides valuable insight into the mechanisms regulating KATP channels, which have been implicated for cardioprotection under ischemic conditions. v Dedicated to My parents & family vi Acknowledgements I would like to pay a tribute to all my teachers (past and present) through this thesis. There are so many people to thank for who have contributed directly or indirectly to this work by, influencing my thinking, discussions, advice and of course blessings and wishes. My deepest gratitude is due to my advisor Dr. Keli Hu for her continued support, guidance and encouragement though out my graduate work; for allowing me to work independently on research areas of my interest. Her attention to detail, focus and fruitful suggestions made my work worthy of presentation. I wish to thank Dr. Jundong Jiao, who started me on patch clamp studies. I also thank Arun Sridhar, Veronique Lacombe for helpful discussions on perforated-patch and voltage-clamp studies. My thanks are due to Dr. Douglas Pfeiffer for discussions regarding mitochondrial studies and allowing me to work in his lab for some of the experiments. I would like to thank my committee members Dr. Lane Wallace, Dr. Terry Elton, Dr. Dale G. Hoyt for their time, effort and sharing their valuable opinions regarding this work. I also thank the faculty members of the pharmacology division especially Dr. Lakhu Keshvara, Dr. Popat N. Patil, Dr. Kari Hoyt, Dr. Cynthia Carnes for their encouragement, conversations, and time to time intellectual stimulation. My training as a research scientist is greatly enriched with your interactions. I thank all of my friends and wonderful colleagues in the division Vaibhav, Zhaogang, Tongzheng, Brandon, Ryan, Rachel, Raeann, Sarmistha who helped me in different ways, be it reagents or discussions regarding individual experiments. I will never forget my friends at OSU. You guys made vii my stay here truly enjoyable. Thanks for sharing times of happiness and celebration; and emotional support in times of stress. I am just humbled by all kinds of contributions and sacrifices made by my parents Dr. Pawan Kumar Garg, Mrs. Kamla Garg and family members Priya, Rishubh, Upma, Anupma, Sanjeev, Tarun, Rohan, Honey, and Aryan. I would have never reached at the stage where I am now without them. Though far, you are always in my thoughts as a perpetual source of inspiration. I wish to thank you all for your unconditional love, blessings and happiness. Last but not the least, I am highly grateful to my wife Priyanka for being supportive and infinitely patient in tolerating my business. Her support and encouragement was in the end what made this dissertation possible in time. She was always there cheering me up in all my endeavors. viii VITA July 18, 1977……………………...Born – Punjab, India 1995 – 1999.………………………Bachelor of Pharmacy Punjab University, India 2000 – 2002………………………..Master of Pharmcy Punjab University, India 2004 – Present……………………Graduate Teaching Associate The Ohio State University, Columbus, Ohio PUBLICATIONS 1. Garg V, Jiao JD, Hu K. (2009) ATP-sensitive K+ channels are regulated by caveolin-enriched microdomains in cardiac myocytes. Cardiovasc Res 82: 51-58. 2. Jiao J, Garg V, Yang B, Elton TS, Hu K. (2008) PKC-epsilon induces caveolin-dependent internalization of vascular ATP-sensitive K+ Channels. Hypertension 52: 499-506. ix 3. Jiao JD*, Garg V*, Yang B, Hu K (2008) Novel functional role of heat shock protein 90 in ATP-sensitive K+ channel-mediated hypoxic preconditioning. Cardiovasc Res 77: 126-33 (*equal contribution). 4. Garg V, Hu K (2007) Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane. Am J Physiol Heart Circ Physiol 293: H322-332. 5. Singh A, Garg V, Gupta S, Kulkarni SK (2002) Role of antioxidants in chronic fatigue syndrome in mice. Indian J Exp Biol 40: 1240-1244. FIELDS OF STUDY Major Field: Pharmacy x TABLE OF CONTENTS PAGE Abstract……………………………………………………………….…………..ii Dedication…………………………………………………………….………….vi Acknowledgements………………………………………………….………….vii Vitae…………………………………………………………………….………..ix List of Figures………………………………………………………….………..xiv Abbreviations………………………………………………………….………...xvi CHAPTER 1. INTRODUCTION………………………………..………..1 1. ATP-sensitive potassium channels……………………………………...1 1.1. Molecular basis……………………………………………...……….......3 1.2. Biophysical properties………………………………………...…………6 1.3. Regulation…………………………………………………………….......7 1.3.1. Nucleotide regulation………………………………........….……7 1.3.2. Pharmacological