STRUCTURAL AND BIOCHEMICAL STUDIES OF THE HUMAN TWO PORE DOMAIN POTASSIUM CHANNEL K2Pl by Alexandria Nuesa Miller A Dissertation Presented to the Faculty ofthe Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy New York, NY April, 2013 ~y;} c;, ZD/3 s~ Date Dissertation Mentor Copyright by Alexandria N. Miller 2013 ABSTRACT Potassium (K+) channels are the largest family of ion channels in eukaryotes with over 70 genes in humans. They have diverse functional roles including controlling the firing duration and frequency of actions potentials in neurons and regulating water retention in the kidneys. K+ channels are highly-selective for K+ over other monovalent cations, can conduct K+ at rates approaching 108 ions per second and, like other ion channels, switch between conductive (open) and non-conductive (closed) states through a process called gating. + Two pore domain (K2P) potassium channels, originally called K background or leak channels, represent a subclass of K+ channels that function to establish and maintain the resting potential in eukaryotic cells. This process primes cells for diverse responses such as action potentials in excitatory cells and cell signaling cascades, which can direct growth and motility in non-excitable cell types. K2P channels have been shown to be gated by a range of cell stimuli and pharmacological agents including temperature, pH, polyunsaturated fatty acids, mechanical stress, and anesthetics. Not surprisingly, it is proposed that they are involved in physiological processes such as pain perception and anesthetic modulation. Structural studies of K2P channels would not only provide insight into how K2P channels are gated by these stimuli, but also may suggest strategies for the generation of K2P specific drugs. In the present work, I focused on both structural and biochemical studies of human K2P1, a member of the K2P family. I demonstrated, through biochemical studies of purified human K2P1, that the channel is dimeric and is glycosylated at residue asparagine 95. This glycosylation mutant was used for subsequent biochemical and structural studies of K2P1. Several limited proteolysis experiments and K2P1 sequence analyses were performed to identify a core unit of human K2P1. A human K2P1 N95Q truncation mutant, containing residues 22-303, was generated iii from these studies and successfully crystallized. Further truncation to the C-terminus and refinement strategies produced crystals of human K2P1 N95Q 22-288 that diffracted to 3.4 Å resolution. The K2P1 crystal structure reveals a number of features that may have general implications on K2P channel selectivity, gating and conductivity. A large extracellular domain is positioned above the extracellular entryway and creates an ion pathway with two side portals that can accommodate + + hydrated K ions. The coordination of K ions within the K2P1 selectivity filter recapitulates the + four-fold symmetry that is present in other K channel structures. However, the non-covalent interactions surrounding the K2P1 filter deviates from what has been previously observed and is two-fold symmetric, which may facilitate outer pore gating. An amphipathic C helix runs parallel to the cytosolic face of the membrane. Its connection and proximity to inner helices, which line + the pore and form the activation gate of other K channels, suggests that the C helix may be involved in inner helical gating. Finally, the K2P1 pore is exposed to the lipid bilayer through openings in the intramembrane molecular surface, accommodating electron density that we attribute to lipid or detergent alkyl chains. We speculate that this may be an access point for endogenous lipids as well as lipophilic compounds such as tetrahexylammonium (THexA), recently shown to be a K2P channel inhibitor. The human K2P1 structure defines the molecular architecture of the K2P channel family, lays a foundation for further investigation of how K2P channels are regulated by diverse stimuli, and provides insight for the development of K2P-specific therapies. Future work to determine a high- resolution structure of K2P1 is ongoing with the hopes of addressing key questions that still remain unanswered, such as the role of the K2P1 selectivity filter in outer pore gating. iv ACKNOWLEDGMENTS I would like to thank the many people who have made this thesis possible: Steve Long, for the support and guidance that he has provided throughout my graduate career. I am finishing my Ph. D. as someone who is more enthusiastic and intrigued about scientific questions and challenges than I was when I started graduate school. I am also more confident with my ability to think independently about my work. For these reasons, I am truly grateful to Steve for taking me on as one of his first graduate students. I am also indebted to the members of the Long lab: Melinda Diver, Xiaowei Hou, Veronica Kane Dickson, and Leanne Pedi. I have had the pleasure of working with these folks throughout my time at the Sloan-Kettering Institute and am forever thankful for both the scientific and personal support that they have given me. My advisory committee, Morgan Huse and Chris Lima, and Stew Shuman, who has been on both my advance to candidacy and thesis exam committees. The insightful questions that these folks posed to me were not only helpful for advancing my progress on my research project, but also allowed me to recognize weak points in my knowledge or my overall experimental strategy. I left these meetings always reminded of what I needed to improve on to mature as a scientist, and will be always grateful for their teaching and guidance through my graduate training. The MSKCC proteomics and monoclonal antibody core, Hediye Erdjument-Bromage, Frances Weis-Garcia, Olivera Grbovic, Paul Montanez and S. Lloyd Bourne, who assisted me with some of the work presented in this thesis. The directors of the X-ray Methods in Structural Biology Course at Cold Spring Harbor Laboratory for giving me the opportunity to learn a bit about X-ray crystallography. The NSLS staff for providing technical assistance on the X25 and X29 beamlines. v I was fortunate to be among the second class of graduate students in the Gerstner Sloan-Kettering (GSK) graduate program. I cannot say enough about the support that GSK offers its students. I would like to thank the staff for handling all aspects of the graduate program with professionalism and diligence. These people include staff of present and past: Associate Dean Linda Burnley, Maria Torres, Ivan Gerena, Iwona Abramek, former Associate Dean C. Gita Bosch, Adriane (Ady) Gupta, Carlene Grant, Maureen Rivera, and Yashira Laurent. Iwona greatly helped with organizing my dissertation and thesis defense. I am also grateful to Dean Ken Marians, Tom Kelly, Harold Varmus and Louis V. Gerstner, Jr. for their efforts in establishing the GSK Graduate School. Bob Goetz, Marion Lederer, Lauren Cowles, and former member Brian Koester, for giving me many years of comradeship through weekly sight-reading of string quartets. Heather Scherr, my former roommate and friend, for her help and insight throughout the first few years of graduate school. Among the many ways in which he has supported me the last few years, I want to thank Bhish for being a constant reminder to me about what is important in life. Finally, I would like to acknowledge my parents for giving me the educational, extracurricular, and travel opportunities that all children deserve, but not a lot get to experience. I would not be the person that I am today without my parents, and for this I am genuinely grateful. vi TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................... x LIST OF FIGURES ........................................................................................................................ xi LIST OF ABBREVIATIONS ....................................................................................................... xiv CHAPTER 1. INTRODUCTION .................................................................................................................... 1 1.1. Transport across the cell membrane .................................................................................. 1 1.2. Ion channels ....................................................................................................................... 3 1.2.1. Membrane potential and ion distribution at rest ......................................................... 3 1.2.2. Properties of ion channels .......................................................................................... 5 1.2.3. Function of ion channels ............................................................................................ 7 1.3. Principles of K+ channel selectivity, gating and conductance from structural studies ...... 8 1.3.1. K+ channel selectivity ................................................................................................. 9 1.3.2. K+ channel gating ...................................................................................................... 16 1.3.3. K+ channel conductance ............................................................................................ 19 1.4. Voltage-gated K+ channels and Ca2+-activated K+ channels ........................................... 22 1.5. Inward-rectifying K+ channels