An Atomic Scale Measurement from the Voltage Sensor in hERG Channels Using Lanthanide-Based Resonance Energy Transfer by Danielle Jeong B.Sc., Simon Fraser University, 2014 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Biomedical Physiology and Kinesiology Faculty of Science Danielle Jeong 2016 SIMON FRASER UNIVERSITY Summer 2016 Approval Name: Danielle Jeong Degree: Master of Science Title: An Atomic Scale Measurement from the Voltage Sensor in hERG Channels Using Lanthanide-Based Resonance Energy Transfer Examining Committee: Chair: Dr. William Cupples Professor Dr. Thomas Claydon Senior Supervisor Associate Professor Dr. Peter Ruben Supervisor Professor Dr. Glen Tibbits Supervisor Professor Dr. Jenifer Thewalt External Examiner Professor Department of Physics Date Defended/Approved: June 16, 2016 ii Ethics Statement iii Abstract The cardiac human ether-a-go-go related gene (hERG) channel is a voltage- gated potassium (Kv) channel that plays a fundamental role in cardiac repolarization. The importance of the hERG channel derives from its unusually slow activation (opening) and deactivation (closing) processes. Like other Kv channels, structural reconfigurations of the hERG voltage sensor upon membrane depolarization and repolarization underlie channel activation and deactivation, respectively. However, specific rearrangements of the voltage sensor that may dictate the unique slow activation and deactivation in hERG channels remain unclear. Lanthanide-based resonance transfer (LRET) is a spectroscopic technique that has previously demonstrated an ability to provide quantitative description of voltage sensor dynamics in an archetypal Kv channel. In this report, we outline a rationalized approach to applying LRET to examine hERG channel voltage sensor dynamics that may be of physiological significance. As a result, we report the first distance measurement from voltage sensors across the hERG channel pore. Keywords: hERG channels; lanthanide-based resonance energy transfer; voltage- gated potassium channels; voltage sensor iv Acknowledgements This project would not have been possible without the selfless assistance of many individuals. As such, I would like to first show my appreciation for those who have notably contributed to its progress. With respect to the donor chelate synthesis, Dr. Rikard Blunck from University of Montreal and Dr. Mattieu Starck, a post-doctorate fellow in Dr. Blunck’s lab, are to be recognized for their generosity in sharing the recipe they have developed. For graciously accommodating me to use their lab space and equipment for synthesizing the chelate, I would like to thank Dr. David Vocadlo from SFU Chemistry department and the members of his lab. Deep gratitude is specifically directed toward Hong Yee Tan, a PhD candidate from Dr. Vocadlo’s lab, and Hongwen Chen, a mass spectrometry specialist at SFU, for all the advice and patience they have provided throughout the synthesis troubleshooting phase. When it comes to addressing technical aspects of the LRET setup, the talent of Pawel Kowalski, an electronic technologist at SFU, is to be admired and appreciated. He has demonstrated utmost ingenuity through various tasks, which includes installing the interlock system, designing and building the delay generator, re-engineering the PMT gating mechanism, and extending the lifespan of the laser used in this project. On matters concerning the optics, I was also fortunate to have an inexhaustible access to the expertise of Dr. Eric Lin, a post-doctorate fellow in Dr. Glen Tibbits’ lab. I cannot be more thankful for his involvement in the alignment of LRET system apparatuses, as well as continued guidance in the maintenance of optical components. As well, I gratefully acknowledge the work of the former post-doctorate fellow in Dr. Tom Claydon’s lab, Dr. Stanislav Sokolov, in initiating the LRET project and establishing a foundation for me. It is upon his shoulders I stand. There are others who have offered more than a helping hand. Alison Li, a PhD candidate in Dr. Tibbits’ lab, is such an example. She not only has proactively arranged multiple sessions for me to use the MBB department spectrofluorometer and taught me how to operate the equipment, but has done so with persistent enthusiasm. The v kindness of Shyam Panchapakesan, a PhD candidate in Dr. Unrau’s lab, should also be noted in making the lab’s Nanodrop 2000 available for me to obtain the absorbance spectra recordings. Furthermore, I owe a heartfelt word of thanks to Ji Qi, a lab technician in Dr. Tom Claydon’s lab, for all her work in molecular biology. Her indefatigable ability to meet the demands to make all the constructs and cRNA used in this project is worthy of the highest praise. Every step forward taken through this project could not have been achieved without the gifts of trust and encouragement from my supervisor, Dr. Tom Claydon. I cannot thank him enough for instilling within me a love and passion for scientific research when I was an undergraduate student, and for making my decision to pursue graduate studies to be un-regrettable. To have Dr. Claydon as a role model in science and leadership has been the greatest source of inspiration for me to become a better science student and person. I know that he will continue to have such an effect on me in the years ahead. On the same note, I would like to show my whole-hearted appreciation for the members of my supervisory committee, Dr. Peter Ruben and Dr. Glen Tibbits, for their fatherly support since my undergraduate years. It is unbelievable how privileged I have been to receive their wise advice (though I seldom took them), and to have mentors who would accept me just as I am- neurotic and obstinate. I am also overwhelmed with gratefulness when I think of members of the Molecular Cardiac Physiology Group. From those who have religiously checked on my happiness quotient to those who have always lent an ear, I could not have asked for better lab mates. Most importantly, a special recognition should be given to my family for the relentless support they have provided behind the scene. I would like to specifically thank my dad for passing on his notorious stubbornness, for the same stubbornness in me was crucial in dealing with roadblocks encountered during the research process. I am also indebted to my mom for praying for me throughout the entire journey. The pillars of prayers that she had built were what had kept me standing strong. I will never be able to adequately express how grateful I am for my sister, Janelle, for injecting bursts of silliness back into my life whenever I was caught up in my work. Lastly, I would like to express my respect for my brother, Samuel, whose considerate nature often reminded me to look around and realize the people I have been blessed with during the duration of this project. vi Table of Contents Approval .......................................................................................................................... ii Ethics Statement ............................................................................................................ iii Abstract .......................................................................................................................... iv Acknowledgements ......................................................................................................... v Table of Contents .......................................................................................................... vii List of Tables ................................................................................................................... x List of Figures................................................................................................................. xi List of Acronyms and Symbols ...................................................................................... xii Chapter 1. Introduction ............................................................................................... 1 1.1. Voltage sensing in voltage-gated ion channels ....................................................... 1 1.1.1. Structural components of voltage-gated ion channels associated with voltage sensing .................................................................................. 2 1.1.2. The principle behind the voltage sensor .................................................... 3 1.1.3. Models of voltage sensor movement ......................................................... 5 1.2. Techniques used to reveal voltage sensor movement ............................................ 6 1.2.1. Description of voltage sensor dynamics from electrophysiological approaches ................................................................................................ 8 1.2.2. Structural insight on the voltage sensor ..................................................... 9 1.2.3. Fluorescence spectroscopy reveals conformational changes of the voltage sensor ......................................................................................... 10 1.3. Lanthanide-based resonance energy transfer (LRET) .......................................... 12 1.3.1. Principle of LRET ..................................................................................... 12 1.3.2. Advantages of LRET ............................................................................... 14 1.3.3. Properties of the lanthanide and donor complex .....................................