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Lynx1 and the Β2v287l Mutation Affect the Stoichiometry of the Α4β2 Nicotinic Acetylcholine Receptor

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Lynx1 and the Β2v287l Mutation Affect the Stoichiometry of the Α4β2 Nicotinic Acetylcholine Receptor Lynx1 and the β2V287L Mutation Affect the Stoichiometry of the α4β2 Nicotinic Acetylcholine Receptor Thesis by Weston A. Nichols In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2014 (Defended May 1, 2014) ii © 2014 Weston A. Nichols All Rights Reserved iii To my mother, father, and Peden. iv Acknowledgements First and foremost, thank you to Henry Lester, my Ph.D. advisor. Henry accepted me into his lab before it was clear that I would be useful as a graduate student, and provided me with a wonderfully appropriate mix of guidance and freedom. Early on, I wanted to start as many projects as possible, and pitched many that made no sense from a practical perspective, and probably several other perspectives. Henry told me not to do them in a way that forced me to learn the process of scientific diligence, and to take ownership of the projects I did pursue. Henry taught me this process by spending a lot of time with me through many meetings, including Fluorescence Club, α Club, and going for coffee at the Red Door. One of the most salient items that Henry taught me is how to plod through data slowly to come to a deeper understanding of the data and how (or if) it fits into a larger picture. I think this is something that is very hard to teach. Henry did it by spending a lot of time with me. Thank you to my thesis committee, Ray Deshaies, Dennis Dougherty, and David Prober. You have been great to me, and provided much helpful advice. Thank you to everyone in the Lester Lab during my time here. Thank you to Eloisa Imel for welcoming me to the lab when I was starting and helping with many issues along the way. Thanks to Purnima for ordering and equipment maintenance, which allowed me to always keep experiments going. Thanks to Elisha Mackey for teaching me how to clone efficiently when I first started in the Lester Lab. Thanks to the other grad students, Crystal Dilworth, Rell Parker, and Teagan Wall for many great discussions, help with experiments, and camaraderie. Thanks to many former members of the lab who taught me much about science, trained me in a wide array of experimental techniques and helped me troubleshoot: Beverly Henley, Rachel Penton, Ying Wang, Cheng Xiao, Charlene Kim, and Jonathan Wang. Thank you to Chris Richards for teaching me the process of science at a high level and how to implement experiments at a technical level. Thank you also for bringing me up to speed very quickly on microscopy and neuronal culture when I was a new hire and you were focused on becoming a professor. Thanks to Chris and Rahul Srinivasan for teaching me the majority of what I know about microscopy. Thanks to Brandon Henderson for teaching me a great deal about pharmacology and chemistry, and for performing the electrophysiology experiments on mammalian cells in this thesis. v Thank you to Shawna Frazier for many things including teaching me how to use the FlexStation and the finer points of dose-response relations. More importantly, thank you for your companionship and causing me to look forward to weekends in lab. Perhaps now is an appropriate time to reveal… that I did not read the manual. Thank you to Bruce Cohen for sharing his vast knowledge of electrophysiology, neuroscience and ADNFLE, and for fun and educational discussions on a wide range of topics including pharmacology, ADNFLE, finance and real estate, cooking, meditation, and many more. Thank you to Julie Miwa (who discovered lynx1) for getting me started in the Lester Lab, and for guidance and leadership on the lynx portion of this thesis. Thank you to Caroline Yu for her extremely competent and diligent help with both executing and planning experiments relevant to both the lynx and β2VL sections of this thesis. I am incredibly lucky to have worked with Caroline, an undergrad with incredible work ethic, excellent training from prior work with Chris Richards and Henry Lester, and the ability to quickly learn new concepts and techniques. I hope I can work with Caroline again someday. Thank you to the members of the Dougherty Lab, and especially to Chris Marotta for collaborating with me on the β2VL paper, and to Tim Miles for a great deal of technical help to Caroline and me with imaging experiments. Thank you to Bruce Hay, especially for support before I joined Henry’s lab. Our meetings during that time meant a great deal to me. Thank you for remaining committed. Thank you to Liz Ayala for making the administrative tasks easy for me, and for knowing and sharing how to get things done. Thank you to all my friends in Biology, and especially Mike Rome, Alex Webster, Eric Erkenbrack, and Jon Valencia. Thank you to Samy Hamdouche, my roommate and great friend. Together, we have set and achieved goals in a wide variety of different fields in the four years we have known each other. The coming years are going to be fun. Finally, thank you to my family: Mom, Dad, and Peden; I love you big time. Table of Contents Acknowledgements ........................................................................................................... iv Table of Contents .............................................................................................................. vi Chapter I: Introduction .................................................................................................. 1 Chapter II: Lynx1 shifts α4β2 nicotinic receptor subunit stoichiometry by affecting assembly in the endoplasmic reticulum ...................................................... 26 Abstract ..................................................................................................................... 27 Introduction ............................................................................................................... 28 Experimental Procedures .......................................................................................... 31 Results ....................................................................................................................... 38 Discussion ................................................................................................................. 52 References ................................................................................................................. 56 Chapter III: The β2V287L mutation suppresses formation of the lower sensitivity stoichiometry of α4β2 and increases α5α4β2 on the plasma membrane ................ 60 Abstract ..................................................................................................................... 61 Introduction ............................................................................................................... 62 Experimental Procedures .......................................................................................... 67 Results ....................................................................................................................... 73 Discussion ................................................................................................................. 92 References ................................................................................................................. 97 Chapter IV: Future Directions ................................................................................... 100 Chapter I: Introduction 2 In his studies of the neuromuscular junction in 1851, Claude Bernard discovered that signals could be transmitted between cells by chemicals that we now call neurotransmitters. He demonstrated that curare, a toxin isolated from plants used to make arrow toxins for hunting, could block this transmission in a reversible manner. Today, we know that curare acts as a competitive antagonist to acetylcholine blocking neurotransmission at the nerve-muscle junction and causing paralysis. Nicotine binds and activates several of these receptor types, which are therefore called the nicotinic acetylcholine receptors (nAChRs). Over the past 25 years, a great deal of progress has been made towards understanding both the molecular and biophysical properties of nAChRs, as well as their relevance towards important biological processes in the brain. The nicotinic acetylcholine receptors The nAChRs are pentameric, ligand-gated ion channels. Their endogenous ligand, acetylcholine, as well as pharmacological agents such as nicotine, activate the channels. The specific proteins that make up the receptors for acetylcholine have been identified, cloned, and several can be expressed in heterologous systems such as Xenopus laevis oocytes or human embryonic kidney (HEK) cells. Heterologous expression enables the use of electrophysiology and microscopy to conduct precise pharmacological and biophysical experiments, several of which are described in this thesis. Neuronal nAChRs are expressed throughout the central and peripheral nervous system, and are made up of nine different α subunits and three different β subunits in both heteropentameric and homopentameric combinations such as α4β2 and α7, respectively. Different subtypes have sensitivities to ACh that vary over 3-4 orders of magnitude. The 3 most widely expressed receptor, α4β2, has two stoichiometries: (α4)2(β2)3 and (α4)3(β2)2, with ACh sensitivities of approximately 1 µM and 80 µM, respectively. Gating of the channel results in permeability to Na+, K+, and, for some receptor subtypes, Ca2+. In general, activation of nAChRs in the brain regulates the amount of other neurotrasmitters realeased into
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