STRUCTURAL AND FUNCTIONAL DYNAMICS OF SEROTONIN TRANSPORTER GENE VARIANTS By Meagan Anne Quinlan Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmacology May 10, 2019 Nashville, Tennessee Approved: Brian Wadzinski, PhD Heidi Hamm, PhD Kevin Schey, PhD Randy Blakely, PhD ACKNOWLEDGEMENTS The completion of this dissertation would not be possible without the unwavering support of several people that have helped me immensely along the way. I first must give my greatest acknowledgment to my Ph.D. mentor Dr. Randy Blakely, without whom this accomplishment would not be possible. Being able to research the serotonin transporter, the protein he cloned back in 1989, has been the highest honor. His depth of knowledge and his unwavering support was unmatched during all the experiments I thought failed. He did always find that silver lining in the blank blots. The enthusiasm he gleaned when I would have exciting new data and even the countless hours we discussed the nuances of serotonin transporter kinetics as we trialed our way through data that didn’t always make sense, will definitely not be forgotten. Needless to say, there was not a better mentor that could have taken me through this journey. I was also lucky to have a supportive thesis committee. To Dr. Brain Wadzinski, Dr. Heidi Hamm, Dr. Ana Carnerio, and Dr. Kevin Schey, all this work definitely would have not been possible without all your insightful questions and constructive critiques. Thank you all for your encouragement and help over the past years. Another enormous thank you must go to the lab members of the Blakely lab, both at Vanderbilt and Florida Atlantic University. I was fortunate enough to have several post-doctoral fellows that took me under their wing as I made my way through graduate school. My first mentor in the lab was Dr. Ran Ye, who showed me everything I know about protein biochemistry and really encouraged me in my early years of graduate school. Dr. Nicole Baganz was always there for a listening ear and a shoulder to cry on during times when it seemed none of my experiments were working. Last, but certainly not least, Dr. Matthew Robson who humored my unbridled optimism and ambition when I was a young grasshopper. He helped with so many of my crazy experimental ideas, including making transparent brains and we danced our way those countless synaptosomes uptakes and head twitch assays. Then there is the tremendous help of the Blakely lab research assistants, who were not only there for science support, but also all became great friends. A massive shout out to Jane Wright, Chris Svitek ii and Qiao Han at Vanderbilt, who were there during my early graduate career as I found stumbled away around in my first year in the lab. An especially big thank you must also go to Rania Katmaish, who I had the greatest pleasure of training while at Florida Atlantic University. She quickly became by far the most productive and helpful person as I was finishing up my Ph.D. Her enthusiasm for science was contagious, and she helped keep my spirits up until the end. I have to also give an enormous thank you to my family that have been there literally from day one. My parents were there for me every step of the way and without their unwavering support, I would definitely not be here. From encouraging me to participate in Science Fairs and Science Olympiads, to the yearly physic inspired Christmas gifts, and to indulging all my wild interests through the years in everything from astronomy to geology, I truly owe the fostering of my love of science to you two. To my older sister, Erin, who I always looked up to since a young age. Even if it seemed like I was always copying everything she did, I really must admit she is the one that initially inspired me to pursue science after see her win the science fair herself in elementary school. She has always been there and has always had faith in my ability. And last, but certainly not least, to my countless friends from around the world that supported me along the way. I am so lucky to have numerous friends in various places that kept in contact with me through the years and many moves. My friends always seem to know when to call or text me when I needed it most during this process. I am eternally grateful for all that I have and know that none of this would have been possible without the help of a number of people. It is not lost on me that a central theme of my dissertation is that the proteins do not act alone, but require a complex set of networks of other proteins to function properly in various states. The completion of my dissertation would not be possible without the immense help of several people that played various roles of support throughout this process. For this reason, I am incredibly thankful to everyone that helped me along the way. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................................... ii LIST OF TABLES ................................................................................................................................ vi LIST OF FIGURES ............................................................................................................................. vii LIST OF ABBREVIATIONS ............................................................................................................... ix CHAPTER I. Introduction........................................................................................................................................ 1 5-HT synthesis and expression ............................................................................................................. 1 5-HT receptors ..................................................................................................................................... 2 SERT: Critical determinant of 5-HT accumulation and clearance ......................................................... 4 Structural characterization of SERT ..................................................................................................... 7 Alternating access model of transport by SERT.................................................................................... 8 Kinase-dependent regulation of SERT ............................................................................................... 13 Receptor-dependent regulation of SERT function............................................................................... 18 Reverse transport of SERT by amphetamine and amphetamine-like derivatives .................................. 22 Protein-protein interactions with SERT .............................................................................................. 24 Post-translational modifications of SERT ........................................................................................... 35 Role of 5-HT and SERT in neuropsychiatric disorders ....................................................................... 39 ASD and autism-associated SERT coding variants ............................................................................. 42 Summary ........................................................................................................................................... 46 II. Biased Conformational State of ASD-Associated SERT Coding Variants ................................... 49 Introduction ....................................................................................................................................... 49 Results ............................................................................................................................................... 52 Discussion ......................................................................................................................................... 66 Methods............................................................................................................................................. 68 III. Proteomic Analysis of Hyperactive SERT Interacting Proteins .................................................. 76 Introduction ....................................................................................................................................... 76 iv Results ............................................................................................................................................... 77 Discussion ......................................................................................................................................... 98 Methods............................................................................................................................................. 99 IV. Genetic Manipulation of Mice to Interfere with Conformation and Signaling Dependent SERT Regulation: Creation and Analysis of the SERT Ala276 and SERT Glu276 Mouse ....................... 121 Introduction ..................................................................................................................................... 121 Results ............................................................................................................................................. 122 Discussion ....................................................................................................................................... 135 Methods..........................................................................................................................................