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DISSERTATION HIGH RESOLUTION OPTICAL ANALYSIS OF NAV1.6 LOCALIZATION AND TRAFFICKING Submitted by Elizabeth Joy Akin Graduate Program in Cell and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Fall 2015 Doctoral Committee: Advisor: Michael Tamkun Co-Advisor: Gregory Amberg Santiago Di Pietro Diego Krapf Susan Tsunoda Copyright by Elizabeth Joy Akin 2015 All Rights Reserved ABSTRACT HIGH RESOLUTION OPTICAL ANALYSIS OF NAV1.6 LOCALIZATION AND TRAFFICKING Voltage-gated sodium (Naᵥ) channels are responsible for the depolarizing phase of the action potential in most nerve cell membranes. As such, these proteins are essential for nearly all functions of the nervous system including thought, movement, sensation, and many other basic physiological processes. Neurons precisely control the number, type, and location of these important ion channels. The density of Naᵥ channels within the axon initial segment (AIS) of neurons can be more than 35-fold greater than that in the somatodendritic region and this localization is vital to action potential initiation. Dysfunction or mislocalization of Naᵥ channels is linked to many diseases including epilepsy, cardiac arrhythmias, and pain disorders. Despite the importance of Naᵥ channels, knowledge of their trafficking and cell-surface dynamics is severely limited. Research in this area has been hampered by the lack of modified Naᵥ constructs suitable for investigations into neuronal Naᵥ cell biology. This dissertation demonstrates the successful creation of modified Naᵥ1.6 cDNAs that retain wild-type function and trafficking following expression in cultured rat hippocampal neurons. The Naᵥ1.6 isoform is emphasized because it 1) is the most abundant Naᵥ channel in the mammalian brain, 2) is involved in setting the action potential threshold, 3) controls repetitive firing in Purkinje neurons and retinal ganglion cells, 4) and can contain mutations causing epilepsy, ataxia, or mental retardation. Using single-molecule microscopy techniques, the trafficking and cell-surface dynamics of Naᵥ1.6 were investigated. In contrast to the current ii dogma that Naᵥ channels are localized to the AIS of neurons through diffusion trapping and selective endocytosis, the experiments presented here demonstrate that Naᵥ1.6 is directly delivered to the AIS via a vesicular delivery mechanism. The modified Naᵥ1.6 constructs were also used to investigate the distribution and cell-surface dynamics of Naᵥ1.6. Somatic Naᵥ1.6 channels were observed to localize to small membrane regions, or nanoclusters, and this localization is ankyrinG independent. These sites, which could represent sites of localized channel regulation, represent a new Naᵥ localization mechanism. Channels within the nanoclusters appeared to be stably bound on the order of minutes to hours, while non-clustered Naᵥ1.6 channels were largely mobile. Novel single-particle tracking photoactivation localization microscopy (spt-PALM) analysis of Naᵥ1.6-Dendra2 demonstrated that the nanoclusters can be modeled as energy wells and the depth of these interactions increase with neuronal age. The research presented in this dissertation represents the first single-molecule approaches to any Naᵥ channel isoform. The approaches developed during the course of this dissertation research will further our understanding of Naᵥ1.6 cell biology under both normal and pathological conditions. iii ACKNOWLEDGEMENTS This journey would not have been possible without the endless support of family, friends, and colleagues. Thank you so much to my mentor, Mike Tamkun, for the constant support through the past years. It was a privilege to be able to follow in your footsteps and contribute to the field of Naᵥ channels. From difficult experiments in the lab to rigorous backpacking escapades to building an igloo, this has been a true adventure. Thank you for taking a risk with both me and my project. Your immense knowledge and willingness to push the boundaries of science are inspiring. I could not be where I am today without such a great role model and mentor, and for that I will be forever grateful. Thank you to my committee members, Greg Amberg, Susan Tsunoda, Diego Krapf, and Santiago Di Pietro. I appreciate your support, great ideas, and for pushing me to always do better. Thank you for taking the time to invest in my graduate training. My fellow Tamkunites, how can I say thank you? You have made my time in Fort Collins absolutely unforgettable. Phil Fox, thank you for putting up with my silly questions and mistakes over the years, and for remaining a source of wisdom and insight. I truly value our friendship and am grateful for our many conversations about science and life. I also learned to never underestimate the value of some good duct tape and pocket food. Laura Solé, llámame, possible? It is an incredible blessing to have such a friend and colleague. Thank you for taking on the difficult challenge of continuing the Naᵥ project. I look forward to many more good times together, both here and in Catalonia. Ben Johnson, Ashley Leek, and Emily Maverick, you are all incredibly talented scientists and I am excited to see where your journeys take you. Keep saving the world, banishing ghosts, and exploring The House on the Hill. We are all counting on you. iv And remember, don’t blink! Aubrey Weigel, as an honorary Tamkunite, thank you for remaining such a good friend despite the miles, and for all the Beers, Bears, and Science. Yaping Moshier, Rob Loftus, Chris Haberkorn, Emily Deutsch, Michaela Schumacher, Tracie VanHorn, Jason Platt, and all the others who have spent time in the Tamkun Lab, thank you for an amazing journey made rich by each of you. To all Tamkunites present and future, above all else make sure you keep the tiki torch! Thank you to Albert Gonzales for the steadfast support, insight, and many volleyball tournaments. To all those that made my graduate career so amazing, Krystle Frahm, An Dang, Mallory Shields, Christina Dennison, Max Vallejos, Ryan Tooker, Reagan Pennock, and so many others that I do not have space to mention. To the Krapf Lab, Diego Krapf, Sanaz Sadegh, Eric Xinran Xu, and Jenny Higgins, thank you for sharing your expertise on all things biophysics. I would be lost without your help and I have learned an immense amount from all of you. Thank you to our collaborators, Steve Waxman and Sulayman Dib-Hajj for the Naᵥ1.6 clone to start my project, and for the advice and support through the arduous journey of publishing. None of this would have been possible without the tireless encouragement of my family. Thank you to my mom, Nancy Akin, for being an unwavering pillar of support and love. Thank you for instilling in me the courage to overcome all obstacles. Thank you to my dad, Dave Akin, for kindling a sense of curiosity in me and an awe of nature. Thank you for teaching and coaching me through the years. Both of you have pushed me to follow my dreams and always believed in me, and I certainly would not have made it this far without you. To Andrew Akin, my brother and friend, thank you for being such a great role model and inspiration. No one could ask for anyone better to share growing up with. Thanks to both you and Mr. Douglas for help with the dissertation writing. Jeff and Aimee Jensen, my adopted parents, I cannot even begin to v express my gratitude for sharing this journey with me. Thank you for sharing your home, pets, food, cars, and love with me. It was above and beyond what anyone could ask for and is a testament to the amazing people you are. To my Grandma Dorothy for starting the tradition of education in our family, I love you and miss you dearly. Uncle Don Akin, thank you for being such an inspiration. You showed that it is possible to live life completely and selflessly, and that it is important to always be present for those you love. I will miss your jokes, but am glad I inherited your determination to never back down from a challenge. To all my grandparents, aunts, uncles, and cousins, thank you for being such an amazing family and for your support. vi DEDICATION In loving memory of Dorothy Dunn Durham (1926-2013) and Donald Ray Akin (1947-2015). vii TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iv DEDICATION .............................................................................................................................. vii Chapter 1: Introduction ................................................................................................................... 1 1.1 Excitable Membranes ............................................................................................................ 1 1.2 Structure/Function of Voltage-Gated Sodium Channels ....................................................... 8 1.3 Naᵥ1.6 .................................................................................................................................. 14 1.4 Axon Initial Segment .......................................................................................................... 19 1.5 High-resolution microscopy in living cells ......................................................................... 24 1.6 Overview of this dissertation ..............................................................................................
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  • Kcne4 Deletion Sex- and Age-Specifically Impairs Cardiac

    Kcne4 Deletion Sex- and Age-Specifically Impairs Cardiac

    The FASEB Journal • Research Communication Kcne4 deletion sex- and age-specifically impairs cardiac repolarization in mice † ‡ Shawn M. Crump,*,1 Zhaoyang Hu, ,1 Ritu Kant,* Daniel I. Levy, ,2 Steve A. N. Goldstein,§ and Geoffrey W. Abbott*,3 *Bioelectricity Laboratory, Department of Pharmacology and Department of Physiology and Biophysics, † School of Medicine, University of California, Irvine, Irvine, California, USA; Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, ‡ Chengdu, People’s Republic of China; Section of Nephrology, Department of Medicine, University of Chicago, Chicago, Illinois, USA; and §Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA ABSTRACT Myocardial repolarization capacity varies channels comprise both pore-forming a subunits and with sex, age, and pathology; the molecular basis for this regulatory proteins including b subunits such as the single- variation is incompletely understood. Here, we show that pass transmembrane KCNE proteins (2) (Fig. 1A). the transcript for KCNE4, a voltage-gated potassium In human ventricular myocardium, the delayed rec- b fi fi (Kv)channel subunit associated with human atrial - ti er currents IKr and IKs are the primary repolarizing a brillation, was 8-fold more highly expressed in the male currents. Generated by hERG and KCNQ1 Kv sub- left ventricle compared with females in young adult units, respectively, these K+ currents repolarize ven- P < C57BL/6 mice ( 0.05). Similarly, Kv current density tricular myocytes after a plateau phase lasting several was 25% greater in ventricular myocytes from young hundred milliseconds (1). In adult mouse ventricles, adult males (P < 0.05). Germ-line Kcne4 deletion elimi- ventricular myocyte action potentials are much shorter; fi nated the sex-speci cKv current disparity by diminishing IKr is absent, and IKs is, at most, weakly expressed and I b ventricular fast transient outward current ( to,f) and detectable only after -adrenergic stimulation (3).