Secretin-Modulated Potassium Channel Trafficking As a Novel Mechanism for Regulating Cerebellar Synapses Michael Williams University of Vermont
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University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2013 Secretin-Modulated Potassium Channel Trafficking as a Novel Mechanism for Regulating Cerebellar Synapses Michael Williams University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Recommended Citation Williams, Michael, "Secretin-Modulated Potassium Channel Trafficking as a Novel Mechanism for Regulating Cerebellar Synapses " (2013). Graduate College Dissertations and Theses. 241. https://scholarworks.uvm.edu/graddis/241 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. SECRETIN-MODULATED POTASSIUM CHANNEL TRAFFICKING AS A NOVEL MECHANISM FOR REGULATING CEREBELLAR SYNAPSES A Dissertation Presented by Michael R. Williams to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Neuroscience October, 2012 Accepted by the Faculty of the Graduate College, The University of Vermont, in partial fulfillment of the requirements for the degree of Doctor of Philosophy, specializing in Neuroscience. Dissertation Examination Committee: ____________________________________ Advisor Anthony Morielli, Ph.D. ____________________________________ Alan Howe, Ph.D ____________________________________ Victor May, Ph.D ____________________________________ Chairperson John Green, Ph. D ____________________________________ Dean, Graduate College Domenico Grasso, Ph.D Date: August 24, 2012 ABSTRACT The voltage-gated potassium channel Kv1.2 is a critical modulator of neuronal physiology, including dendritic excitability, action potential propagation, and neurotransmitter release. However, mechanisms by which Kv1.2 may be regulated in the brain are poorly understood. In heterologous expression systems Kv1.2 is regulated by endocytosis of the channel from the plasma membrane, and this trafficking can be modulated by adenylate cyclase (AC). The goal of this dissertation was to determine whether AC modulated endocytic trafficking of endogenous Kv1.2 occurred in the mammalian nervous system. Within the brain, Kv1.2 is expressed at its highest levels in the cerebellar cortex. Specifically, Kv1.2 is expressed in dendrites of Purkinje cells (PC), the sole efferent neurons of the cerebellar cortex; Kv1.2 is also expressed in axon terminals of Basket cells (BC), which make inhibitory synapses to Purkinje cells. The loss of functional Kv1.2 in PC dendrites or BC axon terminals causes profound changes in the neurophysiology of Purkinje cells, and aberrant loss of Kv1.2 produces cerebellar ataxia. Therefore, the cerebellum offers a brain structure where Kv1.2 is abundant and has known and important roles in synaptic physiology. A candidate regulator of Kv1.2 trafficking in cerebellar synapses is the secretin peptide receptor: the receptor is also located in both PC dendrites and BC axon terminals, and ligand binding to the secretin receptor stimulates AC. Although secretin affects cerebellar neurophysiology and cerebellar dependent behavior, the mechanisms are not well resolved. By cell-surface protein biotinylation and subsequent immunoblot quantitation of secretin treated rat cerebellar slice lysates, secretin was found to decrease cell-surface Kv1.2. This effect could be mimicked by stimulating AC with forskolin, and could be occluded by inhibition of the secretin receptor, AC, or protein kinase A. The secretin receptor stimulated loss of surface Kv1.2 was not accompanied by decreased total Kv1.2 protein levels, but did involve enhanced channel endocytosis. Microscopy studies using two novel independent techniques provided evidence that both BC axon terminals and PC dendrites are sites of AC-stimulated Kv1.2 endocytosis. The physiological significance of secretin mediated suppression of Kv1.2 was supported by collaborative studies which found infusions into the cerebellar cortex of either a toxin that inhibits Kv1.2, or of secretin, enhanced eyeblink conditioning, a form of cerebellar dependent learning, in rats. These studies provided the first evidence that Kv1.2 is regulated by endocytic trafficking in the brain. However, to address the role of that trafficking in synaptic physiology requires knowledge about the determinants of Kv1.2’s endocytic potential, and non-destructive assays to measure Kv1.2 endocytosis in neural circuits. This dissertation therefore concludes with preliminary studies that explore an ancient motif regulating Kv1.2 trafficking, and that discuss a novel dual fluorescent fusion protein reporter of Kv1.2’s subcellular localization. CITATION Material from this dissertation has been published in the following form: Williams, M. R., J. R. Fuchs, et al. (2012). "Cellular mechanisms and behavioral consequences of Kv1.2 regulation in the rat cerebellum." J Neurosci 32(27): 9228-9237. ii ACKNOWLEDGEMENTS At the risk of omitting the deserving, I happily thank several people in particular. Chronologically, though in magnitude as well, my parents Steve and Julie, my sister Aaren, and my wife Heather deserve and have my peerless gratitude for their loving support and patience, including all the time they have waited while I “finish up one more thing” before spending an evening or weekend with them. By the same token, the people I have enjoyed the privilege of calling friends have been irreplaceable sources of support and occasional though welcome reminders of sources of satisfaction outside of work. I must thank Dr. Terry Rabinowitz for kindly providing both my first research venture in Vermont and for being a catalyst for my training. I owe a great debt of gratitude to Dr.’s Felix Eckenstein and Rae Nishi for answering my many calls and e- mails when they welcomed me into the SNURF program. The opportunity, and their kindness, was remarkable, as has been their continued advocacy and efforts in furthering my training. It has also been a personal and professional joy to work with Dr. John Green and Jason Fuchs; the labs of Dr. Jom Hammack and of Dr. Paul Newhouse; Dr.’s Steve Lidofsky and Lis Barfod, as well as Ann Moore. Likewise, my peers in the NGP, and my many teachers, have greatly enriched my experience here. So too have my lab mates Gene Cilento, Jeff Shortway, Jason Fuchs, Emilee Connors, Megan Doczi, Lee Stirling, Ed Nesti, Brandon Fields, and John Markey. iii Any success I have had or will have is a product of many people’s support and training, including from Todd Clason, Dr. Sheryl White, Thomm Buttolph, all the faculty and staff of the COBREs, and indeed of UVM at large, such as Ed McIntyre. A great deal of aid or administrative support has also come from Dr, Cynthia Forehand, Allison Sturtevant, Margo Green, Carrie Perkins, Hallie Davis-Penders, Emily McLaughlin, Sandy Bossick, and Sylvie Frisbie. Financial support has been generously provided through many avenues, including Dr. Dave Warshaw, Dr. Mark Nelson, and Dr. Anthony Morielli. Speaking of Dr. Morielli, it would be a lengthy tome in and of itself to give due thanks for his unwavering support financially, professionally, intellectually, and personally. I will endeavor to embarrass him further with thanks in person, and spare him here but for stating the hope to return the favor even if only to my future mentees. Lastly, it would be a great omission were I not to sincerely thank my defense committee, Dr.’s: Alan Howe, Anthony Morielli, John Green, and Victor May; their time, aid, and insights have been greatly appreciated. iv TABLE OF CONTENTS CITATION .......................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF FIGURES ............................................................................................................ x CHAPTER 1: COMPREHENSIVE LITERATURE REVIEW ......................................... 1 Introduction ..................................................................................................................... 1 Structural Classes Of Potassium Channels...................................................................... 1 Kv Channels .................................................................................................................... 2 The Diverse Roles Of Kv1.2 ........................................................................................... 4 Function Of Kv1.2 In Neurons ........................................................................................ 5 Kv1.2 Controls Transmitter Release In The Brain .......................................................... 8 Change In Kv1.2 Function Is Important For Changes In Neurophysiology ................... 9 Kv1.2 Can Be Regulated In The Brain ......................................................................... 10 Trafficking Of Ion Channels As A Mechanism Of Plasticity ....................................... 10 Regulation Of Kv1.2 By Channel Trafficking At The Plasma Membrane ................... 12 Changes In cAMP Can Regulate Kv1.2 Channel Trafficking ...................................... 13 Evidence That Presynaptic Kv1.2 May Be Regulated Via cAMP ...............................