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And Ligand-Gated Calcium Channels By DUAL REGULATION OF VOLTAGE- AND LIGAND-GATED CALCIUM CHANNELS BY COLLAPSIN RESPONSE MEDIATOR PROTEIN 2 Joel Matthew Brittain Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Program of Medical Neuroscience, Indiana University December 2012 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Rajesh Khanna, Ph.D., Chair Theodore R. Cummins, Ph.D. Doctoral Committee Gerry S. Oxford, Ph.D. Lawrence A. Quilliam, Ph.D. November 12, 2012 Debbie C. Thurmond, Ph.D. ii © 2012 Joel Matthew Brittain ALL RIGHTS RESERVED iii DEDICATION This thesis is dedicated to my parents for all of the love and support they have given me. Knowing that they are there for me and that I can always count on them has given me a calm and strength to work hard to achieve my goals. iv ACKNOWLEDGEMENTS This thesis and the work therein would not be possible without the contributions and support of many people I first thank my mentor Dr. Rajesh Khanna for guiding and challenging me in the laboratory. Dr. Khanna has been a mentor who truly cares about my future and strives to make me a better scientist. The experience of working in his laboratory and under his guidance has led me to mature as both a person and scientist. All of my past and future success is due to your diligence and hard work. I thank Dr. Cummins for convincing me to attend Indiana University and exposing me to the field of ion channel biology. Without your effort and invitation to visit the IUPUI campus I would not have the opportunity to come to IUPUI and become the person I am today. I thank Dr. Oxford, Dr. Thurmond, and Dr. Quilliam for their support and pushing me in the right direction. I thank Dr. Nick Brustovetsky for assistance with technical aspects of several experiments and for many enlightening conversations. I thank Dr. Fletcher White for his insightful conversations and unique perspectives that greatly aided in my research. I thank Dr. Cynthia Hingtgen, Dr. Michael Vasko, and Dr. Grant Nicol along with the rest of the sensory neuron lab meeting participants for challenging me to improve as a scientist and for the tough questions they routinely asked me. I thank Dr. Andy Hudmon for many discussion regarding experimental approach and assistance with aspects of my experiments. I thank Nastassia Belton and Brittany Veal of Stark Neuroscience Research Institute for everything they have done to make my life as a graduate student easier. I thank Tracy Donhardt for helping me to improve my writing skills. I thank Dr. Djane Duarte, Dr. Weiguo Zhu, Dr. Haitao You, Dr. Andrew Piekarz, Matthew Ripsch, Dr. Naikui Liu, and Tatiana Brustovesty for the experiments they performed that have been included in thesis. I thank Dr. May Khanna for her technical assistance with protein purification and surface plasmon resonance. I thank Kashif Kirmani and Eric Thompson for work and assistance with virus production. I thank Dr. Andrei v Molosh, Dr. Michael Due, Dr. Brian Jarecki, and Dr. Patrick Sheets for their assistance with experiments and many enlightening conversations. I thank the graduate students both on the 4th floor of R2 as well as throughout campus that have made graduate school fun even when the science did not work as planned. I thank Dr. Nicole Asphole, Dr. Michael Kalwat, Melissa Walker, Ben Thirlby, Natalie Case, Justin Babcock, Hoa Nyugen, and Sherry Pittman for their friendship and stimulating scientific conversations. I thank members of the Khanna lab for making the laboratory a fun and prosperous environment. I thank Sarah Wilson, Alicia Garcia, Omotore Eruvwetere, Kisan Shah, Stephanie Martinez, Weina Ju, Erik Dustrude, and Xiao-Fang Yang for all of their assistance and for laughing even when my jokes were not funny. I thank all of my friends and family for hanging with me through the ups and downs that graduate school can bring. I lastly thank Alyssha Duncan who has been supportive of me throughout the all- encompassing process that is writing a thesis. vi ABSTRACT Joel Matthew Brittain DUAL REGULATION OF VOLTAGE- AND LIGAND-GATED CALCIUM CHANNELS BY COLLAPSIN RESPONSE MEDIATOR PROTEIN 2 Synaptic transmission is coordinated by a litany of protein-protein interactions that rely on the proper localization and function of pre- and post-synaptic Ca2+ channels. The axonal guidance/specification collapsin response mediator protein-2 (CRMP-2) was identified as a potential partner of the pre-synaptic N-type voltage-gated Ca2+ channel (CaV2.2). CRMP-2 bound directly to CaV2.2 in two regions; the channel domain I-II intracellular loop and the distal C-terminus. Both proteins co-localized within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to EGFP caused a significant increase in Ca2+ channel current density whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2–overexpressing neurons. Both activity- and CRMP-2- phosphoryation altered the interaction between CaV2.2 and CRMP-2. I identified a CRMP-2- derived peptide (called CBD3) that bound CaV2.2 and effectively disrupted the interaction between CaV2.2 and CRMP-2. CBD3 peptide fused to the HIV TAT protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, and reversed neuropathic hypersensitivity produced by an antiretroviral drug. Unchecked Ca2+ influx via N-methyl-D-aspartate receptors (NMDARs) has been linked to activation of neurotoxic cascades culminating in cell death (i.e. excitotoxicity). CRMP-2 was suggested to affect NMDAR trafficking and possibly involved in neuronal survival following excitotoxicity. Based upon these studies, I hypothesized that a peptide from CRMP2 could vii preserve neurons in the face of excitotoxic challenges. Lentiviral–mediated CRMP2 knockdown or treatment with TAT-CBD3 blocked neuronal death following glutamate exposure likely via blunting toxicity from NMDAR-mediated delayed calcium deregulation. TAT-CBD3 induced internalization of the NMDAR subunit NR2B in dendritic spines without altering somal surface expression. TAT-CBD3 reduced NMDA-mediated Ca2+-influx and currents in cultured neurons. The presented work validates CRMP-2 as a novel modulator of pre- and post-synaptic Ca2+ channels and provides evidence that the TAT-CBD3 peptide could be useful as a potential therapeutic for both chronic neuropathic pain and excitotoxicity following stroke or other neuronal insults. Rajesh Khanna, Ph.D., Chair viii TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................ xiv LIST OF FIGURES ....................................................................................................................... xv FIGURE CONTRIBUTIONS: .................................................................................................... xviii LIST OF ABBREVIATIONS ....................................................................................................... xix FOREWORD ................................................................................................................................ xxi CHAPTER 1. INTRODUCTION .................................................................................................... 1 1.1. CaV2.2 ................................................................................................................................ 2 1.1.1. Identification of an “N-type” calcium current ....................................................... 2 1.1.2. Relationship of Ca2+ influx via CaV2.2 to synaptic release ................................... 5 1.1.3. CaV2.2 expression and localization ....................................................................... 7 1.1.4. Developmental regulation of CaV2.2 .................................................................... 8 1.1.5. Structure and function of CaV2.2 .......................................................................... 9 1.1.6. Activation and inactivation of CaV2.2 ................................................................ 12 1.1.7. Pharmacology of CaV2.2 ..................................................................................... 13 1.1.8. Trafficking of CaV2.2 .......................................................................................... 14 1.1.9. The CaV2.2 interactome ...................................................................................... 18 1.1.10. CaV2.2 and pain ................................................................................................. 27 1.2. CRMP-2 ........................................................................................................................... 28 1.2.1. Identification of CRMP-2 .................................................................................... 28 1.2.2. Structure of CRMP-2 ........................................................................................... 29 1.2.3. Expression and developmental regulation of CRMP-2 ........................................ 31 1.2.4. CRMP-2 signaling ............................................................................................... 33 1.2.5. CRMP-2: protein interactions .............................................................................
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