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Alternative Splicing Controls G Protein Inhibition of Cav2.2 Calcium

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Alternative Splicing Controls G Protein Inhibition of Cav2.2 Calcium Alternative Splicing Controls G Protein Inhibition of CaV2.2 Calcium Channels Cecilia Goldsmith Phillips BA, Reed College, 2003 THESIS Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Department of Neuroscience at Brown University Providence, Rhode Island May 2012 © 2012 Cecilia Goldsmith Phillips This dissertation by Cecilia Goldsmith Phillips is accepted in its present form by the Department of Neuroscience as satisfying the dissertation requirement for the degree of Doctor of Philosophy. Date Dr. Diane Lipscombe, Advisor Recommended to the Graduate Council Date Dr. Gilad Barnea, Reader Date Dr. Julie Kauer, Reader Date Dr. Stephen Ikeda, Outside Reader Approved by the Graduate Council Date Dr. Peter M. Weber, Dean of the Graduate School iii CURRICULUM VITAE 23 Elton St, Providence, RI 02906 (503) 705-7387 [email protected] EDUCATION Brown University, Providence, RI PhD, Neuroscience, May 2012 Reed College, Portland, OR BA, Biology, May 2003 Senior Thesis: Processing of GFP-tagged ELH Prohormone in PC12 Cells Advisor: Dr. Stephen Arch RESEARCH POSITIONS Research Assistant to Dr. Stephen M Smith, Division of Molecular Medicine, Oregon Health Sciences University, Portland, OR. September 2003 – August 2005 Internship with Dr. Peter Gillespie, Vollum Institute, Oregon Health Sciences University, Portland, OR. June 2003 – August 2003 Research Assistant to Dr. Maryanne McClellan, Department of Biology, Reed College, Portland, OR. June 2002 – August 2002 Research Assistant to Dr. David McKinnon, Department of Neuroscience, SUNY Stony Brook. June 2001 – August 2001 and June 2000 – August 2000 PUBLICATIONS Allen SE*, Phillips CG*, Raingo J, and D Lipscombe. The neuronal splicing factor Fox- 2 controls Gs protein inhibition of CaV2.2 calcium channels. In submission. *Equal contributions by these authors. Phillips CG, Harnett MT, Chen W, and SM Smith (2008) Calcium-sensing receptor activation depresses synaptic transmission. Journal of Neuroscience 28(46): 12062- 12070. iv ABSTRACTS Phillips CG, Allen SE, and D Lipscombe (2010) The neuronal splicing factor Fox-2 regulates an exon in CaV2.2 that controls the sensitivity of N-type calcium channels to inhibition by Gs proteins. Society for Neuroscience Annual Meeting, San Diego, CA. Phillips CG, Allen, SE, and D Lipscombe (2010) Neuronal splicing factor Fox-2 regulates an exon in CaV2.2 that controls sensitivity of N-type calcium channels to inhibition by Gs G proteins. New Horizons in Calcium Signaling, China. Allen SE, Phillips CG, Lipscombe D (2010) The splicing factor Fox-2 controls N-type calcium channel activity in sympathetic neurons. Second Annual International Calcium Channel meeting, Belize. Phillips CG and D Lipscombe (2009) G protein-coupled receptor inhibition of N-type calcium channel splice variants. Society for Neuroscience Annual Meeting, Chicago, IL. Vyleta NP, Chen W, Phillips CG, Harnett MT, and SM Smith (2009) Regulation of nerve terminal function by the extracellular calcium-sensing receptor. Federation of European Physiological Societies Annual Meeting, Slovenia. Phillips C and D Lipscombe (2008) Modulation of N-type calcium channels associated with β2a subunits. Society for Neuroscience Annual Meeting, Washington, DC. Raingo J, Phillips C, Denome S, and D Lipscombe (2007) Cell-specific alternative splicing specifies G protein signaling to the N-type calcium channel. Society for Neuroscience Annual Meeting, San Diego, CA. Smith SM, Phillips C, Harnett MT, and W Chen (2006) Decreases in extracellular Ca2+ activate a non-selective cation channel and facilitate cortical synaptic transmission via the Ca2+ receptor. Society for Neuroscience Annual Meeting, Atlanta, GA. Phillips C, Harnett MT, and SM Smith (2005) Decreases in extracellular Ca2+ activate a NSCC and facilitate cortical synaptic transmission via the CaR. Spring Brain Annual Meeting,Sedona, AZ. GRANTS & FELLOWSHIPS National Institute of Neurological Disorders and Stroke Predoctoral Fellowship 1F31NS066712-01A1, Does alternative splicing regulate G protein inhibition of calcium channels? 1/2010-12/2012 Sidney E. Frank Graduate Fellowship for Biology and Medicine, Brown University, Providence, RI, 2006-7. Awarded to a single first-year graduate student from the Brown v Division of Biology and Medicine. HHMI Undergraduate Biological Science Education Program Grant #71100-529603, 2002- 3. Used to fund undergraduate thesis research. HONORS & AWARDS Best Brown graduate student research poster, Brown-NIH Graduate Partnership Program retreat, 2009 Reed College Commendation for Academic Excellence, 2002-3 PRESENTATIONS Research talk at Physiology, Molecular Biology and Neuroscience Institute (IFIBYNCE), Buenos Aires, Argentina, 2011 Research talk at Multidisciplinary Institute of Cell Biology (IMBICE), La Plata, Argentina, 2011 Yearly research talk at neuroscience department in-house seminar, Brown University, 2007-2010 Invited speaker, Rockwell Levy Foundation Board meeting, Brown University, 2007 Invited speaker, graduate student recognition luncheon, Brown University, 2007 Research talk at Molecular Medicine department seminar, Oregon Health Sciences University, 2004 Research talk at Pulmonary & Critical Care Grand Rounds, Oregon Health Sciences University, 2004 TEACHING Instructor, The Neuron: Form and Function, Summer@Brown, Brown University, June 2009 I independently designed and taught the curriculum for this introduction to molecular neuroscience. The class had a 15 student enrollment and included lectures, videos, group and independent problem solving, research papers and class presentations. Teaching Assistant to Dr. Carlos Aizenman, Principles of Neurobiology, Brown University, 2008 I taught a weekly review section for 20-40 students covering the previous week’s lectures and led review sessions for exams. Teaching Assistant to Dr. Maryanne McClellan, Cellular Biology, Reed College, 2003 I prepared experimental tools for class lab, including reagents and cell cultures. I provided direction and guidance to students during a weekly laboratory period as well as during completion of independent research projects. UNIVERSITY SERVICE vi Graduate student representative to the Division of Biology and Medicine Faculty Council, 2010-11 Graduate student representative to the Neuroscience Faculty, 2009-10 Member of Division of Biology and Medicine Grad Student Advisory Panel to Dean, 2007-8 OUTREACH Science Fair Consultant, St. Mary’s Middle School, R.I., 2007 Brain Awareness Week presentation to Girl Scout Troops, Baylor College, R.I. 2007 Brain Awareness Week presentation to Cranston East High School, R.I. 2007 PROFESSIONAL SOCIETY MEMBERSHIP Society for Neuroscience, 2006-present vii PREFACE CaV2.2 voltage-gated calcium channels (CaVs) are expressed at presynaptic terminals in most central and peripheral neurons where they control the calcium (Ca2+) entry that triggers exocy- tosis. CaV2.2 channels are under tight regulatory control by many intra- and extracellular mol- ecules. One of the best-documented forms of regulation is inhibition by presynaptic G protein- coupled receptors (GPCRs). In the past three decades since this was first discovered we have learned much about inhibition of CaV2.2 by neurotransmitters, neurohormones, and drugs, and also about the structure, function, and binding partners of CaVs, G proteins, and GPCRs. GPCR inhibition of CaV2.2 channels varies by GPCR-type and by tissue. In this thesis I present data that suggest a new perspective in understanding the diversity in coupling between GPCRs and CaV2.2 channels: that alternative splicing of the target channel determines the ability of specific receptors to inhibit Ca2+ currents. I build off of previous work from our lab showing that alterna- tive splicing of a set of mutually exclusive exons in CaV2.2 pre-mRNA controls the ability of Gi/o protein-coupled receptors (Gi/oPCRs) to inhibit CaV2.2 (Raingo et al., 2007). I first show differen- tial coupling of GPCRs to splice variants of CaV2.2 (Chapter 2), then I show that alternative exon 18a (e18a) modulates inhibition by Gs protein-coupled receptors (GsPCRs; Chapter 3), and finally I present preliminary data gathered to identify the signaling cascade between GsPCRs and e18a- containing CaV2.2 channels (Chapter 4). viii Thank You To the mentors who have taught, supported, and encouraged me: I have been incredibly fortunate to have all of you in my life. Especially, and in chronological order: Harriet Sheridan Anthony Phillips David McKinnon Barbara Rosati Stephen Arch Jesica Raingo (my committee) Gilad Barnea Julie Kauer Stephen Ikeda and Diane Lipscombe: my exceptional, creative, and inspiring advisor. To the past and present members of the Lipscombe Lab: Arturo Andrade Summer Allen Sylvia Denome Tom Helton Rachel Jiang Kiauntee Murray Andrew Pintea Jesica Raingo Johnathan Tran Kristin Webster Valerie Yorgan To the Sidney E. Frank Foundation and National Institute of Neurological Disorders and Stroke for supporting my education and research. To the entire Brown University Neuroscience Graduate Program and Department. ix TABLE OF CONTENTS 1. Introduction |1 I. CaV2.2 channels are presynaptic and control neurotransmitter release |2 II. CaV channel family |4 III. Alternative Splicing of CaV2.2 pre-mRNA |5 IV. Alternative exon 18a (e18a) |6 V. Regulation of CaV2.2 channels by G proteins|10 VI. E18a sequence suggests posttranslational processing |17 VII. Accessory subunits |19 2. Gq- and Gs-coupled receptor inhibition of CaV2.2 splice
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