Functional Characterization of Drosophila Bestrophin 1 Public

Abstract

Mutations in the human bestrophin-1 gene are genetically linked to Best vitelliform macular degeneration, which often leads to childhood blindness. The pathogenesis is unclear because the function of hBest1 is not fully understood. In this dissertation, I report the functional characterization of endogenous bestrophin currents and the identification of the physiological role of bestrophins in Drosophila. Here I show that (1) bestrophins clearly are the pore-forming subunit of a Cl^- channel; (2) the bestrophin Cl^- channel is regulated by both cytoplasmic Ca²⁺ and hyposmotic cell swelling; and (3) dBest1 is very likely involved in salt balance in the fly. The Drosophila S2 cell line expresses four bestrophins and exhibits endogenous Cl^- currents (ICl) that are stimulated by Ca²⁺ or swelling. The ICaCl and IClswell exhibit similar electrophysiological properties and are both suppressed by non-overlapping RNAis to dBest1, but not dBest2-4. The ICl in the dBest1 RNAi-treated S2 cells are rescued by expressing dBest1 cDNA. To determine if dBest1 is the pore-forming subunit but not just an accessory subunit of a Cl^- channel, we rescue the S2 ICl with a mutant dBest1 with altered biophysical properties. As expected for an ion channel with a mutant pore, biophysical properties of the rescued dBest1-F81C current differ significantly from the native ICl. Moreover, when F81 is substituted with a negatively charged glutamate, the anion permeability is reversed. The fact that mutating F81 alters intrinsic properties of the ICl suggests that dBest1 forms the pore of the S2 Cl^- channel. At the cellular level, dBest1 is characterized to regulate cell volume by mediating regulatory volume decrease in response to hyposmotic stimuli. Dysfunctional dBest1 is further found to associate with increased fly mortality on salty food. These studies provide important insights into the possible role of bestrophins in macular degeneration.

Table of Contents

CHAPTER 1 Introduction and Background

Ion Channel in Biological Membranes ....................................................................... 2 Chloride channel Overview ........................................................................................ 5 Ligand-Gated Cl- Channels......................................................................................... 7 Voltage-Gated Cl- Channels........................................................................................ 8 Ca2+-activated Cl- Channels (CaCCs)....................................................................... 10 Volume-Regulated Anion Channels (VRACs).......................................................... 14 Bestrophin, a novel family of Cl- channels? ............................................................. 18 Macular Degeneration and Clinical Presentation ............................................. 18 Familial Macular Degeneration ........................................................................ 19 Symptoms and Progression of Best Disease..................................................... 20 Pathophysiology of Best Vitelliform Macular Degeneration............................ 21 Diagnosis of Best Disease................................................................................. 22 Discovery of the Bestrophin Gene.................................................................... 23 Characterization of the Function of Bestrophin Proteins.................................. 26 Mouse Bestrophins............................................................................................ 32 Limitations Associated with the Present Study of Bestrophins ........................ 36 Drosophila melanogaster and its Cell Line as Model Systems for Bestrophin Study ................................................................................................................. 39 Rationale and Organizational Overview........................................................... 41 CHAPTER 2 Engogenous Drosophila Bestrophin Mediates Calcium-Activated Chloride Currents Summary ................................................................................................................... 44 Introduction............................................................................................................... 45 Results....................................................................................................................... 47 Characterization of Native CaC Currents in Drosophila S2 Cells ................... 47 Mechanisms of Slow Activation by Intracellular Ca2+ ..................................... 49 Pharmacology of Drosophila S2 CaC Currents................................................ 49 Identification of Endogenous Bestrophin Expression in S2 Cells.................... 50 Interfering RNA Knocks Down Both dBest Expression and S2 CaC Currents 51 Heterologous Currents Associated with dBest1 ............................................... 53 Mutation in dBest1 Altered the Biophysical Properties of the Cl- Current ...... 55 Discussion................................................................................................................. 55 Endogenous Drosophila Bestrophins are CaC Channels.................................. 55 Other Endogenous Cl- currents expressed by Drosophila S2 Cells.................. 58 Comparison of endogenous dBest currents and heterologous dBest1 currents 59 Comparison of CaC currents mediated by Drosophila and vertebrate Bestrophins ........................................................................................................................... 59 Materials and Methods.............................................................................................. 62 Cell Culture....................................................................................................... 62 Solutions for Whole Cell Recording................................................................. 62

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Mot-clé	electrophysiology bestrophin ion channel chloride conductance
Committee Chair / Thesis Advisor	Hartzell Jr., Criss, Emory University
Committee Members	Hall, Randy A, Emory University Moberg, Kenneth H, Emory University Traynelis, Stephen, Emory University Peng, Junmin, Emory University

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