Functional Characterization of Anoctamins, a Novel Family of Transmembrane Proteins Public

Abstract

The vital roles of Ca²⁺-activated chloride channels (CaCCs) in various physiological processes are well documented, yet there has been considerable confusion regarding the molecular identity of "classical" outwardly rectifying CaCCs. When I began my thesis work, bestrophins represented the most promising molecular candidates for mediating these currents. However, the discovery of anoctamins (Anos) as CaCCs in 2008 necessitated reassessment of the relative contribution of bestrophins and anoctamins to native CaCC currents. The goal of this work was to examine the structure, function, and localization of anoctamins, and their relative contribution to endogenous CaCCs. Using a multi-disciplinary approach, this work shows that Ano1 and Ano2 function as plasma membrane CaCCs, while Anos 3-7 likely have intracellular roles. Although the channel function of intracellular anoctamins is unknown, the findings presented in this dissertation lay the groundwork for future studies examining the channel function of other anoctamins by identifying highly conserved residues within Ano1 critical for its channel function. Specifically, we have identified residues near the sixth transmembrane domain that are critical for Ca²⁺ and voltage sensitivity of Ano1. This work also sheds light on the context dependent function of CaCCs and the discrete physiological roles of Ano1 and Best1 by examining endogenous CaCCs in Drosophila S2 cells and mouse retinal pigment epithelium. These findings reveal that S2 cell CaCCs are primarily mediated by Best1, and are activated via a Ca²⁺/calmodulin dependent kinase II dependent mechanism. In contrast, Ano1 represents the most likely candidate for CaCCs in mouse retinal pigment epithelium. In summation, the work presented here furthers our understanding of the functions of anoctamins and their roles in physiology and disease.

Functional Characterization of Anoctamins, a Novel Family of
Transmembrane Proteins

B.S., Meredith College 2006
Advisor: Criss Hartzell, Ph.D
A dissertation submitted to the Faculty of the
James T. Laney School of Graduate Studies of Emory University
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Graduate Division of Biological and Biomedical Sciences
Neuroscience
2012

Table of Contents

Table of Contents


CHAPTER 1: Introduction and Background

Ion transport in biological membranes ……………………………………….………………………..….2
Early advances in ion channel research
Ion channels are selective and open in response to specific stimuli

Chloride channel overview……………………………………………………………..………………..………4
Cl- channels have important physiological functions
The molecular identity of some Cl- channels is uncertain
Cl- channels exhibit low selectivity among anions
Cl- channels exhibit a complex gating scheme
The discovery of anoctamins provides new directions in Cl- channel research

Ca2+-activate Cl- channels: channels with diverse properties………...……….………………….10
Bestrophins are bona fide CaCCs
Regulation of Best1 by Ca2+
Best1 functions as a CaCC in specific cell types
Best vitelliform macular dystrophy
BVMD: unsanswered questions

Anoctamins mediate "classical" CaCCs……………………………………..……………..……………..20
Anoctamins 1 and 2 are CaCCs
Anoctamin structure function
Alternative splicing may contribute to heterogeneity of native CaCCs
Gating mechanisms of Ano1

Physiolgoical roles and diseases of Anoctamins……………………………………………..…………26
Physiological functions of Ano1 and Ano2
Some anoctamins may function as intracellular channels
Diseases associated with anoctamin mutations
The Cl- channel function of Anos 3-10 is unknown

Rationale and Significance………………………………………………………………………………..…...35


CHAPTER 2: Anoctamins 3-7 are Intracellular Proteins

Summary………………………………………………………………………………….………………………….43

Introduction…………….………………………………………………………………………..………………...44

Results…..…………………..………………………………………………………………………………………..45
Expression of Anos3-7 do not generate Ca2+-activated Cl- currents in HEK293
Cells
Plasma membrane trafficking of Anos in various cell lines
Subcellular localization of anoctamins
Localization of endogenous Ano7 in prostate
Sequence homology across Anos

Chimeras between Ano1 and Ano5 or Ano7 do not generate Ca2+-activated Cl-
currents

Discussion………………………..…………………………………………………………………………………..51
Anoctamin family homology
Ano5 and Ano7 may have significant structural and functional differences from
Ano1
Are Ano5 and Ano7 CaCCs?

Materials and Methods……………….………………………………………………………………………....55


CHAPTER 3: Epitope Accessibility and Mutagenesis Studies Implicate the
Sixth Transmembrane Domain and Adjacent Residues in Regulation of
Anoctamin 1 by Ca2+ and Voltage

Summary………….………….……………….……………………………………………………………………..75

Introduction………..….……………………….……………………………………………………………..……76

Results…..…………….………………………………………………………………………………………………77
Determinants of anion:cation permeability of Ano1
E702 and E705 contribute to Ca2+ gating
Mutations within TM6 affect channel gating by voltage

Discussion……....…..………………………………………………………………………………………………82
Ca2+-dependent gating of Ano1
E702 and E705 are critical for Ca2+-regulation of Ano1
The role of TM6 in voltage sensitivity of Ano1
Revised topology places Ca2+ and voltage sensors near pore
Ano1 may have a complex transmembrane topology
Determinants of Ano1 Cl- selectivity are still unknown

Materials and Methods………….…….………….…………………………………………………………….89


CHAPTER 4: Drosophila Bestrophin-1 Currents are Regulated by
Phosphorylation via a CaMKII Dependent Mechanism

Summary…………………….…………………………………………………………………………………..….103

Introduction…………………….……………………………………………………………..………………….104

Results……………………………….……………………………………………………………………………….105
Drosophila anoctamin orthologs do not contribute to S2 cell CaCC current
dBest1 currents are regulated by phosphorylation
Regulation of dBest currents by phosphorylation is CaMKII-dependent

Discussion……………………..…………………………………….…………………………………………..…109
Drosophila anoctamins do not mediate S2 cell CaCCs
dBest1 currents are regulated via a CaMKII dependent mechanism

About this Dissertation

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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 Biology, Cell
Mot-clé	anoctamin chloride channels electrophysiology bestrophin ion channels
Committee Chair / Thesis Advisor	Hartzell Jr., Criss, Emory University
Committee Members	Kahn, Richard A, Emory University Iuvone, P Michael, Emory University Traynelis, Stephen, Emory University Faundez, Victor, Emory University McCarty, Nael, Emory University

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