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Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel

Bestrophins have been proposed to constitute a new family of Cl channels that are activated by cytosolic Ca. We showed previously that mutation of serine-79 to cysteine in mouse bestrophin-2 (mBest2) altered the relative permeability and conductance to SCN. In this paper, we have overexpressed vario...

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Autores principales: Qu, Zhiqiang, Hartzell, Criss
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2233901/
https://www.ncbi.nlm.nih.gov/pubmed/15452198
http://dx.doi.org/10.1085/jgp.200409108
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author Qu, Zhiqiang
Hartzell, Criss
author_facet Qu, Zhiqiang
Hartzell, Criss
author_sort Qu, Zhiqiang
collection PubMed
description Bestrophins have been proposed to constitute a new family of Cl channels that are activated by cytosolic Ca. We showed previously that mutation of serine-79 to cysteine in mouse bestrophin-2 (mBest2) altered the relative permeability and conductance to SCN. In this paper, we have overexpressed various mutant constructs of mBest2 in HEK-293 cells to explore the contributions to anion selectivity of serine-79 and other amino acids (V78, F80, G83, F84, V86, and T87) located in the putative second transmembrane domain (TMD2). Residues selected for mutagenesis were distributed throughout TMD2, but mutations at all positions changed the selectivity. The effects on selectivity were rather modest. Replacement of residues 78, 79, 80, 83, 84, 86, or 87 with cysteine had similar effects: the permeability of the channel to SCN relative to Cl (P(SCN)/P(Cl)) was decreased three- to fourfold and the relative SCN conductance (G(SCN)/G(Cl)) was increased five- to tenfold. Side chains at positions 78 and 80 appeared to be situated close to the permeant anion, because the electrostatic charge at these positions affected permeation in specific ways. The effects of charged sulfhydryl-reactive MTS reagents were the opposite in the V78C and F80C mutants and the effects were partially mimicked by substitution of F80 with charged amino acids. In S79T, switching from Cl to SCN caused slow changes in G(SCN)/G(Cl) (τ = 16.6 s), suggesting that SCN binding to the channel altered channel gating as well as conductance. The data in this paper and other data support a model in which TMD2 plays an important role in forming the bestrophin pore. We suggest that the major determinant in anion permeation involves partitioning of the permeant anion into an aqueous pore whose structural features are rather flexible. Furthermore, anion permeation and gating may be linked.
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spelling pubmed-22339012008-03-21 Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel Qu, Zhiqiang Hartzell, Criss J Gen Physiol Article Bestrophins have been proposed to constitute a new family of Cl channels that are activated by cytosolic Ca. We showed previously that mutation of serine-79 to cysteine in mouse bestrophin-2 (mBest2) altered the relative permeability and conductance to SCN. In this paper, we have overexpressed various mutant constructs of mBest2 in HEK-293 cells to explore the contributions to anion selectivity of serine-79 and other amino acids (V78, F80, G83, F84, V86, and T87) located in the putative second transmembrane domain (TMD2). Residues selected for mutagenesis were distributed throughout TMD2, but mutations at all positions changed the selectivity. The effects on selectivity were rather modest. Replacement of residues 78, 79, 80, 83, 84, 86, or 87 with cysteine had similar effects: the permeability of the channel to SCN relative to Cl (P(SCN)/P(Cl)) was decreased three- to fourfold and the relative SCN conductance (G(SCN)/G(Cl)) was increased five- to tenfold. Side chains at positions 78 and 80 appeared to be situated close to the permeant anion, because the electrostatic charge at these positions affected permeation in specific ways. The effects of charged sulfhydryl-reactive MTS reagents were the opposite in the V78C and F80C mutants and the effects were partially mimicked by substitution of F80 with charged amino acids. In S79T, switching from Cl to SCN caused slow changes in G(SCN)/G(Cl) (τ = 16.6 s), suggesting that SCN binding to the channel altered channel gating as well as conductance. The data in this paper and other data support a model in which TMD2 plays an important role in forming the bestrophin pore. We suggest that the major determinant in anion permeation involves partitioning of the permeant anion into an aqueous pore whose structural features are rather flexible. Furthermore, anion permeation and gating may be linked. The Rockefeller University Press 2004-10 /pmc/articles/PMC2233901/ /pubmed/15452198 http://dx.doi.org/10.1085/jgp.200409108 Text en Copyright © 2004, The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Article
Qu, Zhiqiang
Hartzell, Criss
Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel
title Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel
title_full Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel
title_fullStr Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel
title_full_unstemmed Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel
title_short Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel
title_sort determinants of anion permeation in the second transmembrane domain of the mouse bestrophin-2 chloride channel
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2233901/
https://www.ncbi.nlm.nih.gov/pubmed/15452198
http://dx.doi.org/10.1085/jgp.200409108
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