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Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel

Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel MthK is a Ca 2+ -gated K + channel whose activity is inhibited by cytoplasmic H + . To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H + inhibition and Ca 2+ -dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different Ca 2+ (ranging from nominally 0 to 30 mM) at a given H + , in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between Ca 2+ and open probability (Po), with a mean Hill coefficient (n H ) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor n H changed significantly as a function of H + over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca 2+ was not H + sensitive over pH ranging from 7.3 to 9.0. However, increasing H + raised the EC 50 for Ca 2+ activation by ∼4.7-fold per tenfold increase in H + , displaying a linear relation between log(EC 50 ) and log(H + ) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H + binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca 2+ binding and channel opening. We can account for the Ca 2+ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca 2+ allosterically activates MthK, whereas H + opposes activation by destabilizing the binding of Ca 2+ . Footnotes Abbreviations used in this paper: BK large-conductance Ca 2+ -activated K + CHES 2- N -cyclohexylaminoethanesulfonic acid DM decyl maltoside Po open probability MWC Monod-Wyman-Changeux Submitted: 18 December 2009 Accepted: 13 April 2010 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 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ ). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of General Physiology Rockefeller University Press

Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel

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References (54)

Publisher
Rockefeller University Press
Copyright
© 2010 Pau et al.
ISSN
0022-1295
eISSN
1540-7748
DOI
10.1085/jgp.200910387
pmid
20421375
Publisher site
See Article on Publisher Site

Abstract

MthK is a Ca 2+ -gated K + channel whose activity is inhibited by cytoplasmic H + . To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H + inhibition and Ca 2+ -dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different Ca 2+ (ranging from nominally 0 to 30 mM) at a given H + , in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between Ca 2+ and open probability (Po), with a mean Hill coefficient (n H ) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor n H changed significantly as a function of H + over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca 2+ was not H + sensitive over pH ranging from 7.3 to 9.0. However, increasing H + raised the EC 50 for Ca 2+ activation by ∼4.7-fold per tenfold increase in H + , displaying a linear relation between log(EC 50 ) and log(H + ) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H + binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca 2+ binding and channel opening. We can account for the Ca 2+ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca 2+ allosterically activates MthK, whereas H + opposes activation by destabilizing the binding of Ca 2+ . Footnotes Abbreviations used in this paper: BK large-conductance Ca 2+ -activated K + CHES 2- N -cyclohexylaminoethanesulfonic acid DM decyl maltoside Po open probability MWC Monod-Wyman-Changeux Submitted: 18 December 2009 Accepted: 13 April 2010 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 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ ).

Journal

The Journal of General PhysiologyRockefeller University Press

Published: May 1, 2010

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