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An electrostatic interaction between TEA and an introduced pore aromatic drives spring-in-the-door inactivation in Shaker potassium channels

Ahern, Christopher A. and Eastwood, Amy L. and Dougherty, Dennis A. and Horn, Richard (2009) An electrostatic interaction between TEA and an introduced pore aromatic drives spring-in-the-door inactivation in Shaker potassium channels. Journal of General Physiology, 134 (6). pp. 461-469. ISSN 0022-1295. PMCID PMC2806421.

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Slow inactivation of Kv1 channels involves conformational changes near the selectivity filter. We examine such changes in Shaker channels lacking fast inactivation by considering the consequences of mutating two residues, T449 just external to the selectivity filter and V438 in the pore helix near the bottom of the selectivity filter. Single mutant T449F channels with the native V438 inactivate very slowly, and the canonical foot-in-the-door effect of extracellular tetraethylammonium (TEA) is not only absent, but the time course of slow inactivation is accelerated by TEA. The V438A mutation dramatically speeds inactivation in T449F channels, and TEA slows inactivation exactly as predicted by the foot-in-the-door model. We propose that TEA has this effect on V438A/T449F channels because the V438A mutation produces allosteric consequences within the selectivity filter and may reorient the aromatic ring at position 449. We investigated the possibility that the blocker promotes the collapse of the outer vestibule (spring-in-the-door) in single mutant T449F channels by an electrostatic attraction between a cationic TEA and the quadrupole moments of the four aromatic rings. To test this idea, we used in vivo nonsense suppression to serially fluorinate the introduced aromatic ring at the 449 position, a manipulation that withdraws electrons from the aromatic face with little effect on the shape, net charge, or hydrophobicity of the aromatic ring. Progressive fluorination causes monotonically enhanced rates of inactivation. In further agreement with our working hypothesis, increasing fluorination of the aromatic gradually transforms the TEA effect from spring-in-the-door to foot-in-the-door. We further substantiate our electrostatic hypothesis by quantum mechanical calculations.

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Dougherty, Dennis A.0000-0003-1464-2461
Additional Information:© 2009 Ahern et al. 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 After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at Christopher Miller served as editor. Submitted: 14 May 2009. Accepted: 20 October 2009. Published November 16, 2009. We thank Mary Y. Ryan for help with oocytes and molecular biology and Carol Deutsch for helpful discussions. This work is supported by grants from the National Institutes of Health (grants GM079427 to R. Horn and NS34407 to D.A. Dougherty). C.A. Ahern is supported by the Canadian Institutes of Health Research (grant 56858), the Heart and Stroke Foundation of Canada, and the Michael Smith Foundation for Health Research.
Funding AgencyGrant Number
Canadian Institutes of Health Research (CIHR)56858
Heart and Stroke Foundation of CanadaUNSPECIFIED
Michael Smith Foundation for Health ResearchUNSPECIFIED
Issue or Number:6
PubMed Central ID:PMC2806421
Record Number:CaltechAUTHORS:20100106-112241738
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Official Citation:Christopher A. Ahern, Amy L. Eastwood, Dennis A. Dougherty, and Richard Horn An electrostatic interaction between TEA and an introduced pore aromatic drives spring-in-the-door inactivation in Shaker potassium channels J. Gen. Physiol. 134: 461-469; published online before print as 10.1085/jgp.200910260
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:17077
Deposited By: Tony Diaz
Deposited On:07 Jan 2010 00:23
Last Modified:08 Jul 2020 19:09

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