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J. Biol. Chem., Vol. 282, Issue 44, 31972-31981, November 2, 2007

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Activation Gating of hERG Potassium Channels

S6 GLYCINES ARE NOT REQUIRED AS GATING HINGES^*

Rachael M. Hardman, Phillip J. Stansfeld, Sarah Dalibalta, Michael J. Sutcliffe^¶, and John S. Mitcheson¹

From the Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom, the Structural Bioinformatics and Computational Biochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom, and the ^¶Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7ND, United Kingdom

The opening of ion channels is proposed to arise from bending of the pore inner helices that enables them to pivot away from the central axis creating a cytosolic opening for ion diffusion. The flexibility of the inner helices is suggested to occur either at a conserved glycine located adjacent to the selectivity filter (glycine gating hinge) and/or at a second site occupied by glycine or proline containing motifs. Sequence alignment with other K⁺ channels shows that hERG possesses glycine residues (Gly⁶⁴⁸ and Gly⁶⁵⁷) at each of these putative hinge sites. In apparent contrast to the hinge hypotheses, substitution of both glycine residues for alanine causes little effect on either the voltage-dependence or kinetics of channel activation, and open state block by intracellular blockers. Substitution of the glycines with larger hydrophobic residues causes a greater propensity for the channel to open. We propose that in contrast to Shaker the pore of hERG is intrinsically more stable in the open than the closed conformation and that substitution at Gly⁶⁴⁸ or Gly⁶⁵⁷ further shifts the gating equilibrium to favor the open state. Molecular dynamics simulations indicate the S6 helices of hERG are inherently flexible, even in the absence of the glycine residues. Thus hERG activation gating exhibits important differences to other K_v channels. Our findings indicate that the hERG inner helix glycine residues are required for the tight packing of the channel helices and that the flexibility afforded by glycine or proline residues is not universally required for activation gating.

Received for publication, July 16, 2007 , and in revised form, August 16, 2007.

^* This work was supported in part by a career establishment award from the Medical Research Council (to J. S. M.), by a Pfizer-BBSRC CASE studentship (to S. D.), and a Novartis-MRC CASE studentship (to P. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S3.

¹ To whom correspondence should be addressed: Dept. of Cell Physiology and Pharmacology, Medical Sciences Bldg., University of Leicester, University Road, Leicester LE1 9HN, UK. Tel.: 44-116-2928360; Fax: 44-116-252-5045; E-mail: jm109{at}le.ac.uk.

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