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J. Biol. Chem., Vol. 284, Issue 2, 1000-1008, January 9, 2009

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The Pore Domain Outer Helix Contributes to Both Activation and Inactivation of the hERG K⁺ Channel^*

Pengchu Ju¹, Guilhem Pages^¶1, R. Peter Riek, Po-chia Chen^||, Allan M. Torres^**, Paramjit S. Bansal, Serdar Kuyucak^||, Philip W. Kuchel^¶2, and Jamie I. Vandenberg, Recipient of National Health and Medical Research Council Senior Research Fellowship 459001^¶3

From the Division of Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia, St. Vincent's Clinical School, University of New South Wales, Darlinghurst, New South Wales 2010, Australia, the ^¶School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia, the ^||School of Physics, University of Sydney, New South Wales 2006, Australia, the ^**School of Biomedical and Health Sciences, University of Western Sydney, New South Wales 1797, Australia, and the Australian Nuclear Science and Technology Organisation, PMB1, Menai, New South Wales 2234, Australia

Ion flow in many voltage-gated K⁺ channels (VGK), including the (human ether-a-go-go-related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K⁺ channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approximately every third residue, from Phe⁵⁵¹ to Trp⁵⁶³, affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on activation (W563A, F559A, and F551A) face out toward the voltage sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala⁵⁶¹, His⁵⁶², Ala⁵⁶⁵, Trp⁵⁶⁸, and Ile⁵⁷¹), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.

Received for publication, August 19, 2008 , and in revised form, October 10, 2008.

^* This work was supported by Project Grant 459002 from the National Health and Medical Research Council (to J. I. V. and S. K.) and Project Grant DP0450808 from the Australian Research Council (to P. W. K. and J. I. V.). 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 Tables S1 and S2 and Figs. S1 and S2.

¹ These authors contributed equally to this work.

² Recipient of Australian Research Council Australian Professorial Fellowship DP0345961.

³ To whom correspondence should be addressed: Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, 405 Liverpool St., Darlinghurst, NSW 2010, Australia. Tel.: 61-2-9295-8771; Fax: 61-2-9295-8770; E-mail: j.vandenberg{at}victorchang.edu.au.

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