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J. Biol. Chem., Vol. 283, Issue 35, 24118-24127, August 29, 2008

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A Pore-blocking Hydrophobic Motif at the Cytoplasmic Aperture of the Closed-state Na_v1.7 Channel Is Disrupted by the Erythromelalgia-associated F1449V Mutation^*

Angelika Lampert^¶12, Andrias O. O'Reilly^||1, Sulayman D. Dib-Hajj^¶, Lynda Tyrrell^¶, B. A. Wallace^||3, and Stephen G. Waxman^¶4

From the Department of Neurology and Center for Neuroscience & Regeneration Research, Yale University School of Medicine, New Haven, Connecticut 06510, the ^¶Rehabilitation Research Center, Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut 06516, and the ^||Department of Crystallography, Birkbeck College, University of London, London WC1E 7HX, United Kingdom

Sodium channel Na_v1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Na_v1.7 produce painful disorders, whereas loss-of-function Na_v1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Na_v1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Na_v1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Na_v1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Na_v1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Na_v1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Na_v1.7.

Received for publication, April 15, 2008 , and in revised form, June 11, 2008.

^* This work was supported in part by grants from the Rehabilitation Research Service and Medical Research Service, Department of Veterans Affairs, the National Multiple Sclerosis Society, and the Erythromelalgia Association. The work in the laboratory of B. A. W. was supported by grants from the UK Biotechnology and Biological Sciences and Research Council. The Center for Neuroscience and Regeneration Research is a Collaboration of the Paralyzed Veterans of America and the United Spinal Association with Yale University. 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.

¹ Both authors contributed equally to this work.

² Supported by a Postdoctoral Research Training Fellowship of the Epilepsy Foundation of America and UCB Pharma, Inc. Present address: Institute of Physiology, Dept. of Physiological Genomics, Ludwig-Maximilians-University, Schillersstrasse 46, 80336 Munich, Germany.

³ To whom correspondence may be addressed: Tel.: 44-207-631-6800; Fax: 44-207-631-6803; E-mail: b.Wallace{at}mail.cryst.bbk.ac.uk.

⁴ To whom correspondence may be addressed: Dept. of Neurology, Yale Medical School, LCI 707, P. O. Box 208018, New Haven, CT 06520. Tel.: 203-785-6351; Fax: 203-785-2238; E-mail: Stephen.Waxman{at}yale.edu.

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