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ORIGINAL ARTICLE

A Multifunctional Aromatic Residue in the External Pore Vestibule of Na⁺ Channels Contributes to the Local Anesthetic Receptor

The Johns Hopkins University School of Medicine, Baltimore, Maryland (S.Y.T., G.F.T., R.A.L.); and Departments of Physiology and Medicine, Heart & Stroke Richard Lewar Centre, and Division of Cadiology University Health Network (R.G.T., P.H.B.), University of Toronto, Toronto, Ontario, Canada

Abstract

Voltage-gated Na⁺ (Na_v) channels are responsible for initiating action potentials in excitable cells and are the targets of local anesthetics (LA). The LA receptor is localized to the cytoplasmic pore mouth formed by the S6 segments from all four domains (DI–DIV) but several outer pore-lining residues have also been shown to influence LA block (albeit somewhat modestly). Many of the reported amino acid substitutions, however, also disrupt the inactivated conformations that favor LA binding, complicating the interpretation of their specific effects on drug block. In this article, we report that an externally accessible aromatic residue in the Na_v channel pore, DIV-Trp1531, when substituted with cysteine, completely abolished LA block (e.g., 300 µM mexiletine induced a use-dependent block with 65.0 ± 2.9% remaining current and –11.0 ± 0.6 mV of steady-state inactivation shift of wild-type (WT) channels versus 97.4 ± 0.7% and –2.4 ± 2.1 mV of W1531C, respectively; p < 0.05) without destabilizing fast inactivation (complete inactivation at 20 ms at –20 mV; V_1/2 = –70.0 ± 1.6 mV versus –48.6 ± 0.5 mV of WT). W1531C also abolished internal QX-222 block (200 µM; 98.4 ± 3.4% versus 54.0 ± 3.2% of WT) without altering drug access. It is interesting that W1531Y restored WT blocking behavior, whereas W1531A channels exhibited an intermediate phenotype. Together, our results provide novel insights into the mechanism of drug action, and the structural relationship between the LA receptor and the outer pore vestibule.

Address correspondence to: Dr. Ronald Li, The Johns Hopkins University, 720 Rutland Avenue, Ross 1165, Baltimore MD 21205. E-mail: ronaldli{at}jhmi.edu

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