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Original Article

^a Department of Medicine, Emory University, Atlanta, Georgia 30322
^b Department of Physiology, Emory University, Atlanta, Georgia 30322
^c Atlanta Veterans Administration Medical Center, Atlanta, Georgia 30033
^d Department of Anesthesiology, Brigham and Woman's Hospital, Boston, Massachusetts 02115
^e Department of Neurobiology, Pharmacology and Physiology, Chicago, Illinois 60637
^f Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
^g Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1
Assistant Professor of Medicine and Physiology, Division of Cardiology, Emory University/VAMC, 1670 Clairmont Road, Room 111B, Decatur, GA 30033.(404) 329-2211

sdudley{at}emory.edu

Voltage-gated Na⁺ channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na⁺ channel (µ1) outer vestibule and selectivity filter using µ-conotoxin GIIIA (µ-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute –1.0 ± 0.1, –0.9 ± 0.3, and –1.4 ± 0.1 kcal/mol of coupling energy to the native toxin–channel complex, respectively. µ-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.

Key Words: electrophysiology • site-directed mutagenesis • molecular models • kinetics • binding sites

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