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J. Biol. Chem., Vol. 282, Issue 17, 12687-12697, April 27, 2007

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Molecular Interactions of the Gating Modifier Toxin ProTx-II with Na_v1.5

IMPLIED EXISTENCE OF A NOVEL TOXIN BINDING SITE COUPLED TO ACTIVATION^*

Jaime J. Smith, Theodore R. Cummins, Sujith Alphy, and Kenneth M. Blumenthal¹

From the Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214 and Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202

Voltage-gated Na⁺ channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na_v and Ca_v, but not K_v, channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na_v1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K_v channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na_v1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na_v channels with potential to shed new light on this process.

Received for publication, November 9, 2006 , and in revised form, February 15, 2007.

^* 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 1.

¹ To whom all correspondence and reprint requests should be addressed: Dept. of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, 3435 Main St., Buffalo, NY 14214. Tel.: 716-829-2727; Fax: 716-829-2725; E-mail: kblumen{at}buffalo.edu.

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