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Originally published In Press as doi:10.1074/jbc.M111862200 on April 24, 2002

J. Biol. Chem., Vol. 277, Issue 27, 24653-24658, July 5, 2002
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A "Minimal" Sodium Channel Construct Consisting of Ligated S5-P-S6 Segments Forms a Toxin-activatable Ionophore*

Zhenhui ChenDagger , Carmen Alcayaga§, Benjamin A. Suárez-Isla§, Brian O'Rourke, Gordon Tomaselli, and Eduardo Marbán

From the Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 and the § Program of Physiology and Biophysics, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago 6530499, Chile

The large size (six membrane-spanning repeats in each of four domains) and asymmetric architecture of the voltage-dependent Na+ channel has hindered determination of its structure. With the goal of determining the minimum structure of the Na+ channel permeation pathway, we created two stable cell lines expressing the voltage-dependent rat skeletal muscle Na+ channel (µ1) with a polyhistidine tag on the C terminus (µHis) and pore-only µ1 (µPore) channels with S1-S4 in all domains removed. Both constructs were recognized by a Na+ channel-specific antibody on a Western blot. µHis channels exhibited the same functional properties as wild-type µ1. In contrast, µPore channels did not conduct Na+ currents nor did they bind [3H]saxitoxin. Veratridine caused 40 and 54% cell death in µHis- and µPore-expressing cells, respectively. However, veratridine-induced cell death could only be blocked by tetrodotoxin in cells expressing µHis, but not µPore. Furthermore, using a fluorescent Na+ indicator, we measured changes in intracellular Na+ induced by veratridine and a brevotoxin analogue, pumiliotoxin. When calibrated to the maximum signal after addition of gramicidin, the maximal percent increases in fluorescence (Delta F) were 35 and 31% in cells expressing µHis and µPore, respectively. Moreover, in the presence of 1 µM tetrodotoxin, Delta F decreased significantly to 10% in µHis- but not in µPore-expressing cells (43%). In conclusion, S5-P-S6 segments of µ1 channels form a toxin-activable ionophore but do not reconstitute the Na+ channel permeation pathway with full fidelity.

* This work was supported by National Institutes of Health Grant RO1 HL52768 (to E. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of the Michel Mirowski Fellowship from the National Association of Pacing and Electrophysiology (NASPE).

The Michel Mirowski, M.D. Professor of Cardiology of The Johns Hopkins University. To whom correspondence should be addressed: Inst. of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 N. Rutland Ave./Ross 844, Baltimore, MD 21205. Tel.: 410-955-2776; Fax: 410-955-7953; E-mail: marban@jhmi.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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