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J. Biol. Chem., Vol. 280, Issue 47, 39417-39422, November 25, 2005

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Whole-cell Voltage Clamp Measurements of Anthrax Toxin Pore Current^*

Joshua T. Wolfe, Bryan A. Krantz, G. Jonah A. Rainey, John A. T. Young, and R. John Collier1

From the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 and the Infectious Disease Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037-1099

Protective antigen (PA) of anthrax toxin binds cellular receptors and forms pores in target cell membranes, through which catalytic lethal factor (LF) and edema factor (EF) are believed to translocate to the cytoplasm. Using patch clamp electrophysiological techniques, we assayed pore formation by PA in real time on the surface of cultured cells. The membranes of CHO-K1 cells treated with activated PA had little to no electrical conductivity at neutral pH (7.3) but exhibited robust mixed ionic currents in response to voltage stimuli at pH 5.3. Pore formation depended on specific cellular receptors and exhibited voltage-dependent inactivation at large potentials (>60 mV). The pH requirement for pore formation was receptor-specific as membrane insertion occurs at significantly different pH values when measured in cells specifically expressing tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), the two known cellular receptors for anthrax toxin. Pores were inhibited by an N-terminal fragment of LF and by micromolar concentrations of tetrabutylammonium ions. These studies demonstrated basic biophysical properties of PA pores in cell membranes and served as a foundation for the study of LF and EF translocation in vivo.

Received for publication, August 16, 2005 , and in revised form, September 21, 2005.

^* 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 To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1930; Fax: 617-432-0115; E-mail: john_collier{at}hms.harvard.edu.

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