title: Whole-cell voltage clamp measurements of anthrax toxin pore current.
abstract: 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.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Wolfe
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Krantz
authors.forename: G Jonah A
authors.initials: GJ
authors.lastname: Rainey
authors.forename: John A T
authors.initials: JA
authors.lastname: Young
authors.forename: R John
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism* / Q000633:toxicity*; D001427:Bacterial Toxins / Q000378:metabolism* / Q000633:toxicity*; D055592:Biophysical Phenomena; D001703:Biophysics; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D018000:Receptors, Peptide; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C473287:ANTXR1 protein, human; C447150:ANTXR2 protein, human; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D000644:Quaternary Ammonium Compounds; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C030325:anthrax toxin; C009405:tetrabutylammonium