Wolfe Joshua T, Krantz Bryan A, Rainey G Jonah A, Young John A T, Collier R John
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2005 Nov 25;280(47):39417-22. doi: 10.1074/jbc.M509049200. Epub 2005 Sep 23.
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.
炭疽毒素的保护性抗原（PA）与细胞受体结合，并在靶细胞膜上形成孔道，据信催化致死因子（LF）和水肿因子（EF）可通过这些孔道转运至细胞质。我们运用膜片钳电生理技术，实时检测了PA在培养细胞表面形成孔道的情况。用活化的PA处理的CHO-K1细胞的膜在中性pH（7.3）下几乎没有或完全没有导电性，但在pH 5.3时对电压刺激表现出强烈的混合离子电流。孔道的形成依赖于特定的细胞受体，并且在高电位（>60 mV）时表现出电压依赖性失活。形成孔道所需的pH值具有受体特异性，因为在特异性表达肿瘤内皮标志物8（TEM8）或毛细血管形态发生蛋白2（CMG2）（炭疽毒素的两种已知细胞受体）的细胞中进行测量时，膜插入发生在显著不同的pH值下。孔道受到LF的N端片段和微摩尔浓度的四丁基铵离子的抑制。这些研究证明了细胞膜中PA孔道的基本生物物理特性，并为体内LF和EF转运的研究奠定了基础。
