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 (>60mV). 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.Anthrax toxin, secreted by Bacillus anthracis, belongs to the A-B family of bacterial toxins and consists of three protein components. The "B" component, protective antigen (PA), 2 binds target cell receptors and is responsible for translocation of the two "A" components, lethal factor (LF) and edema factor (EF), to the cytoplasm of the affected cell (1). The action of LF and EF, a metalloprotease (2) and a soluble adenylyl cyclase (3), respectively, have various effects on cells, eventually leading to death of the infected host. PA is an 83-kDa protein that binds either of two known cellular receptors, capillary morphogenesis protein 2 (CMG2) (4) and tumor endothelial marker 8 (TEM8) (5). Cleavage of PA at a furin-sensitive site follows receptor binding and releases an N-terminal 20-kDa fragment, leaving a mature 63-kDa protein bound to the receptor (6). This allows interaction with other cleaved PA molecules and leads to the formation of ring-shaped, heptameric "prepore" structures on the cell surface (7). LF and EF bind competitively to the prepore, and the resulting complexes are internalized by endocytosis (8, 9). Endosome acidification by a vesicular membrane proton pump induces a conformational change in the prepore that allows it to form a membrane-spanning pore (10). LF and EF are believed to translocate through this pore to the cytosol. Because LF and EF are nontoxic in the absence of PA, a better understanding of the translocation process may reveal useful drug targets for disrupting the damaging effects of the toxin after anthrax infection.In vivo systems for the study of translocation in real time remain elusive, and instead, most relevant data on the process come from indirect biochemical studies (11) and from experiments in planar lipid bilayers (12, 13). The bio...

Selection and evaluation of the immunogenicity of protective antigen mutants as anthrax vaccine candidates

“…Each mutant is impaired in one of the following steps of PA-mediated anthrax intoxication action: (i) binding of PA to a cell surface receptor [16,17], (ii) cleavage of PA by a furin-type enzyme into two fragments (PA 63 and PA 20 ) [12], (iii) oligomerization of cell-bound PA 63 to (PA 63 ) 7 heptamers [14], and (iv) conformational change of (PA 63 ) 7 upon endocytosis and in acidic endosomes [18] to form transmembrane pores for LF/EF translocation [19,20]. Using these different non-functional mutants, we desired to identify PA mutants that are devoid of toxicity, yet highly efficient in inducing protective antibodies for use as future anthrax vaccine candidates.…”

Toxicity of Anthrax Toxin Is Influenced by Receptor Expression

“…PA has been reported to have nearly 1,000-fold greater affinity for CMG2 than for TEM8 (32). PA insertion is thought to first require dissociation of PA from the receptor (5,27), and insertion for PA associated with the CMG2 complex requires more extreme conditions (pH ϭ 5.2) than PA associated-TEM8 complex (pH ϭ 6.0 to 6.5) (27,41). We observed spontaneous pore formation for the CHO-TEM8 cells grown in culture medium at pH 7.3 (Fig.…”

“…Membrane insertion of PA is thought to first require dissociation from the receptor, and the higher affinity of PA for CMG2 appears to make dissociation more difficult. For example, dissociation from CMG2 requires a very low pH (5.2), while membrane insertion for the PA-associated TEM8 complex has been reported to occur at a much higher pH (6.0 to 6.5) (27,41). In addition to differences in the extracellular domains, these receptors also differ in their cytoplasmic domains.…”

“…The AVA vaccine is extremely reactogenic, and adverse reactions to the anthrax vaccine have been reported (9,10,12,22,25,36,39). In the present study, we examined the influence of receptor expression on anthrax toxin toxicity utilizing wild-type K1 Chinese hamster ovary cells (CHO-K1), CHO cells lacking functional anthrax toxin receptors (CHO-R1.1), and a well-characterized series of CHO-R1.1 cells expressing one of the two human anthrax toxin receptors, TEM8 or CMG2 (31,33,41,42). Our studies suggest that the CMG2 receptor might signal in response to cAMP and that, unexpectedly, PA in the absence of EF or LF may mediate toxicity in a receptor-dependent manner.…”

Effect of the Endosomal Acidification on Small Ion Transport Through the Anthrax Toxin PA63 Channel

“…The endosomal acidification triggers substantial conformational changes of PA 63 oligomers leading to their insertion into endosomal membranes and ion channel formation [25,26]. The pH threshold for PA 63 channel formation is different depending on which cellular receptor PA 83 binds; pH 6.2 is sufficient to weaken the PA contact with TEM8, whereas pH 5.2 is required to weaken the stronger PA/CMG2 binding [11][12][13]. This indicates a possibility for the PA channel formation already at the early endosomal stage when the complex is bound to TEM8, however the lower late endosomal pH is required when the complex is bound to CMG2 [14,15].…”