Probing the structure of the anthrax toxin channel using voltage-dependent blockers

Abstract

The pathogenesis of Bacillus anthracis, the causative agent of anthrax, depends on two important virulence factors: an antiphagocytic poly(scD-glutamic acid) capsule and "anthrax toxin." The latter term refers collectively to three proteins encoded by a plasmid--edema factor (EF; 89 kDa), lethal factor (LF; 83 kDa), and protective antigen (PA; 85 kDa). These three toxin components have no known biological effect when administered individually to experimental animals, but they act in binary combinations to produce two distinct reactions: edema (PA + EF) and death (PA + LF). Although the mechanism(s) by which EF and LF are translocated to the cytoplasm is poorly understood, PA is believed to play a central role.;In this thesis I show that nicked PA (that is PA which has been treated by mild trypsinization), but not unnicked PA, forms cation-selective channels which exhibit both voltage-dependent and pH-dependent activity; LF and EF are devoid of channel-forming activity. I have further explored the biophysical properties of the channel using a series of symmetric tetraalkylammonium (TAA) ions to probe the channel's structure. I show that at micromolar concentrations, these ions, ranging in size from tetramethylammonium to tetraheptylammonium, induce a voltage-dependent flickery block of the channel by binding to a site within the channel lumen, preventing the passage of the major current-carrying ion. Analysis at the single-channel level shows these blocking ions to be driven into the site by modest positive voltages, but driven off the site and through the channel by larger positive voltages, thus relieving the block, and providing a novel way of determining channel size; the permeability to tetrahexylammonium, the largest ion studied, implies that the narrowest part of the channel has a diameter of at least 11 A. An interesting finding that emerges from analysis of the voltage dependence of mean blocked and unblocked times is that the blocking rate, with tetrabutylammonium present on the cis side of the membrane, plateaus at large positive voltages to a voltage-independent value consistent with its entry rate being diffusion-limited.