Staphylococcal alpha-toxin: repair of a calcium-impermeable pore in the target cell membrane

Staphylococcal alpha-toxin forms heptameric pores that render membranes per meable for monovalent cations. The pore is formed by an amphipathic beta-ba rrel encompassing amino acid residues 118-140 of each subunit of the oligom er. Human fibroblasts are susceptible to alpha-toxin but are able to repair the membrane lesions. Thereby, toxin oligomers remain embedded in the plas ma membrane and exposed to the extracellular medium. In this study, we soug ht to detect structural changes occurring in the pore-forming sequence duri ng lesion repair. Single cysteine substitution mutants were labelled with t he environmentally sensitive fluorochrome acrylodan and, after mixing with wild-type toxin, incorporated into hybrid heptamers on fibroblast membranes . Formation of the lipid-inserted beta-barrel was accompanied by characteri stic fluorescence emission shifts. After lesion repair, the environment of the residues at the outer surface of the beta-barrel remained unchanged, in dicating continued contact with lipids. However, the labelled residues orie nted towards the channel lumen underwent a green to blue shift in fluoresce nce, indicating reduced exposure to water. Pore closure proceeded in the pr esence of calmodulin inhibitors and of microtubule disruptors; however, it was prevented by cytochalasin D and by inhibitors of lipid metabolism. Our findings reveal the existence of a novel mechanism of membrane repair that may consist in constriction of the inserted proteinaceous pore within the l ipid bilayer.