Pore formation by Staphylococcus aureus alpha-toxin in lipid bilayers. Dependence upon temperature and toxin...

Staphylococcus aureus alpha-toxin forms ionic channels of large size in lipid bilayer membranes. We have developed two methods for studying the mechanism of pore formation. One is based on measurement of the ionic current flowing through a planar lipid membrane after exposure to the toxin; the other is based on measuring the release of the fluorescent complex Tb-Dipicolinic acid from large unilamellar vesicles under similar conditions. Both methods indicate that the pore formation process is complex, showing an initial delay followed by non-linear kinetics. The power dependence of the pore formation rate on the toxin concentration in planar bilayers indicates that an aggregation mechanism underlies the channel assembly. Arrhenius plots, obtained with both techniques, show no deviation from linearity up to 50 degrees C and the derived activation energies are found to be comparable to those for the binding and the lysis of rabbit erythrocytes by the same toxin. The temperature dependence of the conductance induced in planar bilayers by a large number of toxin channels indicates that the pores are filled with aqueous solution. The analysis of single conductance events shows that a heterogeneous population of pores exist and that smaller channels are preferred at low temperature. We attribute this heterogeneity to the existence of pores resulting from the aggregation of different numbers of monomers.