Why Escherichia coli α-hemolysin induces calcium oscillations in mammalian cells -: the pore is on its own

Escherichia coli alpha-hemolysin ( HlyA), archetype of a bacterial pore-forming toxin, has been reported to deregulate physiological Ca2+ channels, thus inducing periodic low-frequency Ca2+ oscillations that trigger transcriptional processes in mammalian cells. The present study was undertaken to delineate the mechanisms underlying the Ca2+ oscillations. Patch-clamp experiments were combined with single cell measurements of intracellular Ca2+ and with flow-cytometric analyses. Application of HlyA at subcytocidal concentrations provoked Ca2+ oscillations in human renal and endothelial cells. However, contrary to the previous report, the phenomenon could not be inhibited by the Ca2+ channel blocker nifedipine and Ca2+ oscillations showed no constant periodicity at all. Ca2+ oscillations were dependent on the pore-forming activity of HlyA: application of a nonhemolytic but bindable toxin had no effect. Washout experiments revealed that Ca2+ oscillations could not be maintained in the absence of toxin in the medium. Analogously, propidium iodide flux into cells occurred in the presence of HlyA, but cells rapidly became impermeable toward the dye after toxin washout, indicating resealing or removal of the membrane lesions. Finally, patchclamp experiments revealed temporal congruence between pore formation and Ca2+ influx. We conclude that the nonperiodic Ca2+ oscillations induced by HlyA are not due to deregulation of physiological Ca2+ channels but derive from pulsed influxes of Ca2+ as a consequence of formation and rapid closure of HlyA pores in mammalian cell membranes.