The Effects of Calcium and Other Polyvalent Cations on Channel Formation by Escherichia coliα‐Hemolysin in Red Blood Cells and Lipid Bilayer Membranes

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Abstract
Channel formation by Escherichia coliα-hemolysin (HlyA) was studied in lipid bilayer membranes and in erythrocytes as a function of the concentration of divalent and trivalent cations. Hemolysin showed full channel-forming activity in artificial lipid bilayers, even in the presence of 5 mM EDTA and when the E. coli cells were grown in calcium-depleted media (< 1 μM Ca2+). The addition of divalent cations decreased the single-channel conductance by about 50% with half-saturation constants of S mM and less, while the mean lifetime of the HlyA channel was not affected. The addition of trivalent cations, such as Fe3+ or La3+, had a similar effect on the channel conductance, but the half-saturation constant was 1 μM or below. These effects may be caused by the binding of the cations to negatively charged groups at the channel mouth and have probably nothing to do with the possible binding of these cations to the repeat domain of the toxin, which is essential for target cell recognition. When cells were grown in calcium-depleted media, the supernatants showed absolutely no hemolytic activity. Addition of small amounts of Ca2+ to the supernatant led to toxin-mediated hemolysis. Its half-saturation constant was 120 μM. Of the other earth alkaline cations only strontium (Sr2+), which has an ion radius similar to Ca2+, led to full activation of HlyA with a Km of 1.5 mM. Ba2+ induced only weak hemolytic activity, while Mg2+ and several heavy metal cations had no effect. These results led to the conclusion that the target cell recognition sites formed by the repeat region of HlyA have defined sizes and bind only ions with defined radii.