STRUCTURE-FUNCTION RELATIONSHIP OF THE ION-CHANNEL FORMED BY DIPHTHERIA-TOXIN IN VERO CELL-MEMBRANES

Diphtheria toxin (DT) forms cation selective channels at low pH in cel l membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-cla mp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). The se residues might interact electrostatically with cations flowing thro ugh the channel, and were therefore exchanged for uncharged amino acid s or lysine. The increase in whole-cell conductance induced by toxin, Delta g(m), was initially determined. DT-AB induced a similar to 10-fo ld lower Delta g(m) than DT-B. The mutations DT-B E327Q, DT-B D352N an d DT-B E362K did not affect Delta g(m), whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Delta g(m). Single channel analysi s of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then perfo rmed in outside-out patches. No differences were found for the single- channel conductances, but the mutants varied in their gating character istics. DT-B D295K exhibited only a very transient channel activity. D T-AB as well as DT-B D318K displayed significantly lower open probabil ity and mean dwell times than DT-B. Hence, the lower channel forming e fficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work s hows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as ag, and the gating kinetics of s ingle channels. This indicates clearly that the channel activity obser ved in DT-exposed Vero cells at low pH is inherent to DT itself and no t due to DT-activation of an endogenous channel.