STRUCTURE-FUNCTION-RELATIONSHIPS IN DIPHTHERIA-TOXIN CHANNELS .2. A RESIDUE RESPONSIBLE FOR THE CHANNELS DEPENDENCE ON TRANS PH

Ion-conducting channels formed in lipid bilayers by diphtheria toxin a re highly pH dependent. Among other properties, the channel's single c hannel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH -dependent single channel properties as that of the intact toxin. This region corresponds to an alpha-helical hairpin in the recently publis hed crystal structure of DT in solution; the hairpin contains two alph a-helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysin e has no effect on the DT channel's single channel conductance or sele ctivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3 cis/7.2 trans (in 1 M KCl), c onsistent with this group interacting electrostatically with ions in t he channel. The cation selectivity of these mutant channels is also re duced from that of wild-type channels, a direction consistent with res idue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 ( in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0 cis/7.2 trans (with a high c oncentration of permeant buffer in the cis compartment) imply that res idue 352 is on or near the traits side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positiv e voltages supports this conclusion. The trans location of position 35 2 severely constrains the number of possible membrane topologies for t his region.