STRUCTURE-FUNCTION-RELATIONSHIPS IN DIPHTHERIA-TOXIN CHANNELS .3. RESIDUES WHICH AFFECT THE CIS PH-DEPENDENCE OF CHANNEL CONDUCTANCE

The conductance of channels formed by diphtheria toxin (DT) in lipid b ilayer membranes depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient t o form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductan ce on trans pH, whereas another, Glutamate 349, has no effect. Here, w e report that of the seven remaining charged residues in the TH8-9 reg ion, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conduc tance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesti ng that this residue lies within the channel; it is more accessible, h owever, to cis than to trans protons. These results are consistent wit h the membrane-spanning topology previously proposed for the TH8-9 reg ion, and suggest a geometric model for the DT channel.