Structure-function relationships in diphtheria toxin channels: III. Residues which affect the cis pH dependence of channel conductance.
The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membrane depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to 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 conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, 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 conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.
Mindell
J A
JA
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461.
Silverman
J A
JA
Collier
R J
RJ
Finkelstein
A
A
D001224:Aspartic Acid / Q000032:analysis; D004167:Diphtheria Toxin / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D005971:Glutamates / Q000032:analysis; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D008051:Lipid Bilayers / Q000737:chemistry; D008564:Membrane Potentials / Q000502:physiology*; D008567:Membranes, Artificial; D009154:Mutation; D013329:Structure-Activity Relationship
D004167:Diphtheria Toxin; D005971:Glutamates; D007473:Ion Channels; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D001224:Aspartic Acid; D018698:Glutamic Acid