BOTULINUM, TETANUS, AND DIPHTHERIA TOXIN CHANNELS IN PLANAR LIPID BILAYERS (ACIDIC VESICLES, PH-GATING, VOLTAGE-DEPENDENT CHANNELS, CHANNEL-SIZING, SIGNAL HYPOTHESIS)
HOCH, DAVID HARRIS
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Here I report that the heavy chains of both botulinum and tetanus toxins form channels in planar bilayer membranes. These channels have pH and voltage-dependent properties remarkably similar to those of the diphtheria toxin channel. Channel formation is maximal when the protein-containing side of the bilayer is at acidic pH and the opposite side is at physiological pH, a pH gradient comparable to that across acidic vesicles. Selectivity experiments with large cations and anions demonstrate that the channels can accommodate ions at least the size of nicotinamide adenine dinucleotide (NAD). The pH dependence of channel formation and the large size of the channels are both compatible with the idea that the channels function as "tunnel proteins" for the translocation of fully extended active fragments across the vesicle membrane. These findings are relevant to the mechanism of protein transfer across membranes.;I have further characterized the properties of all three channels. Their selectivity and single channel conductances are sensitive functions of pH; selectivity also depends on the charge of the lipid. The increase with pH of both the single channel conductance and the cation selectivity of the diphtheria toxin channel could be diminished by the carbodiimide modification procedure, which neutralizes carboxyl groups. In contrast to diphtheria and tetanus toxin channels, botulinum toxin channels can form in membranes composed exclusively of neutral lipids. The rate of conductance increase induced by tetanus and diphtheria toxin varies with the square of the toxin concentration, suggesting that the channels are dimeric structures. Channel-forming properties of the whole toxins and of fragments containing the amino-terminus of their heavy chain have been compared. The voltage dependence of the diphtheria and tetanus toxin channels is shifted by pH in a manner that suggests that a pH gradient across the membrane acts like an applied electric field--representing a novel regulatory mechanism for gating of channels.