The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltagedependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as "tunnel proteins" for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.Diphtheria toxin (1), botulinum neurotoxin (2), and tetanus toxin (3) are proteins that are similar in origin and macrostructure. All three toxins are synthesized by bacteria (Corynebacterium diphtheriae, Clostridium botulinum, and Clostridium tetani) as single polypeptide chains (diphtheria toxin, "'60 kDa; clostridial neurotoxins, ==150 kDa). When exposed to trypsin or trypsin-like enzymes, they are cleaved to yield two-chain molecules in which a heavy-chain polypeptide is linked by a disulfide bond to a light-chain polypeptide. The two-chain structure is the active form of the three toxins.Various techniques have been used to generate polypeptide fragments from the toxins. The most straightforward of these is disulfide-bond reduction, which releases the heavy chain from the light chain. Alternatively, the clostridial neurotoxins have been exposed to limited proteolysis (e.g., with papain) to generate a fragment B and fragment C. Finally, traditional techniques have been used to select mutant organisms that synthesize incomplete toxins, such as the CRM45 fragment of diphtheria toxin, from which the B45 fragment can be formed. The various toxins and their fragments are illustrated in Fig. 1 Previous studies have shown that the amino terminus of the heavy chain from diphtheria toxin (18) and whole diphtheria toxin (19) form channels in lipid bilayers, and it has been proposed (18) that these channels provide the pathway for the light chain to cross membranes. Here we report that the heavy chains of both botulinum neurotoxin type B and tetanus toxin also form channels in lipid bilayers. Furthermore, for all three toxins, channel formation is maximal when the protein-containing (cis) side of the artificial membrane is at low pH (-4.0) and the opposite (trans) side is at pH -7.0, a pH gradient comparable to that across the membranes of acidic vesicles in cells. The channels for all three toxins are very large, as determined by selectivity experiments with large anions and cations, and this finding is compatible with the idea that the channels function as "tunnel proteins" for translocation of fully extended active fragments. In addition, tetanus toxin channels display a voltage dependence similar to that of diphtheria toxin channels, opening when positive voltages are applied to the cis side of artificial membranes and closing when nega...