A 50‐kDa fragment from the NH2‐terminus of the heavy subunit of Clostridium botulinum type A neurotoxin forms channels in lipid vesicles

Abstract

1. A 50-kDa fragment representing the NH₂-terminus of the heavy subunit of botulinum type A neurotoxin was found, at low pH, to evoke the release of K⁺ from lipid vesicles loaded with potassium phosphate. Similar K⁺ release was also observed with the intact neurotoxin, its heavy chain and a fragment consisting of the light subunit linked the 50-kDa NH₂-terminal heavy chain fragment. The light subunit alone, however, was inactive. 2. In addition to K⁺, the channels formed in lipid bilayers by botulinum neurotoxin type A or the NH₂-terminal heavy chain fragment were found to be large enough to permit the release of NAD (M_r 665). 3. The optimum pH for the release of K⁺ was found to be 4.5. Above this value K⁺ release rapidly decreased and was undetectable above pH 6.0. 4. The binding of radiolabelled botulinum toxin to a variety of phospholipids was assessed. High levels of toxin binding were only observed to lipid vesicles with an overall negative charge; much weaker binding occurred to lipid vesicles composed of electrically neutral phospholipids. 5. A positive correlation between the efficiency of toxin binding and the efficiency of K⁺ release from lipid vesicles was not observed. Whereas lipid vesicles containing the lipids cardiolipin or dicetyl phosphate bound the highest levels of neurotoxin, the toxin-evoked release of K⁺ was low compared to vesicles containing either phosphatidyl glycerol, phosphatidyl serine or phosphatidyl inositol. 6. The implications of these observations to the mechanism by which the toxin molecule is translocated into the nerve ending are discussed.