INHIBITION OF VACUOLAR ADENOSINE-TRIPHOSPHATASE ANTAGONIZES THE EFFECTS OF CLOSTRIDIAL NEUROTOXINS BUT NOT PHOSPHOLIPASE-A2 NEUROTOXINS

Bafilomycin A1, an inhibitor of vacuolar adenosine triphosphatase, was tested for its ability to antagonize botulinum neurotoxins (serotypes A-G), tetanus toxin and phospholipase A2 neurotoxins (notexin, beta-b ungarotoxin, taipoxin and textilotoxin) on the mouse phrenic nerve-hem idiaphragm preparation. Bafilomycin itself produced concentration-depe ndent blockade of neuromuscular transmission without blocking nerve ac tion potentials or muscle action potentials. This effect may have been due to inhibition of the proton pump that regulates acetylcholine tra nsport into vesicles. At submaximal concentrations, bafilomycin was ve ry effective in delaying the onset of paralysis due to all clostridial neurotoxins, but it had no protective effect against phospholipase A2 neurotoxins. Experiments were done to determine which of the three st eps in clostridial neurotoxin action was antagonized by bafilomycin (e .g., binding, internalization and intracellular poisoning). Both pharm acological experiments and ligand-binding experiments showed that the drug did not block toxin binding to the plasma membrane. Similarly, ph armacological experiments on the time-dependent effects of bafilomycin showed that the drug did not antagonize the intracellular actions of toxins. The data indicated that bafilomycin acted at the intermediate step of internalization. This is in keeping with the facts that: 1) ba filomycin inhibits vacuolar adenosine triphosphatase, which in turn le ads to inhibition of acidification in endosomes and 2) clostridial neu rotoxins depend upon acidification of endosomes for translocation to t he cytosol. The finding that bafilomycin antagonizes tetanus toxin may provide important clues for understanding how this toxin can act loca lly to produce flaccid paralysis. The finding that bafilomycin is a un iversal antagonist that protects against all clostridial neurotoxins m ay have important implications for developing therapeutic drugs.