Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization

Abstract

Botulinum neurotoxin (BoNY) type A, a causative agent of botulism, is a di-chain protein (molecular weight 140,000) from Clostridium botulinum, and the most neurotoxic substance known¹. Some cases of sudden infant cot deaths have been attributed to such a neuroparalytic condition². BoNT inhibits irreversibly the release of acetylcholine from peripheral nerves in a highly selective manner^3–6. Hence, it is potentially an invaluable probe for studying the mechanism of transmitter release^7,8. Here we demonstrate specific labelling of murine motor nerve terminals with neurotoxic, ¹²⁵I-labelled BoNT (type A) by autoradiography. We observed saturable, temperature-sensitive binding of BoNT to sites which reside solely on the nerve terminal membrane; these were distributed on all unmyelinated areas, at an average density of 150–500 per µm² of membrane. The binding was mediated by the larger summit of the toxin and was inhibited partially by tetanus toxin, another microbial protein⁹. No specific binding was detectable on any other cell types examined, including noradrenergic terminals. Following binding, internalization of radioactivity was observed; this process was energy-dependent as it could be prevented totally by aride or dinitrophenol (DNP). This direct demonstration of separable steps, including highly selective binding and acceptor-mediated internalization, is reconcilable with the unique potency and the multiphasic inhibitory action of BoNT on transmitter release, as shown electrophysiologically⁸.