Diffusion barrier for acetylcholine at the frog neuromuscular junction

Abstract

INACTIVATION of acetylcholinesterase (AChE) causes the half-decay times of the endplate currents (e.p.cs) and miniature e.p.cs (m.e.p.cs) to increase from about 1.5–2.0ms to 3.0–10.00 ms, times far too long to be accounted for by removal of the transmitter by free diffusion1–3. To explain this, Katz and Miledi1 postulated that, when the AChE is blocked, each acetylcholine (ACh) molecule undergoes numerous collisions with the post-synaptic membrane, leading to several attachments with the ACh receptors and a consequent delay in transmitter removal. This was supported by the observation that ( + )-tubocurarine, which reduces the number of free receptors, markedly decreases the half-decay of m.e.p.cs and e.p.cs after treatment with neostigmine1,3. In addition to this delaying mechanism, due to interaction with the ACh receptors the results described below suggest the existence of a second diffusion barrier for the transmitter at the frog neuromuscular junction. This barrier seems to be formed by non-depolarising sites which bind the ester moiety of ACh. Saturation of these sites with substances which possess –CO–O– or –CO–O–CO– groups accelerates the diffusion of externally applied ACh towards the ACh receptor, enhancing its depolarising effects. The barrier also hinders the outward diffusion of neurally released ACh in neostigmine-treated muscles, but this effect can only be demonstrated if the motor nerve is stimulated at high frequencies (200 Hz).