Diamide, temperature and spontaneous transmitter release at the neuromuscular junction: stimulation of exocytosis by a direct effect on membrane fusion?

The thiol-oxidizing agent diamide markedly increases m.e.p.p. frequency at the frog neuromuscular junction, even at low [Ca2+]0 and also when the mitochondria are uncoupled with DNP. The effect is reversed by dithioerythritol and is very temperature-sensitive, with a marked transition at 16 degrees C; m.e.p.p. frequency is raised 2- to 5-fold at 13-15 degrees C and 55- to 60-fold at 17-20 degrees C. Diamide increases the frequency of large amplitude m.e.p.p.s, the effect being explicable as the fusion of two or more vesicles. It is concluded that (a) diamide does not act at the Ca2+ channels of the plasma membrane, nor at the mitochondria. It affects the release system directly via an alteration of membrane protein --SH groups; (b) the eventual decline in m.e.p.p. frequency after DNP treatment is because of the exhaustion of mitochondrial Ca2+ rather than a depletion of quanta; (c) the major effect of temperature is on the release mechanism, perhaps via a phase-change in the phospholipoproteins of the plasmalemma or vesicles, rather than an elevation of [Ca2+]i; (d) either diamide or temperatures above 16 degrees C make Ca2+ more effective in promoting vesicle-plasmalemma fusion.