alpha-latrocrustatoxin increases neurotransmitter release by activating a calcium influx pathway at crayfish neuromuscular junction

alpha-latrocrustatoxin (alpha-LCTX), a component of black widow spider venom (BWSV), produced a 50-fold increase in the frequency of spontaneously occurring miniature excitatory postsynaptic potentials (mEPSPs) at crayfish neuromuscular junctions but did not alter their amplitude distribution. During toxin action, periods of high-frequency mEPSP discharge were punctuated by periods in which mEPSP frequency returned toward control levels. EPSPs were increased in amplitude during periods of enhanced mEPSP discharge. alpha-LCTX had no effect when applied in Ca(2+)-free saline, but subsequent addition of Ca(2+) caused an immediate enhancement of mEPSP frequency even when alpha-LCTX was previously washed out of the bath with Ca(2+)-free saline. Furthermore removal of Ca(2+) from the saline after alpha-LCTX had elicited an effect immediately blocked the action on mEPSP frequency. Thus alpha-LCTX binding is insensitive to Ca(2+), but toxin action requires extracellular Ca(2+) ions. Preincubation with wheat germ agglutinin prevented the effect of alpha-LCTX but not its binding. These binding characteristics suggest that the toxin may bind to a crustacean homologue of latrophilin/calcium-independent receptor for latrotoxin, a G-protein-coupled receptor for alpha-latrotoxin (alpha-LTX) found in vertebrates. alpha-LCTX caused "prefacilitation" of EPSP amplitudes, i.e., the first EPSP in a train was enhanced in amplitude to a greater degree than subsequent EPSPs. A similar alteration in the pattern of facilitation was observed after application of the Ca(2+) ionophore, A23187, indicating that influx of Ca(2+) may mediate the action of alpha-LCTX. In nerve terminals filled with the Ca(2+) indicator, calcium green 1, alpha-LCTX caused increases in the fluorescence of the indicator that lasted for several minutes before returning to rest. Neither fluorescence changes nor toxin action on mEPSP frequency were affected by the Ca(2+) channel blockers omega-agatoxin IVA or Cd(2+), demonstrating that Ca(2+) influx does not occur via Ca(2+) channels normally coupled to transmitter release in this preparation. The actions of alpha-LCTX could be reduced dramatically by intracellular application of the Ca(2+) chelator, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid. We conclude that induction of extracellular Ca(2+) influx into nerve terminals is sufficient to explain the action of alpha-LCTX on both spontaneous and evoked transmitter release at crayfish neuromuscular junctions.