The Journal of Neurophysiology Vol. 82 No. 6 December 1999, pp. 3550-3562
Copyright ©1999 by the American Physiological Society

-Latrocrustatoxin Increases Neurotransmitter Release by Activating a Calcium Influx Pathway at Crayfish Neuromuscular Junction

Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Elrick, Donald B. and Milton P. Charlton. -Latrocrustatoxin Increases Neurotransmitter Release by Activating a Calcium Influx Pathway at Crayfish Neuromuscular Junction. J. Neurophysiol. 82: 3550-3562, 1999. -latrocrustatoxin (-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. -LCTX had no effect when applied in Ca²⁺-free saline, but subsequent addition of Ca²⁺ caused an immediate enhancement of mEPSP frequency even when -LCTX was previously washed out of the bath with Ca²⁺-free saline. Furthermore removal of Ca²⁺ from the saline after -LCTX had elicited an effect immediately blocked the action on mEPSP frequency. Thus -LCTX binding is insensitive to Ca²⁺, but toxin action requires extracellular Ca²⁺ ions. Preincubation with wheat germ agglutinin prevented the effect of -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 -latrotoxin (-LTX) found in vertebrates. -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 Ca²⁺ ionophore, A23187, indicating that influx of Ca²⁺ may mediate the action of -LCTX. In nerve terminals filled with the Ca²⁺ indicator, calcium green 1, -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 Ca²⁺ channel blockers -agatoxin IVA or Cd²⁺, demonstrating that Ca²⁺ influx does not occur via Ca²⁺ channels normally coupled to transmitter release in this preparation. The actions of -LCTX could be reduced dramatically by intracellular application of the Ca²⁺ chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid. We conclude that induction of extracellular Ca²⁺ influx into nerve terminals is sufficient to explain the action of -LCTX on both spontaneous and evoked transmitter release at crayfish neuromuscular junctions.