Does extracellular calcium determine what pool of GABA is the target for α-latrotoxin?

Abstract

Presynaptic neurotoxin α-latrotoxin, from the venom of Latrodectus mactans tredecimguttatus, causes massive [ 3 H ]GABA release from rat brain synaptosomes, irrespective of calcium presence in the extracellular medium. Whether the binding of α-latrotoxin to Ca 2+-dependent (neurexin 1α) or to Ca 2+-independent (latrophilin) receptor triggers [ 3 H ]GABA release by the same mechanisms or different ones, inducing either exocytotic process or outflow by mobile membrane GABA transporter, is unknown. We examined α-latrotoxin-evoked [ 3 H ]GABA release from synaptosomes which cytosolic [ 3 H ]GABA pool was depleted either by applying competitive inhibitors of the GABA transporter, nipecotic acid and 2,4-diaminobutyric acid, or by permeation with digitonin. We also compared the effect of the GABA transporter inhibitors on depolarisation-evoked and α-latrotoxin-evoked [ 3 H ]GABA release using as depolarising agents 4-aminopyridine and high KCl in the Ca 2+-containing and in Ca 2+-free medium, respectively. Incubation of synaptosomes with nipecotic acid induced the essential acceleration of unstimulated [ 3 H ]GABA release and deep inhibition of high KCl-evoked Ca 2+-independent [ 3 H ]GABA release. In contrast, at the similar conditions the effect of α-latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in α-latrotoxin-induced release lays in an analysis of the effects of depolarisation and α-latrotoxin in consecutive order. The preliminary 4-aminopyridine-stimulated [ 3 H ]GABA release attenuated the toxin effect. But when depolarisation occurred in Ca 2+-free medium, no influence on α-latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that α-latrotoxin could stimulate [ 3 H ]GABA release in the medium with 1 mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that α-latrotoxin-released neurotransmitter has the vesicular nature. We assume that the type of the toxin membrane receptor does not determine the mechanisms of [ 3 H ]GABA release evoked by α-latrotoxin.