alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes.

Abstract

The. effect of a-latrotoxin from black widow. spider venom upon guinea pig cerebral cortical synaptosomes is described. Plasma membranepotential t(A.p), in situ mitochondrial membrane potential (Aqkm) (1-4, 11, 12); moreover, it releases acetylcholine, noradrenaline, dopamine, and y-aminobutyrate from cerebral cortical slices (5-7), synaptosomes (8, 10, 13),. and one neurosecretory cell line (9). A small number ofhigh-affinity binding sites for the toxin are located specifically on the presynaptic plasma. membrane (6, 10), and occupancy of these. correlates well with the subsequent release of neurotransmitter (10). In addition, the toxin is able to create stable conductance channels in protein-free black lipid membranes (14).A number of aspects of a-latrotoxin action are -unclear. In particular, there is no consensus as to the role ofCa2+. Whereas .there is agreement that the release of acetylcholine and yaminobutyrate is independent of external Ca2+ (3, 4, 7, 12, 13), the Ca2e dependency of catecholamine release appears dependent upon the experimental conditions and target membrane (5,(7)(8)(9). In addition, there is a contradiction between the results obtained with neurosecretory PC.12 cells, in which a. primary effect ofthe toxin appears to be a massive.uptake of Ca2+ across the plasma membrane (9), and those obtained with rat cerebral cortical synaptosomes, in which.no effect on Ca2+ flux could be detected (13).The possibility of a-latrotoxin-induced massive Ca2+ uptake across the presynaptic membrane raises a number of unanswered questions about the effects of the toxin on the energy levels in the cell. In particular, it is unclear whether the intraterminal mitochondria would be able to cope with this Ca2+ influx. Indeed swollen and disorganized mitochondria can be observed in electron micrographs of presynaptic expansions both at neuromuscular junctions (1-4, 11) and in cortical slices (7) treated with a-latrotoxin in the presence of external Ca2 . Deenergization of the mitochondria resulting in a collapse of the mitochondrial membrane potential (Aqi(m) would not only prevent mitochondrial synthesis ofATP but also would destroy the ability of the mitochondria to regulate the upper limit of the cytosolic free Ca2+ concentration (for reviews, see refs. 15-17), with a resultant risk of cell death (18).Recent developments in the study of isolated synaptosomes have enabled Ca2+ transport, Aim, and the plasma membrane potential (Ai/5p) to be determined simultaneously (15,(18)(19)(20)(21).In this paper we use these techniques .and introduce the application. of.the tetraphenylphosphonium (Ph4P+)-selective electrode (22) for the monitoring of synaptosomal potentials in suspension. EXPERIMENTAL PROCEDURESSynaptosomes.' Synaptosomes were prepared from the cerebral cortices (including corpus striatum) of Duncan-Hartley strain guinea pigs age 4-8 wk as described (19)(20)(21)(22). The synaptosomes were stored as a pellet in 250 mM suqrose/5 mM 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethane sulfonate (T...

Synaptosomal bioenergetics

“…Avm [14] and may therefore be used to detect qualitative changes in potentials (without directly distinguishing which is changed). Control synaptosomes maintain a Ph4P+ gradient in excess of loo0 (Fig.3A), taking no account of compartments.…”

“…Tetraphenylphosphonium (Ph4P+) accumulation was quantified with a Ph4P+-selective electrode [13] as described previously [14]. Synaptosomes (2 mg protein/ml) were incubated in 2 ml of Ca2+-free incubation medium for 5 min.…”

High Affinity Binding of α-Latrotoxin to Recombinant Neurexin Iα

“…␣-Latrotoxin stimulates neurotransmitter release from vertebrate nerve terminals by triggering massive exocytosis of small synaptic vesicles (1,2). ␣-Latrotoxin-stimulated neurotransmitter release is accompanied by presynaptic membrane depolarization and the influx of Ca 2ϩ through ion channels induced by the toxin (3,4). Purified ␣-latrotoxin forms cation channels in black lipid membranes, leading to the hypothesis that the toxin may act as an ionophore although the channel characteristics differ from those observed in intoxicated PC12 cells (5-7).…”

Pardaxin produces postjunctional muscle contraction in guinea‐pig intestinal smooth muscle

“…In previous studies BWSV has been found to induce a massive influx of calcium, resulting in the release of neurotransmitters (Grasso et al, 1980;Meldolesi, 1982;Nicholls et al, 1982). This mechanism might lead to a release of all types of neurotransmitters (Tzeng & Siekevitz, 1978;Fritz et al, 1980).…”