Mode of action of α-latrotoxin

Role of divalent cations in Ca2+-dependent and Ca2+-independent effects mediated by the toxin

Luann Rosenthal, Daniele Zacchetti, Luisa Madeddu, Jacopo Meldolesi

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Abstract

The potent neurotoxin α-latrotoxin (αLTx), from black widow spider venom, induces neurotransmitter release in both Ca2+-containing and Ca2+-free medium, following interaction with a specific cell surface receptor. Binding studies revealed two populations of αLTx binding sites in bovine synaptosomal membranes, showing the same high affinity (Kd, 0.3 × 10-10 M) for αLTx, with approximately 50% of the sites being Ca2+ sensitive and the rest being Ca2+ insensitive. In contrast, in PC12 cells αLTx binding was completely unaffected by the removal of extracellular Ca2+ (Kd, 5 × 10-10 M). The use of La3+ as an inhibitor of αLTx action, previously shown in synaptosomes, was extended to PC12 cells. In this system, La3+ (100 μM) was shown to inhibit Ca2+ influx, both Ca2+-dependent and -independent dopamine release, and polyphosphoinositide (PPI) hydrolysis induced by αLTx. At the same time, La3+ did not block αLTx binding or dopamine release evoked by either the ionophore ionomycin (0.5 μM) or the phorbol ester tetradecanoylphorbol acetate (100 NM). La3+ also blocked the influx of Mn2+ ions through the αLTx-induced cation channel, as measured by quenching of fura-2 fluorescence. In this PC12 cell line, PPI hydrolysis could also be induced by ionomycin, but only when it was present at concentrations that caused an elevation of free intracellular Ca2+ ([Ca2+]i) that was not transient but was as persistent as that evoked by αLTx. Our conclusions with regard to the mode of action of αLTx are as follows, (i) All the effects of αLTx in PC12 cells (dopamine release, PPI hydrolysis, and Ca2+ influx) can be mediated via a single, Ca2+-insensitive αLTx receptor, (ii) αLTx-induced PPI hydrolysis is most likely due to the activation of a Ca2+-sensitive phospholipase C following the persistent rise in [Ca2+]i elicited by the toxin in Ca2+-containing medium, and not via direct coupling of the αLTx receptor to the enzyme. (iii) Toxin-evoked Ca2+-independent dopamine release can be blocked by La3+ at the extracellular level, most likely by prevention of the entry of divalent cations.

Original languageEnglish
Pages (from-to)917-923
Number of pages7
JournalMolecular Pharmacology
Volume38
Issue number6
Publication statusPublished - Dec 1990

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Phosphatidylinositol Phosphates
PC12 Cells
Divalent Cations
Dopamine
Hydrolysis
Ionomycin
Synaptosomes
Fura-2
Ionophores
Neurotoxins
Cell Surface Receptors
Type C Phospholipases
Phorbol Esters
Tetradecanoylphorbol Acetate
Neurotransmitter Agents
Cations
Fluorescence
Binding Sites
Ions
Cell Line

ASJC Scopus subject areas

  • Pharmacology

Cite this

Mode of action of α-latrotoxin : Role of divalent cations in Ca2+-dependent and Ca2+-independent effects mediated by the toxin. / Rosenthal, Luann; Zacchetti, Daniele; Madeddu, Luisa; Meldolesi, Jacopo.

In: Molecular Pharmacology, Vol. 38, No. 6, 12.1990, p. 917-923.

Research output: Contribution to journalArticle

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abstract = "The potent neurotoxin α-latrotoxin (αLTx), from black widow spider venom, induces neurotransmitter release in both Ca2+-containing and Ca2+-free medium, following interaction with a specific cell surface receptor. Binding studies revealed two populations of αLTx binding sites in bovine synaptosomal membranes, showing the same high affinity (Kd, 0.3 × 10-10 M) for αLTx, with approximately 50{\%} of the sites being Ca2+ sensitive and the rest being Ca2+ insensitive. In contrast, in PC12 cells αLTx binding was completely unaffected by the removal of extracellular Ca2+ (Kd, 5 × 10-10 M). The use of La3+ as an inhibitor of αLTx action, previously shown in synaptosomes, was extended to PC12 cells. In this system, La3+ (100 μM) was shown to inhibit Ca2+ influx, both Ca2+-dependent and -independent dopamine release, and polyphosphoinositide (PPI) hydrolysis induced by αLTx. At the same time, La3+ did not block αLTx binding or dopamine release evoked by either the ionophore ionomycin (0.5 μM) or the phorbol ester tetradecanoylphorbol acetate (100 NM). La3+ also blocked the influx of Mn2+ ions through the αLTx-induced cation channel, as measured by quenching of fura-2 fluorescence. In this PC12 cell line, PPI hydrolysis could also be induced by ionomycin, but only when it was present at concentrations that caused an elevation of free intracellular Ca2+ ([Ca2+]i) that was not transient but was as persistent as that evoked by αLTx. Our conclusions with regard to the mode of action of αLTx are as follows, (i) All the effects of αLTx in PC12 cells (dopamine release, PPI hydrolysis, and Ca2+ influx) can be mediated via a single, Ca2+-insensitive αLTx receptor, (ii) αLTx-induced PPI hydrolysis is most likely due to the activation of a Ca2+-sensitive phospholipase C following the persistent rise in [Ca2+]i elicited by the toxin in Ca2+-containing medium, and not via direct coupling of the αLTx receptor to the enzyme. (iii) Toxin-evoked Ca2+-independent dopamine release can be blocked by La3+ at the extracellular level, most likely by prevention of the entry of divalent cations.",
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