Binding of synaptotagmin to the α-latrotoxin receptor implicates both in synaptic vesicle exocytosis

Abstract

A vertebrate neurotoxin, alpha-latrotoxin, from black widow spider venom causes synaptic vesicle exocytosis and neurotransmitter release from presynaptic nerve terminals. Although the mechanism of action of alpha-latrotoxin is not known, it does require binding of alpha-latrotoxin to a high-affinity receptor on the presynaptic plasma membrane. The alpha-latrotoxin receptor seems to be exclusively at the presynaptic plasmamembrane. Here we report that the alpha-latrotoxin receptor specifically binds to a synaptic vesicle protein, synaptotagmin, and modulates its phosphorylation. Synaptotagmin is a synaptic vesicle-specific membrane protein that binds negatively charged phospholipids and contains two copies of a putative Ca(2+)-binding domain from protein kinase C (the C2-domain), suggesting a regulatory role in synaptic vesicle fusion. Our findings suggest that a physiological role of the alpha-latrotoxin receptor may be the docking of synaptic vesicles at the active zone. The direct interaction of the alpha-latrotoxin receptor with a synaptic vesicle protein also suggests a mechanism of action for this toxin in causing neurotransmitter release.

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

“…Nerve terminals imaged by freeze fractu re exhibited large transmembrane particles at release sites (Pumplin et al, 1981;Heuser et al, 1974Heuser et al, , 1979Ceccarelli et al, 1979), which were believed to reflect the transmembrane regions of calcium channels. However, many other putative membrane proteins, including syntaxin (Bennett et al, 1992;O'Connor et al, 1993;Horikawa et al, 1993), neurexin (Petrenko et al, 1991;Petrenko, 1993), and calcium-activated potassium channels (Roberts et al, 1990;Robitaille et al, 1993), are also localized at or near the transmitter release site, and these may also generate particles in the freeze-fracture replica. Release face calcium channels can be identified with light microscopy, but the results obtained with AFM represent a 10-fold improvement in resolution, down to the molecular level.…”

Presynaptic plasticity: The regulation of Ca2+-dependent transmitter release

“…Interestingly, while the above constituents of fusion complexes appear to have homologues in conserved forms of (Ca 2 ~ -independent) secretion like in yeast, no homologues for synaptotagmin are known to date in such systems that do not display regulated exocytosis. Synaptotagmin (p65), contains two repeats of a sequence on the cytoplasmic domain which are homologous to the Ca 2÷-and phospholipid-binding domain of the protein kinase C family and phospholipase A 2 (Perin et al, 1990(Perin et al, , 1991Brose et al, 1992). Multimers of synaptotagmin form complexes with phospholipids and Ca 2+, when the Ca 2 + concentration rises into micromolar range (Brose et al, 1992;Davletov and Sfidhof, 1993).…”

“…The Ca -,+ dependence of this complex fits well with the suggested range of intracellular free Ca 2 ~ underneath the active zone membrane (see above) and Ca 2 ~-association shows similar co-operativity as Ca 2+-dependence of secretion (see Zucker, 1989). Furthermore, synaptotagmin was found to co-precipitate with syntaxin (Bennett et al, 1992a;Yoshida et al, 1992), og-conotoxin binding sites (N-type Ca 2 + channels; Leveque et al,1992;Yoshida et al, 1992) and the receptor for ~-latrotoxin (Petrenko et al, 1991), a toxin that produces massive synaptic vesicle exocytosis.…”

α‐Latrotoxin receptor

“…Synaptotagmin co-purifies with the solubilized a-latrotoxin receptor and this interaction is so specific that synaptotagmin can be purified in one step by affinity chromatography of the synaptic vesicles detergent extract on a column with immobilized a-latrotoxin receptor [41]. Synaptotagmin binds to cytoplasmic domains of different neurexins, which appear to be highly conserved.…”

“…This conclusion is supported by the following lines of evidence: 1. Electrophoretic analysis detects 200K, 160K and 29K polypeptides as the major protein components in the affinity-purified receptor preparations [36,37,40,41]. 2.…”