The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen.

Abstract

Immunoglobulin G fractions from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, immunoprecipitate '2SI-labeled a-conotoxin GVIA-labeled calcium channels solubilized from rat brain. A 58-kDa antigen was detected by probing Western blots of partially purified calcium channels with LEMS plasma and IgG and was shown to be the relevant antigen in w-conotoxin receptor immunoprecipitation. Monoclonal antibody 1D12, produced by immunizing mice with synaptic membranes, has properties similar to these autoimmune IgGs in both immunoprecipitation and Western blotting assays. 1D12 antigen was purified by immunoaffinity chromatography and shown to bind LEMS IgG. The antigen was identified by screening a rat brain cDNA library with 1D12 and was found to have strong homology to the synaptic vesicle membrane protein synaptotagmin. Our results indicate therefore that these antibodies immunoprecipitate w-conotoxin receptors by binding to synaptotagmin that is associated with calcium channels. We suggest that the interaction between synaptotagmin and the voltage-gated calcium channel plays a role in docking synaptic vesicles at the plasma membrane prior to rapid neurotransmitter release and that autoantibody binding to a synaptotagmin-calcium-channel complex may be involved in the etiology of LEMS.

Interaction of a synaptobrevin (VAMP)‐syntaxin complex with presynaptic calcium channels

“…The present data and previous reports suggest that synaptic vesicle docking is mediated by a syntaxin-SNAP25-VAMPsynaptotagmin complex [2,16,20,21], which can also include presynaptic calcium channels [5,6,22]. A recent report has indicated that syntaxin can bind directly to a cytoplasmic loop of the ~zlB calcium channel subunit [23].…”

“…Co-immunoprecipitation experiments and calcium channel purification have indicated that synaptotagmin and syntaxin can associate with N-type calcium channels [5,6]. Certain classes of calcium channel may therefore interact with synaptic vesicle docking complexes, locating calcium sensor proteins, such as synaptotagmin [7], within a microdomain of calcium entry.…”

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

“…The detection of a preferred modal interchannel spacing that is independent of channel density suggests that these channels are anchored to a membrane-associated latticework. This hypothetical scaffold presumably also anchors other components of the release face (e.g., Bennett et al, 1992;L~v~que et al, 1992;O'Connor et al, 1993;Yoshida et al, 1992) and may ensure, among other functions, the correct spacing between the calcium channels and the calcium binding site on the transmitter release mechanism. Such a structural link has been predicted from the finding that the quantity of calcium that enters during single calcium channel activity can be sufficient to gate individual release sites (Stanley, 1993).…”

“…Recent physiological findings on the presynaptic nerve terminal release face by combined patch-clamp and transmitter detection (Stanley, 1993) or the identification of protein components associated with secretory vesicle docking (Bennett et al, 1992;L~v~que et al, 1992O'Connor et al, 1993;Yoshida et al, 1992;Mastrogiacomo et al, 1994;Abe et al, 1993) suggest that calcium channels are an integral part of a multimolecular transmitter release mechanism. Although these channels have been localized to the nerve terminal release face by fluorescent stains (Robitaille et al, 1990;Cohen et al, 1991;Torri Tarelli et al, 1991), patch-clamp recording (Stanley, 1991), and detection of calcium entry (Llin~s et al, 1992;Smith et al, 1993), it has not proved possible to demonstrate their spatial distribution at the single-channel level.…”

α‐Latrotoxin receptor

“…cipitate with the w-conotoxin receptor [47,48], which is a presynaptic calcium channel protein, and with syntaxin [48], another presynaptic membrane protein implicated in membrane fusion [49]. Thus, the interactions involving the a-latrotoxin receptor, or any other neurexins, may be important for the docking of synaptic vesicles at the release sites with proper positioning in relation to calcium channels, which induce neurotransmitter release (Fig.…”