Isolation and biochemical characterization of a Ca super(2+)-independent alpha -latrotoxin-binding protein

alpha -Latrotoxin, a black widow spider neurotoxin, can bind to high affinity receptors on the presynaptic plasma membrane and stimulate massive neurotransmitter release in the absence of Ca super(2+). Neurexins, previously isolated as alpha -latrotoxin receptors, require Ca super(2+) for their interaction with the toxin and, thus, may not participate in the Ca super(2+)-independent alpha -latrotoxin activity. We now report the isolation of a novel protein that binds alpha -latrotoxin with high affinity in the presence of various divalent cations (Ca super(2+), Mg super(2+), Ba super(2+), and Sr super(2+)) as well as in EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatography on immobilized alpha -latrotoxin and concentrated on a wheat germ agglutinin affinity column. The single polypeptide chain of latrophilin is N-glycosylated and has an apparent molecular weight of 120,000. Sucrose gradient centrifugations demonstrated that latrophilin and alpha -latrotoxin form a stable equimolar complex. In the presence of the toxin, anti- alpha -latrotoxin antibodies precipitated iodinated latrophilin, whose binding to immobilized toxin was characterized by a dissociation constant of 0.5-0.7 nM. This presumably membrane-bound protein is localized to and differentially distributed among neuronal tissues, with about four times more latrophilin expressed in the cerebral cortex than in the cerebellum; subcellular fractionation showed that the protein is highly enriched in synaptosomal plasma membranes. Our data suggest that latrophilin may represent the Ca super(2+)-independent receptor and/or molecular target for alpha -latrotoxin.