Polypeptide composition of the α-latrotoxin receptor: High affinity binding protein consists of a family of related high molecular weight polypeptides complexed to a low molecular weight protein

α-Latrotoxin is a vertebrate neurotoxin from black widow spider venom that causes massive neurotransmitter release. In order to gain insight into the mechanism of action of α-latrotoxin, we have studied α-latrotoxin-binding proteins from bovine and rat brain. Proteins purified by affinity chromatography on immobilized α-latrotoxin were investigated. Two sets of proteins were isolated: 1) three polypeptides of M_r 79,000, 65,000, and 43,000 that were eluted from immobilized a-latrotoxin by increasing KCl concentrations in the presence of Ca²⁺, and 2) a family of related proteins ranging in molecular weight from 160,000 to 220,000 and a low molecular weight component of M_r 29,000 that were eluted from immobilized α-latrotoxin only after removal of Ca²⁺. Amino acid sequences of these proteins demonstrated that all of these proteins represent novel proteins except for the M_r 65,000 polypeptide, which is identical with synaptotagmin (Petrenko, A. G., Perin, M. S., Davletov, B. A., Ushkaryov, Y. A., Geppert, M., and Südhof, T. C. (1991) Nature 353, 65-68). Surprisingly, the M_r 79,000 and 43,000 proteins were also found in tissues insensitive to α-latrotoxin action. Since these proteins do not bind ¹²⁵I-α-latrotoxin with high affinity, their purification probably is not physiologically significant. On the other hand, the fractions containing the M_r 160,000-220,000 and 29,000 polypeptides bound α-latrotoxin with high affinity. Sucrose gradient centrifugations and anion exchange chromatography suggested that most of the M_r 160,000-220,000 proteins were complexed with the M_r 29,000 protein. α-Latrotoxin binding correlated with the presence of the M_r 160,000-220,000 proteins and M_r 29,000 polypeptide, and α-latrotoxin formed stable complexes with the M_r 160,000-220,000 proteins. Accordingly, the α-latrotoxin receptor consists of a high molecular weight protein (M_r 160,000-220,000) that is complexed with one or several copies of an M_r 29,000 polypeptide. In addition, the receptor is found in a less tight association with synaptotagmin but not with other polypeptides.