Reconstitution of the purified alpha-latrotoxin receptor in liposomes and planar lipid membranes. Clues to the mechanism of toxin action.
The receptor of alpha-latrotoxin (the major toxin of the black widow spider venom), purified from bovine synaptosomal membranes, was reconstituted into small unilamellar liposomes. These (but not control) liposomes exhibited high-affinity, specific binding of [125I]alpha-latrotoxin. In the receptor-bearing liposomes alpha-latrotoxin induced depolarization and stimulated 45Ca efflux. These responses to alpha-latrotoxin, that were observed only in the presence of external divalent cations, resembled those previously demonstrated in mammalian brain synaptosomes. The alpha-latrotoxin-activated ion fluxes are therefore, at least in part, the result of the direct interaction of the toxin with its receptor. When control and receptor-bearing liposomes were pre-incubated with alpha-latrotoxin and then added to a solution bathing a planar lipid bilayer membrane, single channel cationic conductances were observed. In the presence of the receptor, the conductances induced by alpha-latrotoxin were markedly different from those observed without the receptor, but not identical to those observed without the receptor, but not identical to those recently characterized by patch clamping in the cells of a line (PC12) sensitive to alpha-latrotoxin. These results demonstrate that the reconstituted receptor is functional, and suggest that the cationic channel activated by the toxin-receptor interaction is modulated by additional component(s) in the membrane of synapses and cells.
D000818:Animals; D001180:Arthropod Venoms / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D010713:Phosphatidylcholines; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D018000:Receptors, Peptide; D013111:Spider Venoms / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
D001180:Arthropod Venoms; D002132:Calcium Radioisotopes; D008081:Liposomes; D010713:Phosphatidylcholines; D011950:Receptors, Cholinergic; D018000:Receptors, Peptide; D013111:Spider Venoms; C034800:alpha-latrotoxin receptor; D002784:Cholesterol