Action and binding of omega-conotoxin on the putative calcium channel of synaptosomal plasma membrane from electric organ of Japanese electric ray, Narke japonica.

Actions of omega-conotoxin GVIA on synaptosomes isolated from a Japanese electric ray, Narke japonica, were investigated. omega-Conotoxin inhibited, in a dose-dependent manner, both increases in free calcium concentration in, and acetylcholine release from synaptosomes depolarized with a high concentration of potassium ions. The concentrations of omega-conotoxin required for half-maximal inhibition (IC50) of increase in intrasynaptosomal Ca and acetylcholine release were 8 and 7 microM, respectively. Assay using radioiodinated toxin derivative revealed a specific binding site with a dissociation constant (KD) of 2.8 microM and a density (Bmax) of 45 pmol/mg protein of synaptosome. Binding assay with synaptosomal plasma membrane showed a KD = 7 microM and a Bmax = 200 pmol/mg protein. Autoradiography with sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after covalent cross-linking of the toxin, using disuccinimidyl suberate, revealed the 170,000 mol. wt peptide to be an omega-conotoxin receptor. The present study has directly and clearly shown that omega-conotoxin inhibits acetylcholine release by blocking Ca influx into nerve terminals. The 170,000 mol. wt peptide identified as a receptor of the toxin exists in high density in the plasma membrane of the presynaptic nerve terminal and is likely to be a component of a voltage-dependent Ca channel responsible for the neurotransmitter release.