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.
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
T
T
O'Hori
C Y
CY
Wang
H
H
Tokumaru
L C
LC
Chen
K
K
Hatanaka
N
N
Hirashima
Y
Y
Kirino
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004557:Electric Organ / Q000187:drug effects* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D009479:Neuropeptides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism; D014101:Torpedo; D020960:omega-Conotoxins
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D009479:Neuropeptides; D010455:Peptides; D020960:omega-Conotoxins; C053423:Conus magus toxin; D000109:Acetylcholine; D002118:Calcium