SYNAPTOSOMAL BINDING OF I-125 LABELED DABOIATOXIN, A NEW PLA(2) NEUROTOXIN FROM THE VENOM OF DABOIA-RUSSELLI-SIAMENSIS

Daboiatoxin (DbTx), the PLA, neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocor tical synaptosomes and synaptic membrane fragments. Two families of bi nding sites were detected by equilibrium binding analysis in the prese nce and absence of Ca2+. Scatchard analysis of biphasic plateaus revea led K-dl 5 nM and B-maxl, 6 pmoles/mg protein, and K-d2 80 nM and B-ma x2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of I-125-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, CO2+ and Sr2+ Native DbTx was the onl y strong competitor to I-125-DbTx synaptosomal binding (IC50 12.5 nM, K-I 5.5 nM). Two other crotalid PLA(2) neurotoxins, crotoxin CB and mo jave toxin basic subunit, and non-toxic C. atrox PLA, enzyme, were rel atively weaker inhibitors, while two viperid PLA(2) neurotoxins, ammod ytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a po or inhibitor even at mu M concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA(2) enzymes from N. naja venom , E. schistosa venom, bee venom and porcine pancreas. All other pharma cologically active ligands examined (epinephrine, norepinephrine, hist amine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine , hexamethonium and cl-bungarotoxin) also failed to interfere with I-1 25-DbTx binding. As those competitors that showed partial inhibition w ere effective only at mu M concentration range compared to the K-d (5 nM) of I-125-DbTx synaptosomal binding, DbTx could well recognize a di fferent neuronal binding site. Rabbit anti-DbTx polyclonal antisera co mpletely blocked the specific binding. When a range of Ca2+ and K+ cha nnels modulators were examined, Ca2+ channel blockers (omega-conotoxin s GVIA and MVIIC, taicatoxin, calciseptine and nitrendipene) did not a ffect the binding even at high concentrations, while charybdotoxin was the only Kf channel effector that could partially displace I-125-DbTX synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethy lammonium), suggesting that neither K+ nor Ca2+ channels are associate d with DbTx binding sites.