EVIDENCE THAT A NEREISTOXIN METABOLITE, AND NOT NEREISTOXIN ITSELF, REDUCES NEURONAL NICOTINIC RECEPTORS - STUDIES IN THE WHOLE CHICK CILIARY GANGLION, ON ISOLATED NEURONS AND IMMUNOPRECIPITATED RECEPTORS

Nereistoxin (100 mu M, 2-10 min) blocks nicotinic receptors in the int act chick ciliary ganglion. This effect mimics blockade by the reducin g agent dithiothreitol (2 mM, 20 min), which is not reversed until oxi dation with dithiobisnitrobenzoic acid (1 mM, 5 min). After treating i ntact ganglia with either nereistoxin or dithiothreitol, the affinity alkylating agent bromoacetylcholine causes irreversible blockade that cannot be reversed by dithiobisnitrobenzoic acid. These data suggest t hat nereistoxin, or a metabolite, acts to reduce nicotinic receptors, although nereistoxin differs from dithiothreitol in that agonists only partially protect against nereistoxin reduction. In studies on chick retina, we previously proposed that a metabolite of nereistoxin (such as dihydronereistoxin) is the actual reducing agent for neuronal nicot inic receptors. Current findings in chick ciliary ganglion supporting this hypothesis include: 1) changing pH alters the minimal nereistoxin concentration needed for blockade in intact ganglia, but has little e ffect on the minimal concentration needed for dithiothreitol, 2) appli cation of a quaternary analog of nereistoxin has little effect on inta ct ganglion, but a quaternary analog of dihydronereistoxin blocks nico tinic receptors by reduction, 3) nereistoxin weakly oxidizes rather th an reduces immunoprecipitated receptors from chick brain and 4) in who le-cell patch-clamp studies, nereistoxin clearly does not reduce recep tors on chick ciliary neurons, although dihydronereistoxin mimics rece ptor blockade by dithiothreitol, and requires oxidation by dithiobisni trobenzoic acid for reactivation. Together, these data suggest that ne reistoxin is not a direct reducing agent for neuronal nicotinic recept ors.