BINDING OF PERHYDROHISTRIONICOTOXIN TO INTACT AND DETERGENT-SOLUBILIZED MEMBRANES ENRICHED IN NICOTINIC ACETYLCHOLINE-RECEPTOR

A tritiated derivative of perhydrohistrionicotoxin was prepared and characterized and used in direct binding studies to membrane fragments from Torpedo californica electroplax. Perhydrohistrionicotoxin bound to a site distinct from the α-bungarotoxin and agonist binding sites in a ratio of one perhydrohistrionicotoxin bound per four α-bungarotoxin sites. In the presence of 10 μM carbamylcholine, the dissociation constant for perhydrohistrionicotoxin was lowered from 0.57 to 0.27 μM in Torpedo Ringer's solution and from 0.9 to 0.5,μM in the absence of divalent cations. Perhydrohistrionicotoxin binding activity was solubilized from the membranes by the anionic detergent sodium cholate but was sensitive to detergent concentration. Binding activity was lost reversibly at cholate concentrations above 0.6%, and the addition of Triton X-100 resulted in loss of binding activity at extremely low concentrations. The perhydrohistrionicotoxin and agonist binding sites, which are conformationally linked in the membranes, are also closely associated in cholate extracts. The ratio of perhydrohistrionicotoxin binding sites to a-bungarotoxin binding sites remained constant upon solubilization. Treatment of the extract with a resin made by coupling α -bungarotoxin to Sepharose 2B resulted in the removal of α -bungarotoxin and perhydrohistrionicotoxin binding activities. The fluorescent probe of receptor function, ethidium bromide, was used to measure perhydrohistrionicotoxin binding to membrane fragments and to cholate extracts and yielded I50 values for the quenching of specific fluorescence which were comparable to the dissociation constants obtained by direct binding. © 1979, American Chemical Society. All rights reserved.