Interactions of Perhydrohistrionicotoxin with Acetylcholine Receptors from Torpedo Californica electroplax

Electrophysiological evidence has shown that the histrionicotoxins reversibly block neuromuscular transmission, acting only in the presence of agonists (Albuquerque, E. X., Barnard, E. A., Chiu, T. H., Lapa, A. J., Dolly, J. 0., Jansson, S.-E., Daly, J., and Witkop, B. (1973) Proc. Natl. Acad. Sci. USA 70, 949-953). The use of tritiated perhydrohistrionicotoxin (H12-HTX) and the fluorescent probe ethidium bromide allowed biochemical characterization of the H12-HTX binding site in postsynaptic membranes and after detergent extraction. 3HH12-HTX bound with high affinity (KD ~ 0.5 μM) to acetyl- choline receptor (AcChR) enriched membranes isolated from Torpedo californica electroplax. The ratio of a-bungarotoxin sites to H12-HTX sites was 4:1. This stoichiometry implied that one H12-HTX bound per AcChR dimer. H12-HTX was not competitive with α-bungarotoxin or agonists, but the agonist recognition site and the H12-HTX site were conformationally linked. The KD for 3HH12-HTX was decreased twofold in the presence of carbamylcholine in concentrations sufficient to convert the receptor to the form having high affinity for agonists. Decahydrohistrionicotoxin increased the binding of 3H acetylcholine. H12-HTX had no effect on 3H carbamylcholine binding. In this system the twofold higher affinity of H12-HTX for the membrane in the high affinity state was not sufficient to strongly affect the process of agonist induced conversion from the low affinity state to the high affinity state for agonists (in vitro "desensitization"). 3 μM H12-HTX did not directly desensitize nor did it accelerate the rate of agonist induced desensitization or decelerate the rate of recovery from agonist induced desensitization. 30 μM H12-HTX did not directly desensitize nor did it accelerate the rate of agonist induced desensitization or decelerate the rate of recovery from agonist induced desensitization. 30 μM H12-HTX increased the rate of in vitro desensitization only slightly. Local anesthetics displaced 3HH12-HTX bound to membranes in an apparently competitive fashion. The presence of carbamylcholine sufficient to convert the receptor to the high affinity state for agonists modulated the apparent KIs for some local anesthetics. The differences in their apparent affinities for the low and high affinity states of the receptor roughly paralleled the effects of these local anesthetics on the rate of carbamylcholine induced "desensitization" (Blanchard, S. G., Elliott, J., and Raftery, M. A. (1979) Biochemistry, in press). For example dibucaine, which greatly accelerated the rate of conversion to the high affinity state, bound 17-fold more tightly to the high affinity state, and tetracaine, which slowed the rate of conversion, bound 20-fold more tightly to the low affinity state. This phenomenon may not have been due to a specific binding, since the detergent Triton X-100 also blocked 3HH12-HTX binding and accelerated in vitro "desensitization" at micromolar concentrations. Specific ethidium bromide fluorescence was quenched by H12-HTX with an apparent KI very similar to the KD determined by direct binding studies. This quenching was probably due to a change in the environment of the bound ethidium rather than an actual displacement. 3HH12-HTX binding could be extracted from AcChR enriched membranes by the detergent sodium cholate. Binding activity was sensitive to detergent concentration and was only recovered at low cholate concentrations where some aggregation occurred. The ratio of α-bungarotoxin sites to H12-HTX sites remained 4:1, though the KD (1.6 μM) was higher than with membranes. The cholate extracts were fractionated by several techniques, yielding an enrichment of AcChR. Treatment of cholate extracts with an α-bungarotoxin-Sepharose resin removed H12-HTX and α-bungarotoxin binding activities in parallel. Treatment of AcChR enriched membranes with base selectively removed most non-AcChR polypeptides without affecting 3HH12-HTX binding. This provided positive identification of the H12-HTX binding component with the AcChR, eliminating the possibility that the binding site was located on the 43,000 Mr polypeptide found in native membranes. The local anesthetics dibucaine and tetracaine displaced 3HH12-HTX bound to base treated membranes with unaltered KIs Carbamylcholine modulated the affinity of the membranes for H12-HTX and the local anesthetics. 3HH12-HTX binding could be reconstituted from cholate extracts of treated membranes. Thus the high affinity H12-HTX site appears to be located on one or more of the AcChR polypeptides, and reconstitution of that binding site from detergent extracts does not require the presence of the 43,000 Mr polypeptide. 3HH12-HTX binding was largely unaffected by reduction and alkylation of AcChR enriched membranes, a treatment which reduced α-bungarotoxin binding activity. It was likewise unaffected by treatment of the membranes with the carboxyl-modifying reagent trimethyl oxonium tetrafluoroborate. Divalent cations (Ca2+, Mg2+) increased the affinity of membranes for 3HH12-HTX about twofold. 3HH12-HTX binding was little affected by changes in NaCl concentration of the buffer.