The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid-protein interface

17,21-Dimethyl-19-nor-pregn-4,9-diene-3,20-dione (promegestone) was used to characterize the mechanism of inhibition of nicotinic acetylcholine (ACh) receptors (AChR) by progestin steroids. Promegestone reversibly inhibited ACh-induced currents of Torpedo AChRs expressed in Xenopus oocytes. Between 1-30 mu M promegestone produced a concentration-dependent enhancement of the equilibrium binding affinity of [H-3]ACh to Torpedo AChR-rich membranes. For AChRs in the presence of agonist (desensitized state) promegestone was a more potent inhibitor of the binding of the noncompetitive antagonist [H-3]phencyclidine (IC50 = 9 mu M) than of [H-3]histrionicotoxin (IC50 similar to 100 mu M). To identify AChR domains in contact with the steroid, AChR-rich membranes equilibrated with [H-3]promegestone were irradiated at 312 nm, and H-3-labeled amino acids were identified by amino-terminal sequencing of fragments isolated from subunit proteolytic digests, Within AChR alpha-subunit, 70% of H-3 was covalently incorporated in a 10-kDa fragment beginning at Asn-339 and containing the M4 membrane spanning segment, and 30% was in a 20-kDa fragment beginning at Ser-173 and containing the M1-M3 segments. Fragments containing the M2 channel domains as well as the M4 segments were isolated from proteolytic digests of AChR subunits and subjected to amino-terminal sequence analysis. No evidence of [H-3]promegestone incorporation was detected in any of the M2 segments. The amino acids in the M4 segments labeled by [H-3]promegestone were among those previously shown to be in contact with the lipid bilayer (Blanton and Cohen, 1994). These results indicate that the steroid promegestone is an AChR noncompetitive antagonist that may alter AChR function by interactions at the lipid-protein interface.