Abstract
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 ofTorpedo AChRs expressed in Xenopusoocytes. Between 1–30 μM promegestone produced a concentration-dependent enhancement of the equilibrium binding affinity of [3H]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 [3H]phencyclidine (IC50 = 9 μM) than of [3H]histrionicotoxin (IC50 ∼ 100 μM). To identify AChR domains in contact with the steroid, AChR-rich membranes equilibrated with [3H]promegestone were irradiated at 312 nm, and 3H-labeled amino acids were identified by amino-terminal sequencing of fragments isolated from subunit proteolytic digests. Within AChR α-subunit, 70% of3H 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 [3H]promegestone incorporation was detected in any of the M2 segments. The amino acids in the M4 segments labeled by [3H]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.
Footnotes
- Received August 24, 1998.
- Accepted October 30, 1998.
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Send reprint requests to: Dr. Jonathan B. Cohen. Department of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston MA 02115. E-mail: jonathan-cohen{at}hms.harvard.edu
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↵1 Current address: Department of Pharmacology, Texas Tech University of Health Sciences Center, Lubbock, TX 79430.
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↵2 Current address: Millenium Pharmaceuticals, Cambridge, MA 02139.
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↵3 Current address: Protein Chemistry Laboratory, Department of Chemistry, Texas A&M University, College Station TX 77842.
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This research was supported in part by U.S. Public Health Service Grant GM 15904 and by an award in Structural Neurobiology from the Keck Foundation.
- The American Society for Pharmacology and Experimental Therapeutics
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