Identification of Amino Acids of the Torpedo Nicotinic Acetylcholine Receptor Contributing to the Binding Site for the Noncompetitive Antagonist [3H]Tetracaine
Abstract
[3H]Tetracaine is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds with high affinity in the absence of cholinergic agonist (Keq = 0.5 μM) and weakly (Keq = 30 μM) in the presence of agonist (i.e., to nAChR in the desensitized state). In the absence of agonist, irradiation at 302 nm of nAChR-rich membranes equilibrated with [3H]tetracaine results in specific photoincorporation of [3H]tetracaine into each nAChR subunit. In this report, we identify the amino acids of each nAChR subunit specifically photolabeled by [3H]tetracaine that contribute to the high-affinity binding site. Subunits isolated from nAChR-rich membranes photolabeled with [3H]tetracaine were subjected to enzymatic digestion, and peptides containing3H were purified by SDS-polyacrylamide gel electrophoresis followed by reversed phase HPLC. N-terminal sequence analysis of the isolated peptides demonstrated that [3H]tetracaine specifically labeled two sets of homologous hydrophobic residues (αLeu251, βLeu257, γLeu260, and δLeu265; αVal255 and δVal269) as well as αIle247 and δAla268 within the M2 hydrophobic segments of each subunit. The labeling of these residues establishes that the high-affinity [3H]tetracaine-binding site is located within the lumen of the closed ion channel and provides a definition of the surface of the M2 helices facing the channel lumen.
Footnotes
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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 Present address: Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110.
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This work was supported in part by United States Public Health Service Grant NS19522 and by an award in structural neurobiology from the Keck Foundation.
- Abbreviations:
- nAChR
- nicotinic acetylcholine receptor
- HTX
- dl-perhydrohistrionicotoxin
- PAGE
- polyacrylamide gel electrophoresis
- NCA
- noncompetitive antagonist
- EKC
- endoproteinase Lys-C
- 1-AP
- 1-azidopyrene
- V8 protease
- Staphylococcus aureus glutamyl endopeptidase
- TFA
- trifluoroacetic acid
- OPA
- o-phthalaldehyde
- PTH
- phenylthiohydantoin
- [125I]TID
- 3-(trifluoromethyl)-3-(M-[125I]iodophenyl)diazirine
- TPS
- Torpedo physiological saline
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- Received March 23, 1999.
- Accepted May 18, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
