Identification of amino acids of the torpedo nicotinic acetylcholine receptor contributing to the binding site for the noncompetitive antagonist [(3)H]tetracaine.
[(3)H]Tetracaine is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds with high affinity in the absence of cholinergic agonist (K(eq) = 0.5 microM) and weakly (K(eq) = 30 microM) 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 [(3)H]tetracaine results in specific photoincorporation of [(3)H]tetracaine into each nAChR subunit. In this report, we identify the amino acids of each nAChR subunit specifically photolabeled by [(3)H]tetracaine that contribute to the high-affinity binding site. Subunits isolated from nAChR-rich membranes photolabeled with [(3)H]tetracaine were subjected to enzymatic digestion, and peptides containing (3)H were purified by SDS-polyacrylamide gel electrophoresis followed by reversed phase HPLC. N-terminal sequence analysis of the isolated peptides demonstrated that [(3)H]tetracaine specifically labeled two sets of homologous hydrophobic residues (alphaLeu(251), betaLeu(257), gammaLeu(260), and deltaLeu(265); alphaVal(255) and deltaVal(269)) as well as alphaIle(247) and deltaAla(268) within the M2 hydrophobic segments of each subunit. The labeling of these residues establishes that the high-affinity [(3)H]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.
Gallagher
M J
MJ
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA.
Cohen
J B
JB
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D018733:Nicotinic Antagonists / Q000378:metabolism*; D019920:Photoaffinity Labels; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D017421:Sequence Analysis; D013748:Tetracaine / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium
D000596:Amino Acids; D018733:Nicotinic Antagonists; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; D013748:Tetracaine; D014316:Tritium