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pubmed:abstractText |
Labeling studies of Torpedo marmorata nicotinic acetylcholine receptor with the noncompetitive channel blocker [3H]chlorpromazine have led to the initial identification of amino acids plausibly participating to the walls of the ion channel on the alpha, beta, and delta subunits. We report here results obtained with the gamma subunit, which bring additional information on the structure of the channel. After photolabeling of the membrane-bound receptor under equilibrium conditions in the presence of agonist and with or without phencyclidine (a specific ligand for the high-affinity site for noncompetitive blockers), the purified labeled gamma subunit was digested with trypsin, and the resulting fragments were fractionated by HPLC. Sequence analysis of peptide mixtures containing various amounts of highly hydrophobic fragments showed that three amino acids are labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner: Thr-253, Ser-257, and Leu-260. These residues all belong to the hydrophobic and putative transmembrane region MII of the gamma subunit. Their distribution along the sequence is consistent with an alpha-helical organization of this segment. The [3H]chlorpromazine-labeled amino acids are conserved at homologous positions in the known sequences of other ligand-gated ion channels and may, thus, play a critical role in ion-transport mechanisms.
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