The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: implications for the alpha-helical organization of regions MII and for the structure of the ion channel.
The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: implications for the alpha-helical organization of regions MII and for the structure of the ion channel.
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非竞争性阻断剂 [3H] 氯丙嗪标记乙酰胆碱受体 γ 亚基的三个氨基酸：对 MII 区域的 α 螺旋组织和离子通道结构的影响。
DOI：
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发表时间：
1990
影响因子：
11.1
通讯作者：
J. Changeux
中科院分区：
文献类型：
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作者：
F. Revah;J. Galzi;J. Giraudat;P. Haumont;F. Lederer;J. Changeux
用非竞争性通道阻断剂[3 H]氯丙嗪对条纹电鳐烟碱乙酰胆碱受体进行标记研究，初步确定了α、β和δ亚基上可与离子通道壁结合的氨基酸。我们在这里报告的γ亚基，这带来了额外的信息的通道的结构上获得的结果。在平衡条件下，在激动剂的存在下，并与或不与苯环己哌啶（一种特异性配体的非竞争性阻断剂的高亲和力位点）的膜结合受体的光标记后，纯化的标记的γ亚基用胰蛋白酶消化，并将所得的片段通过HPLC分馏。含有不同量的高度疏水片段的肽混合物的序列分析表明，三个氨基酸被标记的[3 H]氯丙嗪在苯环己哌啶敏感的方式：Thr-253，Ser-257，和Leu-260。这些残基都属于γ亚基的疏水性和推定的跨膜区MII。它们沿序列的沿着分布与该片段的α-螺旋组织一致。[3 H]氯丙嗪标记的氨基酸在其他配体门控离子通道的已知序列中的同源位置是保守的，因此可能在离子转运机制中发挥关键作用。
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.