Identification of the sites of incorporation of [3H]ethidium diazide within the Torpedo nicotinic acetylcholine receptor ion channel.
Identification of the sites of incorporation of [3H]ethidium diazide within the Torpedo nicotinic acetylcholine receptor ion channel.
复制标题
鱼雷烟碱乙酰胆碱受体离子通道内[3H]二叠氮乙锭掺入位点的鉴定。
DOI：
10.1021/bi0011680
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Cohen,JB
中科院分区：
文献类型：
--
作者：
Pratt,MB;Pedersen,SE;Cohen,JB
用[~ 3 H]二叠氮乙锭的光活化衍生物，将烟碱乙酰胆碱受体（nAChR）的非竞争性拮抗剂乙锭的结合位点定位在脱敏状态的电鳐AChR上。在10 μM [3 H]二叠氮乙锭时，苯环己哌啶（PCP）的存在抑制了α-、β-和δ-亚基的掺入。在α亚基内，掺入定位于从αSer-173开始的20 kDa片段，包含前三个跨膜片段αM1、αM2和αM3。该片段的进一步消化产生了两个具有PCP可识别掺入的片段，一个含有αM1，一个含有αM2和αM3。在αM2内，αLeu-251和αSer-252存在特异性掺入，这些残基先前已显示为离子通道内腔的衬里。用S. aureusV 8蛋白酶产生14-kDa和20-kDa的片段，这两个片段都开始于Ile-192，并含有PCP可识别的标记。进一步消化含有δM1和δM2的14-kDa片段，以生成仅含有δM2的3-kDa片段，并掺入PCP。消化含有δM1、δM2和δM3的20-kDa片段，产生两个掺入片段，一个含有δM1片段，另一个含有δM2和δM3。这些结果表明，在脱敏状态的nAChR，高亲和力的结合位点的乙锭的离子通道的内腔内，并结合的药物是在与氨基酸从M1和M2疏水段接触。
The binding sites of ethidium, a noncompetitive antagonist of the nicotinic acetylcholine receptor (nAChR), have been localized in theTorpedonAChR in the desensitized state by use of a photoactivatible derivative, [3H]ethidium diazide. At 10 μM [3H]ethidium diazide, incorporation into the α-, β-, and δ-subunits was inhibited by the presence of phencyclidine (PCP). Within the α-subunit, the incorporation was mapped to a 20-kDa fragment beginning at αSer-173 and containing the first three transmembrane segments, αM1, αM2, and αM3. Further digestion of this fragment generated two fragments with PCP-inhibitable incorporation, one containing αM1 and one containing both αM2 and αM3. Within αM2, specific incorporation was present in αLeu-251 and αSer-252, residues that have been previously shown to line the lumen of the ion channel. Digestion of the δ-subunit withS. aureusV8 protease generated a 14-kDa and a 20-kDa fragment, both of which began at Ile-192 and contained PCP-inhibitable labeling. The 14-kDa fragment, containing δM1 and δM2, was further digested to generate a 3-kDa fragment, containing δM2 alone, with PCP-inhibitable incorporation. Digestion of the 20-kDa fragment, which contained δM1, δM2, and δM3, generated two fragments with incorporation, one containing the δM1 segment and the other containing δM2 and δM3. These results establish that in the desensitized state of the nAChR, the high-affinity binding site of ethidium is within the lumen of the ion channel and that the bound drug is in contact with amino acids from both the M1 and M2 hydrophobic segments.