d-Tubocurarine binding sites are located at alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor.
d-Tubocurarine binding sites are located at alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor.
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DOI：
10.1073/pnas.87.7.2785
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发表时间：
1990-04
影响因子：
11.1
通讯作者：
S. Pedersen;Jonathan B. Cohen
中科院分区：
文献类型：
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作者：
S. Pedersen;Jonathan B. Cohen
使用竞争性烟碱拮抗剂 d-[3H]筒箭毒碱作为来自 Torpedo 的烟碱乙酰胆碱受体 (AcChoR) 上乙酰胆碱结合位点的光亲和标记。用 d-[3H]筒箭毒碱平衡的富含 AcChoR 的膜用 254 nm 紫外光照射，导致共价掺入 α、γ 和 δ 亚基，这些亚基可以被 α-银环蛇毒素或氨基甲酰胆碱阻断。半最大特异性掺入 γ 和 δ 亚基所需的 d-[3H]筒箭毒碱浓度分别为 40 nM 和 0.9 µM，与高亲和力和低亲和力结合位点的解离常数一致（Kd = 35 nM 和 1.2 µM）。掺入 α 亚基的浓度依赖性是双相的，并且与高亲和力和低亲和力 d-筒箭毒碱结合位点的标记一致。每个 AcChoR 亚基的特异性光标记均受到氨基甲酰胆碱的抑制，并具有适当的剂量依赖性。这些结果表明，除了α亚基之外，γ和δ亚基也直接贡献于乙酰胆碱结合位点，并且每个结合位点都位于亚基的界面处。由于 AcChoR 亚基是同源的，并且围绕中心轴假对称排列，因此光标记结果与 alpha beta alpha gamma delta 的 AcChoR 玫瑰花结中的亚基排列不一致，表明 γ 或 delta 亚基位于 α 亚基之间。
The competitive nicotinic antagonist d-[3H]tubocurarine was used as a photoaffinity label for the acetylcholine binding sites on the nicotinic acetylcholine receptor (AcChoR) from Torpedo. Irradiation with 254-nm UV light of AcChoR-rich membranes equilibrated with d-[3H]tubocurarine resulted in covalent incorporation into the alpha, gamma, and delta subunits that could be blocked by alpha-bungarotoxin or by carbamoylcholine. The concentrations of d-[3H]tubocurarine required for half-maximal specific incorporation into the gamma and delta subunits were 40 nM and 0.9 microM, respectively, consistent with the dissociation constants for the high- and low-affinity binding sites (Kd = 35 nM and 1.2 microM). The concentration dependence of incorporation into alpha subunit was biphasic and consistent with labeling of both the high- and low-affinity d-tubocurarine binding sites. The specific photolabeling of each AcChoR subunit was inhibited by carbamoylcholine with appropriate dose dependence. These results establish that, in addition to the alpha subunits, the gamma and delta subunits also contribute directly to the acetylcholine binding sites and that each binding site is at an interface of subunits. Because the AcChoR subunits are homologous and are arranged pseudosymmetrically about a central axis, the photolabeling results are inconsistent with an arrangement of subunits in the AcChoR rosette of alpha beta alpha gamma delta and indicate that either the gamma or delta subunit resides between the alpha subunits.