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首页> 外文期刊>Biochemistry >Structure of the agonist-binding sites of the Torpedo nicotinic acetylcholine receptor: affinity-labeling and mutational analyses identify gamma Tyr-111/delta Arg-113 as antagonist affinity determinants.
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Structure of the agonist-binding sites of the Torpedo nicotinic acetylcholine receptor: affinity-labeling and mutational analyses identify gamma Tyr-111/delta Arg-113 as antagonist affinity determinants.

机译：鱼雷烟碱型乙酰胆碱受体激动剂结合位点的结构：亲和标记和突变分析确定γTyr-111 /δArg-113为拮抗剂亲和力决定因素。

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摘要

Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the gamma-subunit which, along with the analogous residue in delta-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and alpha-conotoxin (alpha Ctx) MI. nAChR gamma-subunit, isolated from nAChR-rich membranes photolabeled with [3H]dTC, was digested with Staphylococcus aureus V8 protease, and a 3H-labeled fragment was purified by reversed-phase high-performance liquid chromatography. Amino-terminal sequence analysis of this fragment identified 3H incorporation in gamma Tyr-111 and gamma Tyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at gamma Trp-55, the primary site of [3H]dTC photoincorporation within gamma-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940-32950]. The Torpedo nAChR delta-subunit residue corresponding to gamma Tyr-111 (delta Arg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the alpha-delta agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]alpha-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and alpha Ctx MI with nAChRs containing gamma Y111R or delta R113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the gamma Y111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of alpha Ctx MI, with delta R113Y enhancing and gamma Y111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of gamma- (or delta-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.
机译：用[3H] d-微管尿素（dTC）对鱼雷烟碱型乙酰胆碱受体（nAChR）进行光亲和性标记，鉴定出了γ-亚基内的一个残基，连同δ-亚基中的类似残基，赋予了二者之间结合亲和力的选择性dTC和α-芋螺毒素（αCtx）MI的激动剂位点。从金黄色葡萄球菌V8蛋白酶消化从富含[3H] dTC的nAChR膜上分离的nAChRγ亚基，并用反相高效液相色谱法纯化3H标记的片段。此片段的氨基末端序列分析确定3H在γTyr-111和γTyr-117中的掺入率约为[3H] dTC光掺入在[3H]的主要位点γTrp-55处的效率的5％和1％。 γ-亚基内的dTC光掺入[Chiara，DC，and Cohen，JB（1997）J. Biol。化学272，32940-32950]。对应于γTyr-111（δArg-113）的鱼雷nAChRδ亚基残基含有一个正电荷，可以赋予某些竞争性拮抗剂在α-δ激动剂位点上看到的较低的结合亲和力。为了检验该假设，我们通过电压钳分析和/或通过[125I]α-真菌毒素竞争结合试验检测了乙酰胆碱（ACh），dTC和αCtx MI与含有表达的YYRα或δR113Y突变亚基的nAChRs的相互作用。在非洲爪蟾卵母细胞中。虽然这些突变既不影响ACh平衡结合亲和力，也不影响通道激活的浓度依赖性，但γY111R突变降低了10倍的dTC亲和力和抑制能力。另外，每个突变赋予αCtx MI平衡平衡1000倍的变化，而ΔR113Y增强而γY111R减弱亲和力。将这些结果与小鼠nAChR的先前结果进行比较后发现，尽管γ-（或δ-）亚基一级结构的相同区域有助于激动剂结合位点，但充当拮抗剂亲和力决定簇的特定氨基酸是物种依赖性的。

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