Site-selective agonist binding to the nicotinic acetylcholine receptor from Torpedo californica.
Site-selective agonist binding to the nicotinic acetylcholine receptor from Torpedo californica.
复制标题
与加州鱼雷烟碱乙酰胆碱受体结合的位点选择性激动剂。
DOI：
10.1021/bi027405b
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Pedersen,SteenE
中科院分区：
文献类型：
--
作者：
Song,Xing-Zhi;Andreeva,IraidaE;Pedersen,SteenE
用荧光能量转移法测定了丹磺酰-C6-胆碱与加利福尼亚电鳐烟碱型乙酰胆碱受体（AChR）结合的αγ和αδ结合位点的结合特征。平衡结合测量表明，αγ位点的荧光低于αδ位点;发射差异是由于结合荧光团的固有荧光的差异，而不是两个位点能量转移的差异。停流荧光动力学表明，在脱敏构象中，丹酰-C6-胆碱从AChR的αγ位点解离的速度比从αδ位点快5 - 10倍。在非竞争性拮抗剂的存在下，以及在广泛的离子强度范围内，解离速率对于不同的蛋白质制剂是稳健的。平衡荧光结合测量表明，当AChR脱敏时，丹磺酰-C6-胆碱与αδ位点（K= 3 nM）的结合亲和力高于与αγ位点（K= 9 nM）的结合亲和力。对乙酰胆碱本身也观察到类似的亲和力差异。不同的解离速率允许在结合的时间过程中在每个位点测量脱敏的程度。这种测量每个激动剂位点处的脱敏状态群体的顺序混合方法可用于详细研究AChR激活和随后脱敏的机制。
Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) fromTorpedocalifornicawere used to determine binding characteristics of the αγ and αδ binding sites. Equilibrium binding measurements show that the αγ site has a lower fluorescence than the αδ site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5−10-fold faster from the αγ site than from the αδ site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the αδ site (K= 3 nM) than to the αγ site (K= 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.