Agonist-mediated changes of the acetylcholine receptor in its membrane environment.
Agonist-mediated changes of the acetylcholine receptor in its membrane environment.
复制标题
激动剂介导的膜环境中乙酰胆碱受体的变化。
DOI：
10.1016/0022-2836(78)90144-4
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发表时间：
1978
影响因子：
5.6
通讯作者：
F. J. Barrantes
中科院分区：
文献类型：
--
作者：
F. J. Barrantes
用动力学和稳态技术相结合的方法，在体外研究了胆碱能激动剂Suberyldicholine与膜结合型乙酰胆碱受体的相互作用。将富含受体的膜暴露于激动剂，可导致受体特性发生一系列依赖于时间和浓度的、饱和的和可逆的变化。在此过程中，受体对该配体的表观亲和力增加了约50倍，通过抑制α-毒素结合动力学间接测量。通过跟踪(内在)蛋白质荧光猝灭的动力学，可以更直接地研究Suberyldicholine对受体的修饰。停流荧光法可观察到毫秒级的起始步骤，其特征与激动剂与低亲和力受体(KAPP~1μM)的结合过程一致，开启和关闭速率分别为9.8×106M−1S−1和10S−1。这一步骤之后是在秒的时间范围内发生的更大的荧光变化(向前速率1.2s到1.5s−1；反向速率0.06到0.1s−1)。最终，该复合体的“高亲和力”状态，其荧光性质不同于受体的游离(初始)和连接形式，暂定归因于活体研究假设的“不敏感”构象(Katz&Tesleff，1957)。琥珀胆碱诱导的该复合体的形成速率高于乙酰胆碱和氨基甲酰胆碱。另一方面，三种激动剂的总荧光猝灭程度大致相同，这表明最终的构象不依赖于配体的性质。特定激动剂诱导的猝灭可能是通过“静态”机制进行的，因为激发态的寿命不因配体的存在而改变。本征荧光团对丙烯酰胺或氮氧化物自旋标记物的可及性，如分别从体相和脂相的荧光猝灭检测到的，也受到琥珀胆碱的影响。荧光和毒素抑制研究都根据活体观察得出的反应机理进行了分析，涉及乙酰胆碱受体在其膜环境中的相互转化构象状态，对激动剂的亲和力不同，以及它们的本征荧光性质。
The interaction of a cholinergic agonist, suberyldicholine, with the membrane-bound acetylcholine receptor fromTorpedo marmoratawas studiedin vitroby a combination of kinetic and steady-state techniques. Exposure of the receptor-rich membranes to the agonist resulted in a time and concentration dependent, saturable and reversible series of changes in receptor properties. Theapparentaffinity of the receptor for the ligand increased about 50-fold in the course of this exposure, as measured indirectly by the inhibition of α-toxin association kinetics.The modifications induced by suberyldicholine in the receptor could be studied in a more direct manner by following the kinetics of the (intrinsic) protein fluorescence quenching. An initial step occurring in the time scale of milliseconds could be observed with stopped-flow fluorimetry, its characteristics being consistent with a binding process of the agonist to a “low affinity” form of the receptor (Kapp~ 1μM) with on and off-rates of 9.8 × 106m−1s−1and 10s−1, respectively. This step was followed by a larger fluorescence change taking place in a time range of seconds (forward rate 1.2 to 1.5s−1; backward rate 0.06 to 0.1s−1). The final, “high-affinity” state of the complex, which differed in its fluorescence properties from both the free (initial) and liganded-forms of the receptor, is tentatively attributed to the “desensitised” conformation postulated fromin vivostudies (Katz & Thesleff, 1957). The rate of formation of this complex elicited by suberyldicholine was higher than those of acetyl- or carbamoylcholine. The extent of the total fluorescence quenching was, on the other hand, approximately the same for the three agonists, suggesting that the final conformation does not depend on the nature of the ligand.The specific agonist-induced quenching probably operatesviaa “static” mechanism, since the lifetime of the excited state did not change in the presence of the ligand. The accessibility of the intrinsic fluorophores to acrylamide or to nitroxide spin labels, as sensed by quenching of fluorescence from the bulk and lipid phases, respectively, was also affected by suberyldicholine.Both fluorescence and toxin-inhibition studies have been analysed in terms of a reaction mechanism derived fromin vivoobservations, involving interconvertible conformational states of the acetylcholine receptor in its membrane environment, differing in affinity for the agonist, and in their intrinsic fluorescence properties.