Halothane interactions with nicotinic acetylcholine receptor membranes. Steady-state and kinetic studies of intrinsic fluorescence quenching.
Halothane interactions with nicotinic acetylcholine receptor membranes. Steady-state and kinetic studies of intrinsic fluorescence quenching.
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氟烷与烟碱乙酰胆碱受体膜的相互作用。
DOI：
10.1097/00000542-199702000-00023
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发表时间：
1997
期刊：
影响因子：
8.8
通讯作者：
McClure,KB
中科院分区：
文献类型：
--
作者：
Raines,DE;McClure,KB
虽然已经提出麻醉剂通过结合蛋白质内部区域的非极性位点来改变蛋白质的构象状态，但麻醉剂穿透膜蛋白的速度和程度尚未被表征。作者报告使用稳态和停流光谱来表征氟烷与受体膜的相互作用。方法采用稳态和停流荧光光谱法，表征氟烷对富烟碱乙酰胆碱受体（nictinicacetylcholine receptor, nAcChoR）膜内固有荧光的猝灭作用和异氟烷诱导的nAcChoR脱敏率。结果在平衡状态下，氟烷只猝灭了54+/-1.4%的色氨酸荧光。乙醚未能减少氟烷对荧光的猝灭，这表明乙醚与氟烷结合的蛋白质位点不同。停止流动的荧光痕迹定义了两种猝灭的动力学成分：一种快速成分，发生在不到1ms的时间内，其次是较慢的双相荧光衰减。用十二烷基硫酸钠展开蛋白质，降低了氟烷的Stern-Volmer猝灭常数，消除了双相衰变，使荧光在1ms内可被氟烷猝灭。功能研究表明，麻醉诱导的nAcChoR脱敏发生在不到2 ms的时间内。结论：不可猝灭的荧光是由埋在蛋白质内部的色氨酸残基产生的，这些残基不受氟烷的影响。十二烷基硫酸钠展开膜蛋白，并允许先前埋藏的荧光蛋白残基被氟烷快速均匀地淬灭。氟烷在发挥其功能作用的相同浓度范围和时间尺度上淬灭nAcChoR膜的蛋白质成分，这一发现通常与蛋白质的作用位点一致。
BackgroundAlthough it has been suggested that anesthetics alter protein conformational states by binding to nonpolar sites within the interior regions of proteins, the rate and extent to which anesthetics penetrate membrane proteins has not been characterized. The authors report the use of steady-state and stopped-flow spectroscopy to characterize the interactions of halothane with receptor membranes.MethodsSteady-state and stopped-flow fluorescence spectroscopy was used to characterize halothane quenching of nicotinic acetylcholine receptor (nAcChoR)-rich membrane intrinsic fluorescence and the rate of isoflurane-induced nAcChoR desensitization.ResultsAt equilibrium, halothane quenched only 54+/-1.4% of all tryptophan fluorescence. Diethyl ether failed to reduce fluorescence quenching by halothane, suggesting that it does not bind to the same protein sites as halothane. Stopped-flow fluorescence traces defined two kinetic components of quenching: a fast component that occurred in less than 1 ms followed by a slower biphasic fluorescence decay. Protein unfolding with sodium dodecyl sulfate reduced halothane's Stern-Volmer quenching constant, eliminated the biphasic decay, and rendered fluorescence accessible to quenching by halothane within 1 ms. Functional studies indicate that anesthetic-induced desensitization of nAcChoR occurs in less than 2 ms.ConclusionsUnquenchable fluorescence arises from tryptophan residues that are buried within the protein and protected from halothane. Sodium dodecyl sulfate unfolds membrane proteins and allows previously buried fluorescence protein residues to be rapidly and homogeneously quenched by halothane. Halothane quenches protein components of nAcChoR membranes over the same concentration range and time scale that it exerts its functional effects, a finding that is generally consistent with a protein site of action.