Kinetics of agonist-induced intrinsic fluorescence changes in membrane-bound acetylcholine receptor
Kinetics of agonist-induced intrinsic fluorescence changes in membrane-bound acetylcholine receptor
复制标题
膜结合乙酰胆碱受体激动剂诱导的内在荧光变化的动力学
作者：
R. Bonner;F. Barrantes;T. Jovin
鱼(电鱼和鱼雷)的富含乙酰胆碱受体(AChR)的膜被广泛用于体外结合和功能研究。这些膜碎片(微囊)特别适合于ACHR-配体相互作用的物理研究，因为它们固有的高受体含量，保留在其自然环境中。许多外源荧光探针已被应用于在离体膜、神经肌肉接头和纯化的受体1-6水平上测定结合的特异性、平衡性和动力学。我们报道了天然神经递质乙酰胆碱在鱼雷电斑的膜碎片中诱导的内源性蛋白质荧光变化。我们使用高灵敏的荧光动力学技术获得了初始结合反应和随后的异构化反应的浓度依赖关系以及一些相应的平衡常数和速率常数。后者可以与电生理数据和突触后膜7-9的功能特性相关，特别是与脱敏过程相关，以及与相同的微囊制备获得的其他定量结果10相关。特别是，我们的数据为胆碱能受体蛋白在原位存在至少三种构象状态提供了物理证据，这些构象通过可逆的动力学过程相互关联。
ACETYLCHOLINE receptor (AChR)-rich membranes derived from fish (Electrophorus and Torpedo) electric organs have been used extensively for in vitro binding and functional studies1. These membrane fragments (microsacs) are particularly suited for physical investigations of AchR–ligand interactions because of their inherently high content of receptor, retained in its natural environment. A number of extrinsic fluorescent probes have been applied to the determination of binding specificity, equilibria and, in a preliminary way, kinetics at the level of the isolated membrane, as well as the neuromuscular junction and purified receptor1–6. We report here the existence of intrinsic protein fluorescence changes, induced in membrane fragments from Torpedo marmorata electroplaques by the natural neurotransmitter acetylcholine. We have used highly sensitive fluorescence kinetic techniques to obtain the concentration dependence and some of the corresponding equilibrium and rate constants for the initial binding and subsequent isomerisation reactions. The latter can be correlated with electrophysiological data and the functional properties of the post-synaptic membrane7–9, particularly with respect to the process of desensitisation, and with other quantitative results obtained with the same microsac preparations10. In particular, our data provide physical evidence for the existence of at least three conformational states of the cholinergic receptor protein in situ, interrelated by reversible kinetic processes.