Sullivan J M, Shukla P
Department of Ophthalmology, State University of New York, Health Science Center at Syracuse, Syracuse, New York 13210 USA.
Biophys J. 1999 Sep;77(3):1333-57. doi: 10.1016/S0006-3495(99)76983-3.
The early receptor current (ERC) is the charge redistribution occurring in plasma membrane rhodopsin during light activation of photoreceptors. Both the molecular mechanism of the ERC and its relationship to rhodopsin conformational activation are unknown. To investigate whether the ERC could be a time-resolved assay of rhodopsin structure-function relationships, the distinct sensitivity of modern electrophysiological tools was employed to test for flash-activated ERC signals in cells stably expressing normal human rod opsin after regeneration with 11-cis-retinal. ERCs are similar in waveform and kinetics to those found in photoreceptors. The action spectrum of the major R(2) charge motion is consistent with a rhodopsin photopigment. The R(1) phase is not kinetically resolvable and the R(2) phase, which overlaps metarhodopsin-II formation, has a rapid risetime and complex multiexponential decay. These experiments demonstrate, for the first time, kinetically resolved electrical state transitions during activation of expressed visual pigment in a unicellular environment (single or fused giant cells) containing only 6 x 10(6)-8 x 10(7) molecules of rhodopsin. This method improves measurement sensitivity 7 to 8 orders of magnitude compared to other time-resolved techniques applied to rhodopsin to study the role particular amino acids play in conformational activation and the forces that govern those transitions.
早期受体电流（ERC）是光感受器光激活过程中发生在质膜视紫红质中的电荷重新分布。ERC的分子机制及其与视紫红质构象激活的关系均尚不清楚。为了研究ERC是否可以作为视紫红质结构 - 功能关系的时间分辨测定方法，利用现代电生理工具的独特灵敏度，对用11 - 顺式视黄醛再生后稳定表达正常人视杆视蛋白的细胞中的闪光激活ERC信号进行测试。ERC的波形和动力学与光感受器中的相似。主要R(2)电荷运动的作用光谱与视紫红质光色素一致。R(1)阶段在动力学上无法分辨，与变视紫红质II形成重叠的R(2)阶段具有快速的上升时间和复杂的多指数衰减。这些实验首次证明了在仅含有6×10^6 - 8×10^7个视紫红质分子的单细胞环境（单个或融合的巨细胞）中，表达的视觉色素激活过程中动力学分辨的电状态转变。与应用于视紫红质的其他时间分辨技术相比，该方法将测量灵敏度提高了7到8个数量级，用于研究特定氨基酸在构象激活中的作用以及控制这些转变的力。
