EARLY RECEPTOR CURRENT OF RHODOPSIN ACTIVATION

在视觉的初始事件中，光触发构象变化 在视紫红质中导致光感受器兴奋。 光谱分析 的突变视紫红质的作用，表明特定的氨基酸在快速 导致活性后视紫红质-II状态的构象变化， 结合并激活转导素。 早期受体电流（Early Receptor Current，ERC） 在激活过程中视紫红质的构象依赖性电荷流，可以是 研究使用全细胞膜片钳技术，是基础 早期感受器电位（ERP）。 紧急救济协调员 提供足够的微秒级时间分辨率， 视紫红质的构象变化，并允许测试的作用， 特定的氨基酸在激活的发色团和远程 蛋白质环境 ERC技术在研究人类基因突变中也是有用的。 视紫红质，例如由常染色体显性视网膜色素变性引起的视紫红质 （adRP），破坏光转导并导致光感受器变性。 ERC技术是合适的，因为在50个突变中， adRP位于视紫红质的膜内区域。 为了更好地了解特定氨基酸在激活 野生型和突变型（adRP）视紫红质，我建议： (1)表征在细胞周期中产生的早期受体电流（ERC）。 在细胞系中表达的人视紫红质的活化。 (2)研究特定的膜内氨基酸对 ERC。 (3)使用ERC检验adRP突变减少的假设 视紫红质的热稳定性，并创造替代途径， 后视紫红质-II 拟议的实验应有助于更好地了解 ERC背后的基本生物物理过程， 视紫红质激活中的特定氨基酸，以及adRP如何导致 视紫红质功能障碍 我建议在细胞中进行这些研究， 表达系统，其中来自正常人视紫红质的ERCs已被 测量并在其中可以表达视紫红质突变体。

During the initial event in vision light triggers conformational changes in rhodopsin that lead to photoreceptor excitation. Spectroscopic assays of mutant rhodopsins suggest roles for specific amino acids in the rapid conformational changes leading to the active Metarhodopsin-II state that binds and activates transducin. The Early Receptor Current (ERC), the conformation-dependent charge flow in rhodopsin during activation, can be studied using whole-cell patch clamp techniques and is the fundamental process underlying the familiar early receptor potential (ERP). The ERC provides sufficient microsecond time resolution for studying fast conformational changes in rhodopsin and permits tests of the role of specific amino acids in activation in both the chromophore and remote protein environments. The ERC technique should also be useful in studying how mutations of human rhodopsin, such as those caused by autosomal dominant retinitis pigmentosa (adRP), disrupt phototransduction and lead to photoreceptor degeneration. The ERC technique is suitable because over half of the fifty mutations in adRP are in the intramembrane region of rhodopsin. To better understand the role of specific amino acids in the activation of wild type and mutant (adRP) rhodopsins, I propose to: (1) Characterize the Early Receptor Current (ERC) produced during the activation of human rhodopsin expressed in a cell line. (2) Investigate the contribution of specific intramembrane amino acids to the ERC. (3) Use the ERC to test the hypothesis that adRP mutations decrease thermal stability of rhodopsin and create alternative paths to Metarhodopsin-II. The proposed experiments should lead to a better understanding of the fundamental biophysical processes underlying the ERC, the roles of specific amino acids in activation of rhodopsin, and how adRP leads to rhodopsin dysfunction. I propose to carry out these studies in a cellular expression system in which ERCs from normal human rhodopsin have been measured and in which rhodopsin mutants can be expressed.