EARLY RECEPTOR CURRENT OF RHODOPSIN ACTIVATION

在视觉的最初事件中，光触发构象变化 导致光感受器兴奋的视紫红质。光谱分析 突变的视紫红质提示特定的氨基酸在快速的 导致变紫红质-II活性状态的构象变化 结合并激活转导蛋白。早期受体电流(ERC)、 激活期间视紫红质中构象相关的电荷流，可以是 使用全细胞膜片钳技术进行研究，是基础 熟悉的早期受体电位(ERP)的潜在过程。环境资源中心 为快速学习提供足够的微秒时间分辨率 视紫红质的构象变化，并允许测试 发色团和远端中激活的特定氨基酸 蛋白质环境。 ERC技术也应该有助于研究人类突变是如何 视紫红质，如常染色体显性遗传性视网膜色素变性引起的视紫红质 (Adrp)，破坏光转导，导致光感受器退化。 ERC技术是适用的，因为在50个突变中，超过一半是 ADRP位于视紫红质的膜内区。 为了更好地了解特定氨基酸在激活肌动蛋白中的作用 野生型和突变型(Adrp)视紫红质，我建议： (1)表征早期受体电流(ERC) 在细胞系中表达的人视紫红质的激活。 (2)研究膜内特定氨基酸对蛋白质合成的影响。 ERC。 (3)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 变视紫质-II。 拟议的实验应该会让我们更好地了解 ERC的基本生物物理过程，作用 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 正常人视紫红质来源的ERCs的表达系统 测量的视紫红质突变体可以在其中表达。

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.