PHOTORECEPTOR LIGHT-MODULATED CHANNEL--MOLECULAR STUDIES

这项研究旨在确定一个单一的离子群体 通道转换外节的光化学信息 转化为离子电流，传递所有预处理的视觉信息。 通道的门控由多个 cGMP分子位于蛋白质的胞质表面。 以前我们 研究了结构功能识别的配体，通过采用 a系列cGMP衍生物。 我们扩展这些研究使用三- 结合位点的三维模型。 用标准的分子模型 方法，我们将这些衍生物对接到结合位点， 计算相互作用的能量。 空间约束将提供 模型的附加测试。 我们还将研究 在单通道水平上的通道激活。 由于沟道电导 已经显示随着每个结合位点的占有率逐步增加， 我们可以测量每个配体占有率的平衡常数 直接通过测量每个电流的通道打开概率 水平 通过这些通道的电流是由Na和Na+的流入产生的。 钙离子。由于细胞质Ca水平的变化需要调节细胞内Ca的浓度， 光感受器对背景光水平变化的敏感性， 关于光传导的基本问题是， 段电流由Ca承载。 通道可以保持固定 内流比钠和钙或改变响应于不同的比例 背景光水平通过细胞溶质cGMP或Ca水平的变化。一 我们研究的目的是确定这些流入比率。 因为离子 不能区分电生理，我们将测量22 Na和 瞬时表达45 Ca内流的哺乳动物细胞 渠道 我们以前表明存在高亲和力的二价 在调节电流的通道的胞质表面的结合位点 当cGMP水平低时，细胞溶质二价水平。 渗透模型 的二价电流表明，这个结合位点是外部的 使用宏观和单通道记录，我们将 探索二价阳离子变构调节 在恒定cGMP水平下的通道功能。定点诱变将 用于定义通道拓扑结构并测试 结合位点。

The research proposed seeks to determine how a single population of ion channels transduces the photochemical information of the outer segment into an ionic current that conveys all pre-processed visual information. Gating of the channel is controlled by cooperative binding of multiple cGMP molecules at the cytosolic face of the protein. Previously we examined the structure-function recognition of the ligand by employing a series a cGMP derivatives. We extend these studies using a three- dimensional model of the binding site. With standard molecular modeling approaches, we will dock these derivatives into the binding site and calculate the energies of interaction. Steric constraints will provide an additional test of the model. We will also examine the cooperativity of channel activation at the single channel level. Since channel conductance has been shown to increase stepwise with occupancy of each binding site, we can measure the equilibrium constants for each ligand occupancy directly by measuring the channel opening probability for each current level. Current through these channels is generated by the influx of both Na and Ca ions. Since changes in cytosolic Ca levels are required to adjust the sensitivity of photoreceptors to changes in background light levels, a fundamental question about phototransduction is what fraction of the outer segment current is carried by Ca. The channel could either maintain fixed influx ratios of Na and Ca or alter the ratio in response to varying levels of background light via changes in cytosolic cGMP or Ca levels. One aim of our research is to determine these influx ratios. Because the ions cannot be distinguished electrophysiologically, we will measure 22Na and 45Ca influx in cultured mammalian cells that transiently express the channels. We showed previously the existence of a high affinity divalent binding site at the cytosolic face of the channel which regulates current at cytosolic divalent levels when cGMP levels are low. Permeation models of the divalent currents suggest that this binding site is outside the permeation path. Using macroscopic and single channel recordings we will explore the possibility that divalent cations allosterically regulate channel function at constant cGMP levels. Site directed mutagenesis will be used to define the channel topology and test the structural models of the binding site.