PHOTORECEPTOR LIGHT-MODULATED CHANNEL--MOLECULAR STUDIES

光感受器光调制通道--分子研究

• 批准号: 2160692
• 负责人: JACQUELINE C TANAKA
• 金额: $ 27.13万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1997-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2160692
• 关键词: Xenopus oocyte; calcium channel; calcium flux; chemical binding; cyclic GMP; electrophysiology; membrane structure; molecular site; protein structure function; receptor binding; single cell analysis; site directed mutagenesis; sodium channel; tissue /cell culture; transfection; visual photoreceptor; visual phototransduction; voltage /patch clamp

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.

拟议的研究旨在确定一个离子群体如何 通道传递外部段的光化学信息 进入一个离子电流，该电流传达了所有预处理的视觉信息。 通道的门控通过多个的合作结合来控制 CGMP分子在蛋白质的胞质面部。 以前我们 通过采用A A系列CGMP衍生物。 我们使用三个研究扩展了这些研究 结合位点的维度模型。 与标准分子建模 方法，我们将将这些衍生物扩展到约束地点，然后 计算相互作用的能量。 空间约束将提供 模型的其他测试。 我们还将研究 单个通道级别的通道激活。 自通道电导以来 已显示出随着每个结合位点的占用率的逐步增加， 我们可以测量每个配体占用的平衡常数 直接通过测量每个电流的通道打开概率 等级。 通过这些通道的电流是由NA和NA和 ca离子。由于需要胞质CA水平的变化以调整 光感受器对背景光水平变化的敏感性，a 关于光转录的基本问题是外部的部分 分段电流由CA携带。 该频道可以维护固定 Na和ca的涌入比或改变比率随着变化而改变的比率 背景光的水平通过胞质CGMP或CA水平的变化。一 我们研究的目的是确定这些涌入比。 因为离子 无法从电生理学上区分，我们将测量22NA，并且 45CA在培养的哺乳动物细胞中涌入，这些细胞瞬时表达 频道。 我们以前展示了高亲和力二价的存在 在调节电流的通道的胞质面上的结合位点 当CGMP水平较低时，在胞质二价水平下。 渗透模型 二价电流表明该结合位点在 渗透路径。 使用宏观和单个通道记录，我们将 探索二价阳离子对变构调节的可能性 恒定CGMP级别的通道函数。站点定向诱变将 用于定义通道拓扑​​并测试结构模型 结合位点。