GATING MECHANISMS OF RETINAL ROD CGMP-ACTIVATED CHANNELS

光感受器的cGMP激活的离子通道是光感受器的基本功能。 分子元件，该分子元件产生对光的电响应， 视网膜。在光传导过程中， 一种视紫红质分子导致细胞内 第二信使cCMP的浓度和cGMP的关闭- 光感受器外节膜中的激活通道。的 响应于光关闭该阳离子选择性通道产生 由视觉系统处理的主要电信号。 光感受器中宏观光敏电流的研究进展 表示这些渠道的作用是高度专业化的 光传导通过协同结合多个cGMP分子， 通道表现为快速分子开关。建议的目标 实验是为了了解这些分子机制的基础， cGMP激活通道的专业化。特别是，建议 重点研究通道蛋白的构象变化， 在cGMP激活期间发生。方法将是研究开放 单个cGMP激活通道的关闭行为和改变 在通道中的位点特异性突变产生的门控行为中， 蛋白将使用单通道膜片钳研究通道 记录牛cDNA克隆表达的cGMP激活通道 在非洲爪蟾卵母细胞或哺乳动物培养细胞系中。分析 单通道行为将评估关闭和打开的数量 通道的构象，允许的构象转变，以及 各种构象的相对能量。该分析将 应用于其结构被定点改变的通道， 诱变通道子单元之间的相互作用将是 通过分析含有正常亚基和突变亚基的通道进行检查。 由于定义的功能改变的性质 结构变化将提供有价值的见解， 渠道功能的机制。

The cGMP-activated ion channel of photoreceptors is the fundamental molecular element that generates the electrical response to light in the retina. During phototransduction, the absorption of a photon of light by a rhodopsin molecule results in the reduction of the cytosolic concentration of the second messenger cCMP and the closing of a cGMP- activated channel in the membrane of the photoreceptor outer segment. The closing of this cation selective channel in response to light produces the primary electrical signal that is processed by the visual system. Previous work on the macroscopic light-sensitive current in photoreceptors has indicated that these channels are highly specialized for their role in phototransduction. By cooperatively binding multiple cGMP molecules, these channels behave as rapid molecular switches. The goal of the proposed experiments is to understand the molecular mechanisms that underlie these specializations in the cGMP-activated channel. In particular, the proposal focus on studies of the conformational changes in the channel protein that occur during activation by cGMP. The approach will be to study the opening and closing behavior of single cGMP-activated channels and the alterations in this gating behavior produced by site-specific mutations in the channel protein. The channels will be studied using single-channel patch-clamp recording on cGMP-activated channels expressed from the bovine cDNA clone in Xenopus oocytes or mammalian cultured cell lines. Analysis of the single-channel behavior will assess the number of closed and open conformations of the channel, the allowed conformational transitions, and the relative energy of the various conformations. This analysis will be applied to channels that have their structure altered by site-directed mutagenesis. The interactions between subunits of the channel will be examined by analyzing channels containing both normal and mutant subunits. The nature of the functional alterations that result from defined structural changes will provide valuable insights into the molecular mechanisms of the channel function.