GATING MECHANISMS OF RETINAL ROD CGMP-ACTIVATED CHANNELS

光感受器中cGMP激活的离子通道是 对光产生电响应的分子元素 视网膜。在光转换过程中，光对光子的吸收 视紫红质分子导致胞浆减少 第二信使CCMP的浓度和cGMP的关闭- 光感受器外节膜上的激活通道。这个 这种阳离子选择性通道的关闭对光的响应产生 由视觉系统处理的主要电信号。 光感受器宏观光敏电流的研究进展 已经表明，这些渠道高度专业化，因为它们在 光转导。通过协同结合多个cGMP分子，这些 通道就像是快速的分子开关。建议的目标是 实验是为了了解这些现象背后的分子机制 CGMP激活渠道中的专业化认证。尤其是，该提案 重点研究通道蛋白的构象变化 发生在cGMP激活过程中。我们的方法将是研究开放 和单个cGMP激活通道的关闭行为及其变化 在这种由通道中特定位置突变所产生的门控行为中 蛋白。将使用单通道膜片钳来研究通道 牛cDNA克隆表达的cGMP激活通道的记录 在非洲爪哇卵母细胞或哺乳动物培养细胞系中。三、分析了 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。这一分析将是 应用于通过站点定向更改其结构的频道 诱变。通道亚基之间的相互作用将是 通过分析包含正常和突变亚基的通道进行检查。 由定义导致的功能改变的性质 结构变化将提供对分子的有价值的见解 经络作用的机制。

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.