GATING MECHANISMS OF RETINAL ROD CGMP-ACTIVATED CHANNELS

视网膜杆 CGMP 激活通道的门控机制

• 批准号: 2164130
• 负责人: William N Zagotta
• 金额: $ 14.78万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2164130
• 关键词: Xenopus; Xenopus oocyte; allosteric site; alternatives to animals in research; aminoacid; animal tissue; binding proteins; conformation; cyclic AMP; cyclic GMP; electrophysiology; inhibitor /antagonist; mathematical model; membrane channels; molecular cloning; polymerase chain reaction; protein structure function; rod cell; site directed mutagenesis; visual photoreceptor; visual phototransduction; voltage /patch clamp

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.

感光体的CGM​​P激活离子通道是基本 分子元素产生对光的电响应 视网膜。在光转导的过程中，通过 视紫红质分子导致胞质的还原 第二信使CCMP的浓度和CGMP的关闭 光感受器外部段的膜中的活化通道。这 响应光的阳离子选择通道的关闭会产生 视觉系统处理的主要电信号。 在光感受器中宏观光敏电流的先前工作 这些渠道在 光转导。通过合作结合多个CGMP分子，这些 通道的行为为快速分子开关。提议的目标 实验是了解这些基于这些的分子机制 CGMP激活的通道中的专业知识。特别是建议 关注通道蛋白中构象变化的研究 在CGMP激活期间发生。该方法将是研究开口 以及单个CGMP激活的通道和更改的结束行为 在这种通道中位点特异性突变产生的门控行为中 蛋白质。将使用单通道贴片钳研究这些通道 在CGMP激活的通道上记录从牛cDNA克隆表达的通道 在爪蟾卵母细胞或哺乳动物培养的细胞系中。分析 单通道行为将评估封闭式和打开的数量 通道的构象，允许的构象过渡和 各种构象的相对能量。该分析将是 应用于其结构通过站点定向改变的通道 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检查。 由定义的功能变化的性质 结构变化将为分子提供宝贵的见解 通道功能的机制。