Molecular Mechanisms of Retinal cGMP-Activated Channels

视网膜cGMP激活通道的分子机制

• 批准号: 7057333
• 负责人: MICHAEL D. VARNUM
• 金额: $ 24.75万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-04 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7057333
• 关键词: RNA splicing; Xenopus oocyte; binding sites; calcium flux; calmodulin; color blindness; confocal scanning microscopy; cyclic GMP; gene mutation; genetic regulation; immunoprecipitation; membrane channels; photoactivation; protein localization; protein protein interaction; protein structure function; retina; retina degeneration; rod cell; stoichiometry; tissue /cell culture; visual photoreceptor; visual photosensitivity; visual phototransduction; voltage /patch clamp

DESCRIPTION (provided by applicant): Cyclic nucleotide-gated (CNG) ion channels play a fundamental role in signal transduction in the retina. In photoreceptor outer segments, they signal the fall in intracellular cGMP concentration that results from receptor activation by absorption of light. At synapses between cone and horizontal cells, they regulate synaptic transmission and mediate presynaptic feedback by nitric oxide. The overall goal of our research is to elucidate the molecular mechanisms underlying the activity of CNG channels that are important for visual signaling and retinal disease. CNG channels are composed of four homologous subunits, each containing a single cyclic nucleotide-binding site. Ligand binding to these sites is coupled to conformational changes that lead to opening of the channel pore. Native cone photoreceptor CNG channels contain two different subunit types, CNGA3 and CNGB3; the assembly of these divergent subunits creates heteromeric CNG channels with properties optimized for their role in phototransduction. In this proposal, we will discover the structural elements and mechanisms regulating the assembly and trafficking of cone CNG channel subunits. Cone CNG channels are especially sensitive to calcium-feedback regulation, and this feature is critical to the extraordinary range of light adaptation in cones. Here, we will examine the structural determinants and interactions that are important for calcium-dependent regulation of cone CNG channels. In addition, we will elucidate the molecular pathology of mutations in cone CNG channel genes that have been linked previously to complete and incomplete achromatopsia and to progressive cone dystrophy. For these experiments, channels will be studied using electrophysiological recording of exogenously expressed cDNA clones in Xenopus oocytes, fluorescent microscopy to localize channels fused to green fluorescent protein, and biochemical protein interaction assays. These experiments will enhance our understanding of the molecular basis for the unique properties of cyclic nucleotide-gated ion channels essential to signal transduction in the retina, and of the molecular mechanisms that lead to color blindness and retinal degeneration.

描述（由申请人提供）：环状核苷酸门控（CNG）离子通道在视网膜的信号转导中起着基本作用。在光感受器的外部段中，它们表明细胞内CGMP浓度下降，这是由于受体吸收而引起的。在锥体和水平细胞之间的突触时，它们调节突触传播并通过一氧化氮介导突触前反馈。我们研究的总体目标是阐明CNG通道活性的分子机制，这对于视觉信号传导和视网膜疾病很重要。 CNG通道由四个同源亚基组成，每个亚基都包含一个循环核苷酸结合位点。配体与这些位点的结合耦合到导致通道孔打开的构象变化。天然锥形光感受器CNG通道包含两种不同的亚基类型CNGA3和CNGB3；这些不同的亚基的组装产生了异元CNG通道，其特性对其在光转移中的作用进行了优化。在此提案中，我们将发现调节锥体CNG通道亚基组装和运输的结构要素和机制。锥体CNG通道对钙反馈调节特别敏感，并且此特征对于锥体中的光适应范围至关重要。在这里，我们将研究对锥体CNG通道的钙依赖性调节很重要的结构决定因素和相互作用。此外，我们将阐明锥体CNG通道基因中突变的分子病理学，这些通道基因以前已将其连接到完整且不完整的Achromomopsia以及进行性锥体营养不良。对于这些实验，将使用外源表达的cDNA克隆在异爪蟾卵母细胞中的电生理记录，荧光显微镜将通道进行研究，以将融合到绿色荧光蛋白和生化蛋白质相互作用分析的通道定位。这些实验将增强我们对循环核苷酸门控离子通道的独特性能的分子基础的理解，这对于视网膜中信号转导和导致色盲和视网膜变性的分子机制所必需。