Molecular Mechanisms of Retinal cGMP-Activated Channels

视网膜cGMP激活通道的分子机制

• 批准号: 7230914
• 负责人: MICHAEL D. VARNUM
• 金额: $ 24.6万
• 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 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7230914
• 关键词: Alternative Splicing; Binding; Binding Sites; Biochemical; Biological Assay; Calcium; Calmodulin; Cations; Cell membrane; Cells; Chemicals; Chromosome Pairing; Closure; Color blindness; Complementary DNA; Coupled; Cyclic GMP; Cyclic Nucleotides; Disease; Elements; Feedback; Functional disorder; Genes; Goals; Green Fluorescent Proteins; Human; Inborn Genetic Diseases; Individual; Ion Channel; Ions; Lead; Ligand Binding; Ligands; Light; Light Adaptations; Link; Localized; Mediating; Microscopy; Molecular; Mutation; Nitric Oxide; Phosphorylation; Photoreceptors; Phototransduction; Play; Process; Property; Proteins; Range; Receptor Activation; Recovery; Regulation; Research; Retina; Retinal; Retinal Cone; Retinal Degeneration; Retinal Diseases; Role; Signal Transduction; Site; Synapses; Synaptic Transmission; Vertebrate Photoreceptors; Visual; Work; Xenopus oocyte; absorption; achromatopsia; base; cell type; cyclic-nucleotide gated ion channels; falls; horizontal cell; molecular pathology; potassium exchanger sodium-calcium; presynaptic; research study; stoichiometry; trafficking; visual information

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）：环核苷酸门控（CNG）离子通道在视网膜中的信号转导中发挥重要作用。在光感受器外节中，它们发出信号，使细胞内cGMP浓度下降，这是由光吸收引起的受体激活引起的。在视锥细胞和水平细胞之间的突触，它们调节突触传递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，这些通道对视觉信号和视网膜疾病非常重要。CNG通道由四个同源亚基组成，每个亚基含有单个环核苷酸结合位点。与这些位点结合的配体与导致通道孔打开的构象变化偶联。天然视锥光感受器CNG通道包含两种不同的亚基类型，CNGA 3和CNGB 3;这些不同亚基的组装产生了具有针对其在光转导中的作用而优化的特性的异聚CNG通道。在这个提议中，我们将发现调节锥CNG通道亚基的组装和运输的结构元件和机制。视锥细胞CNG通道对钙反馈调节特别敏感，这一特征对视锥细胞中光适应的非凡范围至关重要。在这里，我们将研究的结构决定因素和相互作用是重要的钙依赖性调节锥CNG通道。此外，我们将阐明在锥CNG通道基因突变的分子病理学已被链接到以前完全和不完全全色盲和进行性锥营养不良。在这些实验中，将使用电生理学记录的外源表达的cDNA克隆在非洲爪蟾卵母细胞，荧光显微镜定位通道融合到绿色荧光蛋白，和生化蛋白质相互作用测定通道进行研究。这些实验将增强我们对环核苷酸门控离子通道的独特性质的分子基础的理解，这些离子通道对视网膜中的信号转导至关重要，以及导致色盲和视网膜变性的分子机制。