Molecular Mechanisms of Retinal cGMP-Activated Channels

描述(申请人提供)：环核苷酸门控(CNG)离子通道在视网膜的信号转导中起着重要作用。在光感受器外段，它们发出信号，表明细胞内cGMP浓度下降，这是由于光吸收激活了受体。在锥体细胞和水平细胞之间的突触，它们调节突触传递，并通过一氧化氮调节突触前反馈。我们研究的总体目标是阐明CNG通道活动的分子机制，这些通道对视觉信号和视网膜疾病至关重要。CNG通道由四个同源亚基组成，每个亚基都含有一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相耦合。天然锥体光感受器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创建了具有优化其在光转导中的作用的特性的异构体CNG通道。在这项提案中，我们将发现调控锥体CNG通道亚单位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，这一特征对于视锥细胞对光的非凡适应范围至关重要。在这里，我们将研究结构决定因素和相互作用，这是重要的钙依赖调节的锥体CNG通道。此外，我们还将阐明视锥CNG通道基因突变的分子病理学，这些基因以前被认为与完全性和不完全性色盲以及进行性视锥营养不良有关。在这些实验中，将使用电生理记录在非洲爪哇卵母细胞中外源表达的cDNA克隆，荧光显微镜定位融合到绿色荧光蛋白的通道，以及生化蛋白质相互作用分析来研究通道。这些实验将加深我们对环核苷酸门控离子通道在视网膜信号转导中所必需的独特性质的分子基础，以及导致色盲和视网膜变性的分子机制的理解。

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.