Molecular Mechanisms of Retinal cGMP-Activated Channels

描述（由申请人提供）：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。在光感受器外节段，它们发出细胞内cGMP浓度下降的信号，这是由吸收光的受体激活引起的。在锥体细胞和水平细胞之间的突触中，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，这对视觉信号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CNGA3和CNGB3两种不同的亚基类型；这些不同亚基的组装创造了异质CNG通道，其特性针对其在光导中的作用进行了优化。在本提案中，我们将发现调节CNG通道亚单元组装和运输的结构元素和机制。锥体CNG通道对钙反馈调节特别敏感，这一特征对锥体的特殊光适应范围至关重要。在这里，我们将研究对锥形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通道基因突变的分子病理学，这些基因突变先前与完全和不完全色盲以及进行性锥体营养不良有关。在这些实验中，通道将通过电生理记录外源表达的cDNA克隆在非洲爪蟾卵母细胞中，荧光显微镜来定位融合绿色荧光蛋白的通道，以及生化蛋白相互作用分析来研究。这些实验将增强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的理解，以及导致色盲和视网膜变性的分子机制。

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.