MOLECULAR MECHANISMS OF RETINAL CGMP ACTIVATED CHANNELS

环核苷酸门控离子通道在视网膜的信号转导中起着重要的作用。在光感受器外节段，它们信号胞内cGMP浓度下降，这是由视紫红质吸收光引起的。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活性CNG通道的分子机制。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。原生天然气通道被认为包含两种不同的亚基类型：α和β；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，我们将确定负责这些通道组装的结构决定因素，其亚基的精确排列以及调节其环核苷酸特异性的分子特征。此外，我们将研究与杆状细胞单色和视网膜色素变性相关的CNG通道基因突变的分子机制。这些通道将通过电生理记录在非洲爪蟾卵母细胞和哺乳动物细胞系中外源表达的cDNA克隆，荧光显微镜观察转染细胞以定位与绿色荧光蛋白融合的通道，以及生化和遗传蛋白相互作用测定来研究。这些实验将揭示视网膜信号转导的关键通道，以及导致视网膜变性和色盲的分子机制。

Cyclic nucleotide-gated ion channels play a fundamental role in signal transduction in the retina in the retina. In photoreceptor outer segments, they signal the fall in intracellular cGMP concentration that results from absorption of light by rhodopsin. 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 CNG channels. 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 CNG channels are thought to contain two different subunit types, alpha and beta; the assembly of these divergent subunits creates heteromeric CNG channels with properties optimized for their role in phototransduction. In this proposal, we will ascertain the structural determinants responsible for the assembly of these channels, the precise arrangement of their subunits and the molecular features that modulate their cyclic nucleotide specificity. In addition, we will examine the molecular mechanisms underlying mutations in CNG channel genes that have been linked to rod monochromasy and retinitis pigmentosa. The channels will be studied using electrophysiological recording of exogenously expressed cDNA clones in Xenopus oocytes and in a mammalian cell line, fluorescent microscopy of transfected cells to localize channels fused to green fluorescent protein, and biochemical and genetic protein interaction assays. These experiments will channels essential to signal transduction in the retina, and of the molecular mechanisms that lead to retinal degeneration and color-blindness.