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，这是由于视紫红质吸收光线所致。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通道活性的分子机制。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CNG通道被认为包含两种不同的亚基类型，α和β；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性质的异构体CNG通道。在这项建议中，我们将确定负责这些通道组装的结构决定因素，它们的亚基的精确排列，以及调节它们的环核苷酸特异性的分子特征。此外，我们还将研究CNG通道基因突变的分子机制，这些突变与视杆细胞单色性和视网膜色素变性有关。这些通道将通过电生理记录在非洲爪哇卵母细胞和哺乳动物细胞系中外源表达的cDNA克隆，转基因细胞的荧光显微镜来定位融合到绿色荧光蛋白的通道，以及生化和遗传蛋白相互作用分析来研究。这些实验将对视网膜中的信号转导以及导致视网膜退化和色盲的分子机制进行研究。

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.