MOLECULAR MECHANISMS OF RETINAL CGMP ACTIVATED CHANNELS

视网膜 CGMP 激活通道的分子机制

• 批准号: 6350901
• 负责人: MICHAEL D. VARNUM
• 金额: $ 22.45万
• 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 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6350901
• 关键词: SDS polyacrylamide gel electrophoresis; Xenopus oocyte; binding sites; color blindness; confocal scanning microscopy; cyclic GMP; electrophysiology; fluorescence microscopy; gene mutation; human tissue; immunoprecipitation; membrane channels; nucleotides; photoactivation; protein protein interaction; retina; retina degeneration; rod cell; stoichiometry; tissue /cell culture; visual photoreceptor; visual phototransduction; voltage /patch clamp; western blottings; yeast two hybrid system

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.

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。在光感受器外节中，它们发出信号，使细胞内cGMP浓度下降，这是由视紫红质吸收光引起的。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。我们的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CNG通道被认为含有两种不同的亚基类型，α和β;这些不同亚基的组装产生了异聚CNG通道，其特性针对其在光转导中的作用进行了优化。在这个提议中，我们将确定负责这些通道的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，我们将研究与视杆单色性和视网膜色素变性有关的CNG通道基因突变的分子机制。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和哺乳动物细胞系，转染细胞的荧光显微镜定位融合到绿色荧光蛋白的通道，和生化和遗传蛋白质相互作用测定的通道进行研究。这些实验将为视网膜中的信号传导以及导致视网膜变性和色盲的分子机制提供必要的通道。