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.

环状核苷酸门控离子通道在视网膜的视网膜信号转导中起着基本作用。在光感受器的外部段中，它们表明细胞内CGMP浓度下降，这是由视紫红质吸收的吸收而导致的。在锥体和水平细胞之间的突触时，它们调节突触传播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，每个亚基都包含一个循环核苷酸结合位点。配体与这些位点的结合耦合到导致通道孔打开的构象变化。本机CNG通道被认为包含两种不同的亚基类型，即alpha和beta。这些不同的亚基的组装产生了异元CNG通道，其特性对其在光转移中的作用进行了优化。在此提案中，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状核苷酸特异性的分子特征。此外，我们将研究与杆单色和色素性视网膜炎有关的CNG通道基因中的分子机制。将使用对异植虫卵母细胞中外源表达的cDNA克隆的电生理记录和哺乳动物细胞系，转染细胞的荧光显微镜将融合到绿色荧光蛋白融合的通道以及生物化学和遗传蛋白质相互作用分析的荧光显微镜。这些实验将通道对视网膜中的信号转导以及导致视网膜变性和色盲性的分子机制所必需的通道。