Analysis of retina rod photoreceptor GARP and cGMP-gated cation channel

描述（由申请人提供）：研究计划的长期目标是了解视网膜光感受器中的蛋白质结构/功能关系，并将这种理解转化为视网膜色素变性和相关疾病患者的治疗。在人类中，CNGB 1基因缺陷导致遗传性视网膜变性。因此，我们专注于鼠Cngbl基因座，表达，专门在杆外节（ROS），两个相对丰富的蛋白质：质膜定位的cGMP门控阳离子通道的2-亚基和细胞质谷氨酸-脯氨酸丰富的多肽（GARP-2）。视杆细胞cGMP门控阳离子通道通过调节钙离子和钠离子流入视杆细胞而在视觉中起关键作用。此外，2-亚基和GARP-2与外周蛋白2/rds（一种盘膜蛋白）相互作用，因此可能发挥重要的结构作用。为了确定蛋白质在ROS中的贡献，我们产生了纯合的Cngbl光感受器无效（5' KO），并使用光和EM水平分析、ERG和单细胞记录以及Western分析来分析小鼠。纯合5' KO小鼠显示出光敏感性的显著降低（>30倍）和减弱的b波反应，但在视网膜变性发作之前是功能性的。令人惊讶的是，在59 KO中，不仅2-亚基缺失，而且通道1-亚基在ROS中的丰度也大大降低。我们假设GARP区域在光感受器中具有重要的结构作用，并且GARP-2和2-亚基一样也具有重要的功能作用。我们将通过靶向缺失GARP-2表达并将转基因引入59 KO和2-亚基敲除小鼠（39 KO），直接检查β亚基和GARP-2的GARP区域的结构和功能作用。提出的研究将有助于建立Cngbl编码的GARP区域和GARP-2在光感受器中的功能，从而为CNGB 1基因缺陷导致遗传性视网膜变性的机制提供重要的见解。这些研究也可能产生新的干预目标，作为某些形式的遗传性视网膜变性的可能治疗方法。

DESCRIPTION (provided by applicant): The long term objectives of the research program are to understand protein structure/function relationships in retinal photoreceptors, and to translate this understanding to the treatment of patients with retinitis pigmentosa and related disorders. In humans, CNGB1 gene defects cause hereditary retinal degeneration. We have therefore focused on the murine Cngbl locus that expresses, exclusively in the rod outer segment (ROS), two relatively abundant proteins: plasma membrane localized 2-subunit of the cGMP-gated cation channel and cytoplasmic glutamic acid-proline rich polypeptide (GARP-2). The rod cGMP-gated cation channel plays a critical role in vision by regulating the flow of calcium and sodium ions into the rod cell. In addition, both the 2-subunit and GARP-2 interact with peripherin-2/rds, a disk membrane protein, and thus may play essential structural roles. To determine the contribution of the proteins in ROS, we generated a homozygous Cngbl photoreceptor null (5' KO), and are analyzing the mice using light and EM level analysis, ERG and single cell recordings, and Western analysis. Homozygous 5' KO mice show a significant reduction (>30 fold) in light sensitivity and attenuated b-wave response, but are functional prior to the onset of retinal degeneration. Surprisingly, in the 59 KO not only is the 2-subunit absent but the channel 1-subunit is also greatly reduced in abundance in ROS. We hypothesize that the GARP region has an important structural role in the photoreceptor and that GARP-2, like the 2-subunit has also an important functional role. We will directly examine the structural and functional roles of the GARP region of the beta subunit and of GARP-2 by targeted deletion of GARP-2 expression and introduction of transgenes into 59 KO and 2-subunit knockout mice (39 KO). The proposed studies will help to establish the function of the Cngbl encoded GARP region and of GARP-2 in the photoreceptor, and hence provide important insights into the mechanisms whereby CNGB1 gene defects cause hereditary retinal degeneration. The studies may also yield new targets for intervention as possible treatments for certain forms of hereditary retina degeneration.