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

描述(申请人提供)：该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，并将这一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，CNGB1基因缺陷会导致遗传性视网膜变性。因此，我们重点研究了仅在视杆外段(ROS)表达两种相对丰富的蛋白质的小鼠Cngb1基因座：质膜定位的cGMP门控阳离子通道的2-亚基和细胞质中富含谷氨酸-脯氨酸的多肽(GARP-2)。棒状cGMP门控阳离子通道通过调节钙离子和钠离子流入杆状细胞，在视觉中起着关键作用。此外，2-亚基和GARP-2都与磁盘膜蛋白外周蛋白-2/RDS相互作用，因此可能发挥重要的结构作用。为了确定ROS中蛋白质的贡献，我们产生了一个纯合子Cngbl光感受器空基因(5‘KO)，并使用光和EM水平分析、ERG和单细胞记录以及Western分析对小鼠进行了分析。纯合子5‘KO小鼠的光敏感度和b波反应显著降低(-gt；30倍)，但在视网膜变性开始之前是正常的。令人惊讶的是，在59KO中，不仅没有2-亚基，而且ROS中通道1-亚基的丰度也大大减少。我们假设GARP区在光感受器中具有重要的结构作用，GARP-2和2-亚基一样也具有重要的功能作用。我们将通过靶向删除GARP-2表达并将转基因引入59KO和2亚单位基因敲除小鼠(39KO)中，直接研究β亚基和GARP-2的GARP区域的结构和功能作用。这些研究将有助于建立CNGB1编码的GARP区和GARP-2在光感受器中的功能，从而为CNGB1基因缺陷导致遗传性视网膜变性的机制提供重要的见解。这些研究还可能为干预提供新的靶点，作为对某些形式的遗传性视网膜退化的可能治疗。

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.