Analysis of retina rod photoreceptor GARP and cGMP-gated channel

描述(由申请人提供)：该研究计划的长期目标是了解视杆感光细胞中cGMP门控阳离子通道蛋白的结构/功能关系，并将这一理解转化为视网膜色素变性(RP)及相关疾病患者的治疗。我们将集中研究通道�亚基和相关的可溶性富含谷氨酸蛋白(GARP)GARP2，这两个基因都由Cngb1基因编码，研究GARP区域在三个特定目的中的结构和功能作用。我们以前培育了一个纯合子的Cngb1光感受器缺失(X1KO)小鼠，结果不仅导致了通道功能的部分丧失，而且在体内建立了严重的结构扰动，证明这些蛋白质是正常的杆状外节、ROS盘的形态发生和结构完整性所必需的。我们推测�-亚基上的GARP2序列是质膜/盘膜相互作用所必需的。为了测试这一点，我们创造了在X1KO背景上表达N端截短的�亚单位(T�)的转基因小鼠，该亚基没有所有的GARP2序列。尽管没有可溶性的GARP2，整个�亚基的GARP2区域和T�上只剩下一个富含谷氨酸的片段，但仍有显著的但不完全的结构和功能挽救。确定观察到的救援的基础(A)。我们将使用组织学、免疫细胞化学和超微结构分析、透射和冷冻-EM分析这些小鼠的结构，并使用ERG、单细胞生理学和现已建立的视网膜冲压制剂对其进行功能分析。在不表达�亚基的X1KO和X26小鼠中，在X1KO中也缺少可溶性GARP的小鼠，光反应减弱。在过度表达GARP2的WT小鼠中，观察到光传导增益显着增加，这表明GARP2在调控光传导中的作用是以前未知的。(B)我们假设GARP2调节杆状暗噪声，也有助于新的慢伯恩斯适应。为了测试这一点，我们将使用我们建立的生理学工具来比较WT、GARP2过表达、T�转基因小鼠和表达全长�亚基的小鼠在激活、恢复和适应过程中的光传导参数，以进一步确定GARP2和�亚基在调节光反应中的作用。利用新的锌指核酸酶技术，我们建立了GARP2特异的敲除小鼠，其最后一个独特的GARP2外显子缺失，以及一个潜在的缺失GARP2 3‘-UT区域的低晶型。(C.)我们将使用这些小鼠来验证GARP2是杆状细胞结构和功能所必需的假设。我们将进行结构分析，以确定对椎间盘形态发生和整体外节结构的影响，并进行功能分析，以补充目标A和B中提出的研究，并直接检查GARP2在视杆功能中的作用。拟议的研究记录了一项全面的计划，以确定GARP区域的结构重要性，并确定GARP2在调节杆光反应中的作用。这些研究还可能为某些形式的遗传性视网膜变性的干预治疗提供新的靶点。

DESCRIPTION (provided by applicant): The long-term objectives of this research program are to understand cGMP-gated cation channel protein structure/function relationships in rod photoreceptors and to translate this understanding to the treatment of patients with retinitis pigmentosa (RP) and related disorders. We will focus our studies on the channel �-subunit and the associated soluble glutamic acid rich protein (GARP) GARP2, both encoded by the Cngb1 locus, examining the structural and functional roles of the GARP region in three specific aims. We previously generated a homozygous Cngb1 photoreceptor null (X1 KO) mouse resulting not only in partial loss of channel function but also severe structural perturbations establishing in vivo that these proteins are necessary for normal rod outer segment ROS disk morphogenesis and structural integrity. We hypothesized that the GARP2 sequence on the �-subunit is required for plasma membrane/disc membrane interaction. To test this we created transgenic mice expressing an N-terminally truncated �-subunit (T�) devoid of all GARP2 sequence on the X1 KO background. Despite the absence of soluble GARP2, the entire �-subunit GARP2 region and only one glutamate rich segment remaining on T�, there is significant but not complete structural and functional rescue. To determine the basis for the observed rescue (A.) we will analyze these mice structurally using histology, immunocytochemistry and ultrastructure analysis by transmission and Cryo-EM and functionally using ERG, single cell physiology and a now established retina punch preparation. In the X1 KO and X26 mice that do not express the �-subunit and in X1 KO also missing soluble GARPs the photoresponse is attenuated. In WT mice overexpressing GARP2 a significant increase in phototransduction gain was observed demonstrating a previously unknown role for GARP2 in modulating phototransduction. (B.) We hypothesize that GARP2 regulates rod dark noise and also contributes to novel slow Burnsian adaptation. To test this we will use our established physiologic tools to compare phototransduction parameters in WT, GARP2 overexpressing, T� transgenic mice and mice expressing full length �-subunit during activation, recovery and adaptation to further define the role of GARP2 and the �-subunit in modulating the photoresponse. Using novel zinc finger nuclease technology we have established GARP2-specific knockout mice deleted for the last unique GARP2 exon and a potential hypomorph that is missing the GARP2 3'-UT region. (C.) We will use these mice to test the hypothesis that GARP2 is required for structure and function in the rods. We will perform structural analysis to determine effects on disk morphogenesis and overall outer segment structure and functional analysis to complement studies proposed in Aims A. and B. and to directly examine the role of GARP2 in rod function. The proposed studies document a comprehensive plan to define the structural importance of the GARP region and to define the role of GARP2 in modulating the rod photoresponse. The studies may also yield new targets for intervention treatments for certain forms of hereditary retinal degeneration.