Analysis of retina rod photoreceptor GARP and cGMP-gated channel

7.项目摘要/摘要 该研究计划的长期目标是了解cGMP门控阳离子通道蛋白 杆状感光器的结构/功能关系，并将这一理解转化为治疗 患有视网膜色素变性(RP)及相关疾病的患者。我们将重点研究b频道- Cngb1编码的GARP和相关的GARP2亚基 Locus，研究GARP区域在三个具体目标中的结构和功能作用。我们之前 产生了纯合子Cngb1光感受器缺失(X1KO)小鼠，不仅导致部分通道丧失 功能，但也有严重的结构紊乱，在体内建立这些蛋白质是必要的 正常杆状外节、ROS盘形态发生及结构完整性。我们假设GARP2 质膜/盘膜相互作用需要b亚基上的序列。为了测试这一点，我们 建立了表达N端截短的b亚单位(TB)的转基因小鼠，该小鼠不含所有GARP2序列 X1 KO背景。尽管没有可溶性GARP2，但整个b亚基GARP2区和只有 结核上残留的一个富含谷氨酸的片段，有显著但不完整的结构和功能 救援队。确定观察到的救援的基础(A)。我们将对这些老鼠进行结构分析，使用 组织学、免疫细胞化学和超微结构的组织学、免疫细胞化学和超微结构分析 使用ERG、单细胞生理学和现已建立的视网膜穿孔准备。在X1KO和X26小鼠中 在X1KO中也缺少可溶性GARP，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 展示了GARP2在调控光信号转导中以前未知的作用。(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 将使用我们已建立的生理工具来比较WT、GARP2的光传导参数 过表达的TB转基因小鼠和在激活、恢复和恢复过程中表达全长b亚基的小鼠 适应以进一步确定GARP2和b亚基在调节光反应中的作用。使用小说 利用锌指核酸酶技术，我们已经建立了针对GARP2特异的基因敲除小鼠，最后进行了唯一的删除 GARP2外显子和一个潜在的缺失GARP2 3‘-UT区域的低晶型。(C.)我们将用这些老鼠 以验证GARP2是杆状细胞结构和功能所必需的假设。我们将表演结构性的 分析确定对椎间盘形态发生和整体外节结构和功能的影响 分析补充目标A和B中提出的研究，并直接检查GARP2在ROD中的作用 功能。拟议的研究记录了一项全面的计划，以确定 GARP区域，并确定GARP2在调节杆光响应中的作用。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目标。

7. Project Summary/Abstract 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 b- 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 b-subunit is required for plasma membrane/disc membrane interaction. To test this we created transgenic mice expressing an N-terminally truncated b-subunit (Tb) devoid of all GARP2 sequence on the X1 KO background. Despite the absence of soluble GARP2, the entire b-subunit GARP2 region and only one glutamate rich segment remaining on Tb, 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 b-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, Tb transgenic mice and mice expressing full length b-subunit during activation, recovery and adaptation to further define the role of GARP2 and the b-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.