Analysis of retina rod photoreceptor GARP and cGMP-gated channel

视网膜视杆光感受器 GARP 和 cGMP 门控通道的分析

• 批准号: 8579459
• 负责人: STEVEN J PITTLER
• 金额: $ 36.74万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8579459
• 关键词: 3' Untranslated Regions; Address; Affect; Alleles; Attenuated; Binding; Blindness; Cations; Cell membrane; Cell physiology; Cells; Code; Complement; Cyclic GMP; Data; Defect; Disease; Exons; Family; Genes; Genome; Glutamates; Glutamic Acid; Goals; Grant; Hereditary Disease; Histology; Inherited; Intervention; Ion Channel; Knock-out; Knockout Mice; Length; Literature; Membrane; Molecular; Morphogenesis; Mus; N-terminal; Noise; Patients; Photoreceptors; Phototransduction; Physiological; Poly(A) Tail; Population; Preparation; Process; Property; Protein Region; Proteins; Recovery; Research; Retina; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Rod Outer Segments; Role; Structure; Structure-Activity Relationship; Technology; Testing; Transgenic Mice; Translating; Vision; Work; Zinc Fingers; base; design; functional loss; immunocytochemistry; in vivo; mouse genome; mouse model; novel; nuclease; overexpression; programs; protein structure function; public health relevance; retinal rods; tool; transmission process

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.

7.项目总结/摘要 本研究计划的长期目标是了解cGMP门控阳离子通道蛋白 结构/功能的关系，并将这种理解转化为治疗 患有视网膜色素变性（RP）和相关疾病的患者。我们将集中研究B通道- 亚基和相关的可溶性谷氨酸丰富蛋白（GARP）GARP 2，两者都由Cngb 1编码 位点，研究GARP区域在三个特定目标中的结构和功能作用。我们之前 产生了纯合的Cngb 1光感受器缺失（X1 KO）小鼠，不仅导致通道的部分丧失， 功能，但也严重的结构扰动，在体内建立，这些蛋白质是必要的， 正常视杆外节ROS盘形态发生和结构完整性。我们假设GARP 2 B亚基上的序列是质膜/盘膜相互作用所必需的。为了验证这一点， 创建了表达N-末端截短的b-亚基（Tb）的转基因小鼠，其上缺乏所有GARP 2序列。 X1 KO背景尽管不存在可溶性GARP 2，但整个b亚基GARP 2区域和仅 一个富含谷氨酸的片段保留在Tb上，存在显著但不完整的结构和功能改变， 救援确定观察到的救援的依据（A.）我们将分析这些小鼠的结构， 组织学、免疫细胞化学、透射电镜和冷冻电镜超微结构分析以及功能 使用ERG、单细胞生理学和现已建立的视网膜打孔制备。X1 KO和X26小鼠 不表达b-亚基，并且在也缺失可溶性GARP的X1 KO中，光响应减弱。 在过表达GARP 2的WT小鼠中，观察到光转导增益显著增加， 证明了GARP 2在调节光转导中的先前未知的作用。（B.）我们假设 GARP2调节视杆细胞暗噪声，也有助于新的缓慢Burnsian适应。为了验证这一点， 将使用我们建立的生理学工具比较WT、GARP 2中的光转导参数 过表达的Tb转基因小鼠和在活化、恢复和活化过程中表达全长b亚基的小鼠。 适应，以进一步定义GARP 2和b亚基在调节光响应中的作用。使用新颖 锌指核酸酶技术，我们建立了GARP 2特异性敲除小鼠， GARP 2外显子和缺失GARP 2 3'-UT区域的潜在亚型。（C.）我们会用这些老鼠 以检验GARP 2是视杆细胞结构和功能所必需的这一假设。我们将执行结构 分析以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，以补充目标A中提出的研究。和B。并直接检测GARP 2在视杆细胞中的作用， 功能拟议的研究记录了一个全面的计划，以确定结构的重要性， GARP区域的功能，并确定GARP 2在调节视杆细胞光反应中的作用。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目标。