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: 9090247
• 负责人: STEVEN J PITTLER
• 金额: $ 3万
• 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/9090247
• 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; Glutamates; Glutamic Acid; Goals; Grant; Health; 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; base; design; functional loss; genome editing; immunocytochemistry; in vivo; mouse genome; mouse model; novel; overexpression; programs; protein structure function; retinal rods; tool; transmission process; zinc finger nuclease

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.

描述（由申请人提供）：本研究项目的长期目标是了解视杆细胞中cGMP门控阳离子通道蛋白质结构/功能关系，并将此理解转化为视网膜色素变性（RP）和相关疾病患者的治疗。我们将把我们的研究重点放在通道β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基因座编码，检查GARP区域在三个特定目标的结构和功能作用。我们以前产生了一个纯合子Cngb 1光感受器无效（X1 KO）小鼠不仅导致部分通道功能的丧失，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杆外节ROS盘形态发生和结构完整性。我们假设β-亚基上的GARP 2序列是质膜/盘膜相互作用所必需的。为了测试这一点，我们在X1 KO背景下创建了表达N末端截短β-亚基（T β）的转基因小鼠，该亚基缺乏所有GARP 2序列。尽管缺乏可溶性GARP 2，整个β-亚基GARP 2区域和T β上仅剩下一个富含谷氨酸的片段，但存在显著但不完全的结构和功能拯救。确定观察到的救援的依据（A.）我们将使用组织学、免疫细胞化学和透射电镜和冷冻电镜的超微结构分析对这些小鼠进行结构分析，并使用ERG、单细胞生理学和现在建立的视网膜打孔制备进行功能分析。在不表达β-亚基的X1 KO和X26小鼠中，以及在缺失可溶性GARP的X1 KO小鼠中，光反应减弱。在过表达GARP 2的WT小鼠中，观察到光转导增益的显著增加，证明GARP 2在调节光转导中的先前未知的作用。（B.）我们假设GARP 2调节视杆细胞暗噪声，也有助于新的缓慢Burnsian适应。为了测试这一点，我们将使用我们建立的生理工具来比较WT，GARP 2过表达，T β转基因小鼠和表达全长β亚基的小鼠在激活，恢复和适应过程中的光转导参数，以进一步确定GARP 2和β亚基在调节光反应中的作用。使用新的锌指核酸酶技术，我们建立了GARP 2特异性敲除小鼠，其缺失了最后一个独特的GARP 2外显子和缺失GARP 2 3 '-UT区域的潜在亚型。（C.）我们将使用这些小鼠来测试GARP 2是杆中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中提出的研究。和B。并直接检测GARP 2在视杆细胞功能中的作用。拟议的研究文件一个全面的计划，以确定GARP区域的结构重要性，并确定GARP 2在调节视杆细胞光反应的作用。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。