MOLECULAR BIOLOGY OF RETINA-SPECIFIC GABA RECEPTORS

视网膜特异性 GABA 受体的分子生物学

• 批准号: 2163114
• 负责人: Garry R Cutting
• 金额: $ 21.66万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2163114
• 关键词: GABA receptor; Xenopus oocyte; animal genetic material tag; animal tissue; autosomal dominant trait; cell type; complementary DNA; fusion gene; gene expression; gene mutation; genetic markers; genetic promoter element; human genetic material tag; human tissue; immunocytochemistry; in situ hybridization; linkage mapping; messenger RNA; molecular biology; northern blottings; nucleic acid sequence; polymerase chain reaction; protein structure; protein structure function; receptor expression; retina; retina disorder; site directed mutagenesis; structural genes; synthetic nucleic acid; tissue /cell culture

Gamma amino butyric acid (GABA) is a major retinal neurotransmitter than can function at a variety of retinal receptors. Some of these receptors are ligand-gated ion channels, probably similar to the GABA/A receptors prominently expressed in the brain. Complementary DNAs encoding 14 different GABA/A receptor subunits arranged in 4 major classes (alpha, beta, gamma, and delta) have been cloned from the brain. We have recently described a GABA rho subunit class discovered through cDNA cloning. GABA rho1 expression in retina is at least 20-fold greater than its expression in any other tissue. This retina-specific receptor subunit has distinctive structural features and avidly associates into homo-oligomeric receptors displaying unique pharmacologic and physiologic characteristics. A second rho subunit isolated from a retinal cDNA library is expressed at lower levels in retina RNA but displays structural and functional features similar to the rho1 subunit. This application proposes to investigate the molecular biology of this distinct class of retinal GABA receptor subunits and to explore their possible roles in retinal pathology by pursuit of the following aims: 1) Analyzing the genomic organization and relative orientation of the rho genes including detailed examination of the putative promoter regions of each gene. 2) Determining whether mutations in GABA rho1 or rho2 cause an autosomal dominant form of macular dystrophy which has recently been mapped to the same chromosomal region (6q13-q21) as the rho genes. 3) Ascertaining whether additional members of alternatively-spliced forms of the GABA rho subunit family are expressed in retina by PCR amplification of retinal cDNA using primers derived from rho cDNA sequences. 4) Performing structure/function analysis to determine which portions of the GABA rho cDNA sequences confer the pharmacologic and physiologic properties characteristic of this subunit class. 5) Ascertaining which retinal cell types express the GABA rho subunits using Northern and PCR analysis of RNA, in situ hybridization and immunohistochemical techniques.

伽马氨基丁酸 (GABA) 是一种主要的视网膜神经递质 可以作用于多种视网膜受体。 其中一些受体 是配体门控离子通道，可能类似于 GABA/A 受体 在大脑中明显表达。 编码 14 的互补 DNA 不同的 GABA/A 受体亚基分为 4 个主要类别（α、 beta、gamma 和 delta）已从大脑中克隆出来。 我们有 最近描述了通过 cDNA 发现的 GABA rho 亚基类 克隆。 视网膜中 GABA rho1 的表达量至少是视网膜中的 20 倍 它在任何其他组织中的表达。 这种视网膜特异性受体 亚基具有独特的结构特征并热衷于结合成 显示独特药理学和生理学的同源寡聚受体 特征。 从视网膜 cDNA 中分离出第二个 rho 亚基 文库在视网膜 RNA 中表达水平较低，但显示 结构和功能特征与rho1亚基相似。 这 申请建议研究该物质的分子生物学 不同类别的视网膜 GABA 受体亚基并探索其 通过追求以下目标，在视网膜病理学中可能发挥作用： 1) 分析rho的基因组组织和相对方向 基因，包括详细检查推定的启动子区域 每个基因。 2) 确定 GABA rho1 或 rho2 突变是否会导致常染色体遗传病 黄斑营养不良的主要形式，最近已被映射到 与 rho 基因相同的染色体区域 (6q13-q21)。 3) 确定选择性剪接形式是否有其他成员 通过 PCR 在视网膜中表达 GABA rho 亚基家族的 使用源自 rho cDNA 的引物扩增视网膜 cDNA 序列。 4) 进行结构/功能分析以确定哪些部分 GABA rho cDNA 序列赋予药理学和生理学作用 该子单元类的属性特征。 5) 确定哪些视网膜细胞类型表达 GABA rho 亚基 使用 Northern 和 PCR 分析 RNA、原位杂交和 免疫组织化学技术。