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）是主要的视网膜神经递质，而不是 可以在各种视网膜受体上发挥作用。 其中一些受体 是配体门控离子通道，可能类似于GABA/A受体 在大脑中突出表达。 互补的DNA编码14 在4个主要类别中安排的不同GABA/A受体亚基（Alpha， Beta，Gamma和Delta）已从大脑中克隆。 我们有 最近描述了通过cDNA发现的GABA RHO子单位类别 克隆。 视网膜中的GABA RHO1表达至少比 它在任何其他组织中的表达。 该视网膜特异性受体 亚基具有独特的结构特征，并将相关性 具有独特的药理学和生理学 特征。 从视网膜cDNA分离的第二个Rho亚基 库在视网膜RNA中以较低的水平表示，但显示 与Rho1亚基类似的结构和功能功能。 这 应用建议研究这一点的分子生物学 不同类别的视网膜GABA受体亚基并探索它们 通过追求以下目的，可能在视网膜病理学中作用： 1）分析RHO的基因组组织和相对取向 基因包括对推定启动子区域的详细检查 每个基因。 2）确定GABA RHO1或RHO2中的突变是否引起常染色体 黄斑营养不良的主要形式，最近已映射到 与Rho基因相同的染色体区域（6q13-Q21）。 3）确定是否有其他成员的形式 GABA RHO子单元家族的PCR在视网膜中表达 使用源自Rho cDNA的引物对视网膜cDNA进行扩增 序列。 4）执行结构/功能分析以确定哪些部分 GABA RHO cDNA序列赋予了药理学和生理学 该亚基类的属性特征。 5）确定哪种视网膜细胞类型表达GABA RHO亚基 使用RNA的北部和PCR分析，原位杂交和 免疫组织化学技术。