ION TRANSPORT MECHANISMS IN RETINAL PIGMENT EPITHELIUM

视网膜色素上皮中的离子传输机制

• 批准号: 2405217
• 负责人: NANCY J MANGINI
• 金额: $ 17.11万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2405217
• 关键词: ION; TRANSPORT; MECHANISMS; RETINAL; PIGMENT

Substantial changes in CA2+ concentration ([CA2+]) in the fluid surrounding photoreceptors (subretinal space or SRS) follow light/dark transitions. Because Ca2+ affects photoreceptor function, SRS [Ca2+] must be tightly regulated. Though it is known that transport activity in the retinal pigment epithelium (RPE) subserves this regulatory role, the specific mechanisms have not been identified. All cells have only 2 mechanisms that can export Ca2+ across the plasma membrane: Na=:Ca2+ exchangers (NCXs) and plasma membrane Ca2+ ATPases (PMCAs). Except for studies in this application, neither of these proteins, nor the genes that encode them, have been characterized in human RPE. This project targets these two Ca2+ transport proteins. The fundamental hypothesis underlying planned studies is that the combined activities of a PMCA and NCX in RPE regulate light-dependent changes in SRS [Ca2+], and that impaired function of either of these Ca2+ transporters will cause impaired vision. Multiple genes and alternative splicing of primary mRNA transcripts govern the tissue-specific expression and regulation of PMCA and NCX proteins (termed isoforms). Thus, testing these hypotheses requires identifying PMCA and NCX proteins as expressed by RPE, and assaying their roles in RPE physiology. These are goals of this project. Experiments use freshly isolated RPE cells and tissue from human donor eyes and other animal sources. The approach is on 3 levels, molecular (AIM1), structural (AIM2), and biochemical/functional (AIM3). Molecular studies will identify PMCA and NCX isoforms expressed in RPE, and will sequence cDNAs that encode them. Structural studies using immunoEM will identify the membrane location (apical vs. basolateral) of these Ca2+ transport proteins in native RPE tissue. Biochemical studies will identify RPE PMCA and NCX proteins (immunoblotting), and examine their basic properties in freshly isolated RPE cells. Functional studies will examine transepithelial Ca2+ transport (ion flux studies), and regulation of [Ca2+]I (Ca2+ imaging) in freshly isolated RPE tissue. Ion substitution and selective inhibitors will be used to evaluate roles of PMCA and NCX activities in these processes. Proposed studies represent a new initiative in RPE cell biology. The molecular characterization of the PMCA and NCX has been well studied in erythrocytes and excitable cells, respectively. Proposed studies will extend findings to human RPE, and will contribute generally to the field of epithelial Ca2+ transport. Planned studies will identify the physiological significance of PMCA and NCX activities in RPE, and will help to identify ocular pathologies that could be caused by impaired PMCA or NCX function.

流体中Ca2+浓度([Ca2+])的实质性变化 周围的光感受器(视网膜下间隙或SRS)跟随 明/暗转换。因为钙离子影响光感受器 功能，SRS[Ca~(2+)]必须受到严格调控。尽管它是 已知视网膜色素上皮中的转运活性 (RPE)从属于这种调节作用，具体的机制 还没有确定身份。所有细胞只有两个机制， 可以跨质膜输出钙离子：Na=：Ca2+ 交换器(NCX)与质膜Ca~(2+)-ATP酶 (PMCA)。除本申请中的研究外，这两项研究 蛋白质，或编码它们的基因，都已经被表征 在人类的RPE中。该项目针对这两个钙离子转运 蛋白质。计划研究背后的基本假设 是PMCA和NCX在RPE中的组合活动 调节SRS[Ca~(2+)]的光依赖性变化，以及 这两种钙离子转运体中的任何一种功能受损都会导致 视力受损。多基因与多基因选择性剪接 初级信使核糖核酸转录本控制组织特异性表达 以及对PMCA和NCX蛋白(称为异构体)的调控。 因此，检验这些假设需要确定PMCA和 NCX蛋白在RPE中的表达，并分析它们在 RPE生理学。这些都是这个项目的目标。实验 使用新鲜分离的人眼RPE细胞和组织 和其他动物来源。这种方法在三个层面上，分子 (AIM1)、结构(AIM2)和生化/功能(AIM3)。 分子研究将鉴定PMCA和NCX亚型 在RPE中表达，并将对编码它们的cDNA进行测序。 使用免疫电子显微镜的结构研究将鉴定膜 这些钙离子转运蛋白的位置(顶端与基底端) 在天然的RPE组织中。生化研究将确定RPE PMCA和NCX蛋白(免疫印迹)，并检测其 新鲜分离的RPE细胞的基本特性。功能研究 将检查跨上皮细胞的钙离子转运(离子通量研究)，以及 新鲜分离的视网膜色素上皮细胞内钙离子成像的调节 组织。离子替代和选择性缓蚀剂将用于 评估PMCA和NCX活动在这些过程中的作用。 拟议的研究代表了RPE细胞生物学的一项新倡议。 PMCA和NCX的分子表征 分别在红细胞和可兴奋细胞中得到了很好的研究。 拟议的研究将把研究结果扩展到人类RPE，并将 在上皮性钙离子转运领域有广泛的贡献。 计划中的研究将确定 RPE中的PMCA和NCX活动，并将有助于确定 PMCA受损或可能导致的眼部病理改变 NCX函数。