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: 2711163
• 负责人: NANCY J MANGINI
• 金额: $ 16.67万
• 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/2711163
• 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.

体液中CA 2+浓度（[CA 2 +]）的显著变化 周围的光感受器（视网膜下腔或SRS） 亮/暗过渡。 因为Ca 2+影响感光细胞 功能，SRS [Ca 2 +]必须严格调节。 虽然它是 已知视网膜色素上皮中转运活性 (RPE)支持这种调节作用， 尚未被确认。 所有细胞只有两种机制， 可跨质膜输出Ca 2+：Na=：Ca 2 + 交换器（NCXs）和质膜Ca 2 + ATP酶 （PMCAs）。 除了本申请中的研究外， 蛋白质和编码蛋白质的基因 在人类视网膜色素上皮中。 该项目针对这两种Ca 2+运输 proteins. 计划研究的基本假设 在RPE中，PMCA和NCX的联合活动 调节SRS [Ca 2 +]的光依赖性变化， 这些Ca 2+转运蛋白的功能受损将导致 视力受损 多基因和选择性剪接 初级mRNA转录物控制组织特异性表达 以及PMCA和NCX蛋白（称为同种型）的调节。 因此，检验这些假设需要确定PMCA， 通过RPE表达的NCX蛋白，并测定它们在 RPE生理学。 这就是这个项目的目标。 实验 使用来自人类供体眼睛的新鲜分离的RPE细胞和组织 其他动物来源。 该方法是在3个层次上，分子 （AIM 1），结构（AIM 2）和生物化学/功能（AIM 3）。 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。 使用免疫电镜的结构研究将识别膜 这些Ca 2+转运蛋白的位置（顶侧与基底侧） 在天然RPE组织中。 生化研究将确定RPE PMCA和NCX蛋白（免疫印迹），并检查其 新鲜分离的RPE细胞的基本特性。 功能研究 将检查跨上皮钙转运（离子通量研究）， 新鲜分离的RPE中[Ca 2 +]I（Ca 2+成像）的调节 组织. 离子取代和选择性抑制剂将用于 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细胞生物学的新举措。 PMCA和NCX的分子表征已被证实。 分别在红细胞和可兴奋细胞中进行了充分研究。 拟议的研究将把研究结果扩展到人类RPE， 一般有助于上皮钙离子转运领域。 计划中的研究将确定 在RPE中的PMCA和NCX活动，并将有助于确定 可能由PMCA受损引起的眼部病变，或 NCX功能。