Regulation of pHi and Fluid Flux in Corneal Endothelium

描述(申请人提供)：角膜是主要屈光状态 眼睛的元素，因此它必须是光滑和透明的 幻象。透明度受基质水合程度的影响最大 结缔组织。水合作用取决于细胞的代谢活动。 角膜上皮和内皮，然而，是内皮，是 主要负责泵送离子和流体，以维持 基质的稳定水合作用。这个项目的长期目标是 了解角膜内皮是如何完成这项工作的，因此药物治疗 可以被开发来促进角膜的内皮功能，这些角膜已经 因疾病或创伤而受损的。内皮细胞最依赖于HCO3- 因为它的功能。一些与HCO3相关的细胞膜转运机制 已在基底外侧表面(面向 间质)。我们在此应用程序中的重点是建立 一些重要的HCO3-和流体运输机制的作用， 包括：1)基侧Na+：HCO3-共转运体；2)根尖膜 结合碳酸酐酶(CA-IV)，3)阴离子交换器，4)囊 纤维化跨膜调节剂(CFTR)。为了建立这些角色，HCO3-熔剂 而角膜维持其厚度的能力(即水合作用)将 在传输机制被抑制的条件下被测量 药理、蛋白质分解或反义基因敲除方法。地点 其中的运输蛋白将通过免疫荧光和 膜蛋白分离技术。CA-IV在CO2/HCO3-中的作用 交通是最耐人寻味的。CA-IV的功能将得到更深入的探讨 通过测定其活性对根尖表面pH值的影响。我们建议 利用pH敏感绿色荧光蛋白构建新型表面pH探针 (绿色荧光蛋白)，定向到顶膜，并拴在细胞外 表面用糖基磷脂酰肌醇(GPI)锚定。最后，我们将使用 RT-PCR和测序鉴定新的HCO3相关转运蛋白，然后 表征它们的功能，并确定它们的表达，例如，一些阴离子 交易所，取决于CFTR的存在。

DESCRIPTION (provided by applicant): The cornea is the primary refractive element of the eye and therefore it must be smooth and transparent for good vision. Transparency is most affected by the hydration level of the stromal connective tissue. Hydration is dependent on the metabolic activities of the corneal epithelium and endothelium, however it is the endothelium that is primarily responsible for pumping ions and fluid that maintains the steady-state hydration of the stroma. The long-term goal of this project is to understand how the corneal endothelium does this job, so that medical therapies could be developed to boost endothelial function in corneas that have been compromised by disease or trauma. The endothelium is most dependent on HCO3- for its function. A number of cell membrane HCO3- related transport mechanisms have been identified and characterized on the basolateral surface (facing the stroma) of the endothelium. Our focus in this application is to establish the roles of some prominent transport mechanisms for HCO3- and fluid transport, including: 1) the basolateral Na+: HCO3- co-transporter, 2) the apical membrane bound carbonic anhydrase (CA-IV), 3) the anion exchanger and 4) the Cystic Fibrosis Transmembrane Regulator (CFTR). To establish these roles, HCO3- fluxes and the ability of the cornea to maintain its thickness (i.e., hydration) will be measured under conditions where a transport mechanism is inhibited by pharmacological, proteolytic or antisense knockdown approaches. The locations of these transport proteins will be confirmed by immunofluorescence and membrane protein separation techniques. The role of CA-IV in CO2/HCO3- transport is most intriguing. The function of CA-IV will be probed more deeply by measuring the effect of its activity on the apical surface pH. We propose to construct a novel surface pH probe using pH sensitive Green Fluorescent Protein (GFP) that is directed to the apical membrane and tethered to the extracellular surface by a glycosylphosphatidylinositol (GPI) anchor. Lastly, we will use RT-PCR and sequencing to identify new HCO3- related transport proteins, then characterize their function and determine if their expression, e.g., some anion exchangers, is dependent on the presence of CFTR.