Investigating the Action and Physiological Role of Slc4a11 in the Cornea

项目总结/摘要 在美国，与角膜混浊相关的视力丧失影响约300，000人。最厚的层 角膜的主要成分是基质;一种透明的胶原蛋白基质，可以从眼房水中吸取液体。一 基质和房水之间的角膜内皮细胞层对此几乎没有提供物理屏障 液体运动，但主动地将渗透压物质（碳酸氢盐乳酸盐）和因此的液体从基质中挤出回到基质中。 房水内皮功能障碍使液体在基质中积聚，使基质变形， 使其散射光。角膜移植仍然是所有角膜疾病的最终治疗方法， 但我们在疾病的遗传学和内皮细胞的机制的理解进展， 功能已经为生物工程角膜和微创治疗的发展铺平了道路。然而，在这方面， 我们仍然没有完全了解内皮细胞是如何工作的。直到最近， 遗传连锁研究揭示了膜转运蛋白SLC 4A 11对 内皮健康我们最近已经确定，SLC 4A 11是一个pH敏感的H+导体。我们假设 SLC 4A 11作为内皮细胞pH的主要调节剂，感应和对抗内皮细胞中的pH干扰， pH敏感性渗透转运蛋白NBCe 1-B（Na+/2 HCO-协同转运蛋白）和MCT 1（H+/乳酸盐-）附近 3 共转运蛋白）。在我们建议的第一部分中，我们在非洲爪蟾卵母细胞中表达SLC 4A 11，并使用 离子选择性微电极和电压钳电路的组合，以确定动力学 控制SLC 4A 11动作的参数，这些参数是在数学模型中实现SLC 4A 11所必需的 内皮液体转运我们还研究了SLC 4A 11对NBCe 1-B和MCT 1作用的影响。在 在我们的第二部分，我们比较了Slc 4a 11基因敲除小鼠和 我们的新型Nbce 1b基因敲除小鼠研究内皮液体转运紊乱的长期影响。最后， 我们研究了一种新的治疗策略，使用 眼药水。 我们的研究计划的长期目标是了解内皮细胞如何执行和平衡液体 运输与pH稳态，并了解SLC 4A 11对角膜健康和疾病的重要性。 该项目与NEI角膜疾病项目的使命一致，应用来自 基础科学发现，以了解角膜的生理学和角膜的治疗 疾病

Project Summary/Abstract Vision loss associated with corneal clouding affects ~300,000 individuals in the United States. The thickest layer of the cornea is the stroma; a transparent collagen matrix that tends to draw fluid from the aqueous humor. A layer of corneal endothelial cells between the stroma and aqueous humor provides little physical barrier to this fluid movement but actively extrudes osmolytes (bicarbonate lactate), and thus fluid, from the stroma back into the aqueous humor. Endothelial dysfunction allows fluid to accumulate in the stroma, distorting the matrix and causing it to scatter light. Corneal transplant has remained the definitive treatment for all corneal diseases for over a century, but advances our in our understanding of the genetics of disease and mechanisms of endothelial function have paved the way for development of bioengineered corneas and less invasive treatments. However, we still do not have a complete understanding of how the endothelium works. It is only relatively recently that genetic-linkage studies revealed the critical importance of the membrane transport protein SLC4A11 to endothelial health. We have recently established that SLC4A11 is a pH-sensitive H+ conductor. We hypothesize that SLC4A11 acts as a master regulator of endothelial cell pH, sensing and countering pH disturbance in the vicinity of pH-sensitive osmolyte transporters NBCe1-B (Na+/2HCO − cotransporter) and MCT1 (H+/lactate− 3 cotransporter). In the first part of our proposal we express SLC4A11 in Xenopus oocytes and, using a combination of ion-selective microelectrodes and voltage-clamp circuitry in order to determine the kinetic parameters governing SLC4A11 action that are necessary to implement SLC4A11 in mathematical models of endothelial fluid transport. We also examine the influence of SLC4A11 on NBCe1-B and MCT1 action. In the second part of our proposal we compare the progression of corneal disease signs in Slc4a11-knockout mice and our novel Nbce1b-knockout mouse to study the long-term effects of disturbed endothelial fluid transport. Finally, we investigate a novel therapeutic strategy to de-swell the edematous corneas of Slc4a11-knockout mice using eye drops. The long term goal of our research program is to understand how endothelial cells perform and balance fluid transport with pH homeostasis and to understand the importance of SLC4A11 to corneal health and disease. This project is aligned with the mission of the Corneal Disease Program of the NEI, to apply knowledge from basic science discoveries to the understanding of the physiology of the cornea and to the treatment of corneal disease.