Investigating the Action and Physiological Role of Slc4a11 in the Cornea

项目摘要/摘要 在美国，与角膜云雾相关的视力损失影响了大约30万人。最厚的一层 角膜的基质是一种透明的胶原蛋白基质，它往往会从房水中吸出液体。一个 基质和房水之间的角膜内皮细胞层对此几乎没有物理障碍 液体运动但主动地将渗透物质(乳酸碳酸氢钠)挤出，从而将液体从基质中排回 房水的体液。内皮功能障碍使液体积聚在基质中，扭曲基质和 导致它散射光线。角膜移植仍然是所有角膜疾病的最终治疗方法 一个多世纪以来，但促进了我们对疾病的遗传学和内皮细胞的机制的理解 功能已经为开发生物工程角膜和更少侵入性治疗铺平了道路。然而， 我们仍然没有完全了解内皮细胞是如何工作的。只是在最近才发现 遗传连锁研究揭示了膜转运蛋白SLC4A11对 内皮健康。我们最近确定SLC4A11是一种对pH敏感的H导体。我们假设 SLC4A11作为内皮细胞pH的主要调节器，感知和对抗血管内皮细胞的pH紊乱 PH敏感渗透压转运体NbCe1-B(Na/2HCO−共转运体)和MCT1(H/乳酸−)附近 3. 共转运体)。在我们建议的第一部分中，我们在非洲爪哇卵母细胞中表达SLC4A11，并使用 离子选择微电极与电压钳制电路相结合测定动力学 控制在数学模型中实现SLC4A11所必需的SLC4A11操作的参数 内皮细胞液体运输。我们还检测了SLC4A11对NBCe1-B和MCT1作用的影响。在 在我们的建议的第二部分，我们比较了SLC4a11基因敲除小鼠和SLC4a11基因敲除小鼠的角膜疾病症状的进展 我们的新型Nbce1b基因敲除小鼠用于研究内皮液体转运障碍的长期影响。最后， 我们研究了一种新的治疗策略来消肿SLC4a11基因敲除小鼠的水肿性角膜 滴眼液。 我们研究计划的长期目标是了解内皮细胞如何运行和平衡液体 在pH动态平衡的情况下转运，并了解SLC4A11对角膜健康和疾病的重要性。 该项目与NEI的角膜疾病计划的使命相一致，以应用来自 对了解角膜生理学和治疗角膜的基础科学发现 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.