Functional role of transporter Slc4a11 (BTR1/NaBC1) in the cornea

描述（由申请人提供）：先天性遗传性内皮营养不良（CHED）导致角膜混浊导致视力受损。隐性CHED （CHED2）以及某些类型的富氏内皮营养不良和Harboyan综合征最近被证明与编码BTR1/NaBC1的SLC4A11基因突变有关，BTR1/NaBC1是碳酸氢盐转运蛋白家族的成员。该蛋白在角膜中的功能和亚细胞位置尚不清楚，目前也没有足够的动物模型来研究SLC4A11突变引起的CHED。本实验的目的是确定SLC4A11的分子功能及其在角膜中的定位，并建立小鼠CHED2模型。现有的功能信息表明，SLC4A11转运Na+和硼酸盐[B(OH)4-]，尽管没有进行过用该蛋白转运硼的实验。特异性目的1A是使用HEK293细胞和角膜内皮细胞系，通过质谱法确定哺乳动物（小鼠，人）SLC4A11是否共同运输Na+和硼。具体的Aim 1B是使用爪蟾卵母细胞，使用离子选择性微电极，确定SLC4A11是否共同运输Na+和HCO3-。在特异性目的2中，我们将进行免疫组织化学来确定Slc4a11是否位于小鼠角膜内皮的基底外侧膜或顶膜。在Specific Aim 3中，我们将生成并评估通过将已知导致人类CHED2的点突变（位于人类R755Q的位置）引入小鼠Slc4a11制备的CHED2小鼠模型的表型。

DESCRIPTION (provided by applicant): Congenital hereditary endothelial dystrophy (CHED) causes impaired vision resulting from opacification of the cornea. The recessive form of CHED (CHED2) as well as some types of Fuch's endothelial dystrophy and Harboyan syndrome were recently shown to be associated with mutations in the SLC4A11 gene that encodes BTR1/NaBC1, a member of the bicarbonate transporter family. The function and subcellular location of this protein in cornea are unknown, and there is currently no adequate animal model for the study of CHED caused by SLC4A11 mutations. The goals of the proposed experiments are to determine the molecular function of SLC4A11 and its localization in the cornea, and to generate a mouse model for CHED2. The available functional information suggests that SLC4A11 transports Na+ and borate [B(OH)4- ], although no boron transport experiments with this protein have been performed. Specific Aim 1A is to use HEK293 cells and a corneal endothelial cell line, with mass-spectrometry, to determine whether mammalian (mouse, human) SLC4A11 cotransports Na+ and boron. Specific Aim 1B is to use Xenopus oocytes, with ion-selective microelectrodes, to determine whether SLC4A11 cotransports Na+ and HCO3-. In Specific Aim 2 we will perform immunohistochemistry to determine whether Slc4a11 is located in the basolateral or apical membrane of the mouse corneal endothelium. In Specific Aim 3 we will generate and evaluate the phenotype of a mouse model of CHED2 prepared by introducing, to mouse Slc4a11, a point mutation (in the position of human R755Q) that is known to cause CHED2 in humans. PUBLIC HEALTH RELEVANCE: Maintenance of transparency of the cornea is essential for good vision. A variety of conditions can cause the cornea to become cloudy, including an inherited condition known as congenital hereditary endothelial dystrophy (CHED). Some forms of CHED are a result of mutations in a transport protein known as SLC4A11, the function of which has not been well established. The goals of this project are to determine the function of SLC4A11 and to establish a mouse model of CHED.