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。隐性形式的CHED（CHED 2）以及某些类型的Fuch内皮营养不良和Harboyan综合征最近被证明与编码BTR 1/NaBC 1的SLC 4A 11基因突变相关，BTR 1/NaBC 1是碳酸氢盐转运蛋白家族的成员。这种蛋白在角膜中的功能和亚细胞位置尚不清楚，目前还没有足够的动物模型用于研究SLC 4A 11突变引起的CHED。所提出的实验的目标是确定SLC 4A 11的分子功能及其在角膜中的定位，并产生CHED 2的小鼠模型。现有的功能信息表明，SLC 4A 11转运Na+和硼酸盐[B（OH）4- ]，虽然没有硼转运实验与这种蛋白质已经进行。具体目标1A是使用HEK 293细胞和角膜内皮细胞系，用质谱法确定哺乳动物（小鼠，人）SLC 4A 11是否共转运Na+和硼。具体目标1B是使用非洲爪蟾卵母细胞，与离子选择性微电极，以确定是否SLC 4A 11共转运Na+和HCO 3-。在特定目标2中，我们将进行免疫组织化学以确定Slc 4a 11是否位于小鼠角膜内皮的基底膜或顶膜中。在特定目标3中，我们将生成并评估CHED 2小鼠模型的表型，该模型通过向小鼠Slc 4a 11引入已知在人类中引起CHED 2的点突变（在人R755 Q的位置）制备。

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.