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

描述(申请人提供)：先天性遗传性内皮细胞营养不良症(CHED)因角膜混浊导致视力受损。隐性的CHED(CHED2)以及某些类型的Fuch型内皮营养不良和哈博扬综合征最近被证明与编码BTR1/NaBC1的SLC4A11基因突变有关，BTR1/NaBC1是重碳酸盐转运体家族的成员。该蛋白在角膜中的功能和亚细胞定位尚不清楚，目前还没有合适的动物模型来研究SLC4A11突变引起的CHED。该实验的目的是确定SLC4A11的分子功能及其在角膜中的定位，并建立CHED2的小鼠模型。现有的功能信息表明，SLC4A11可以运输钠和硼[B(OH)4-]，尽管还没有用这种蛋白进行硼运输的实验。具体目的是利用HEK293细胞和角膜内皮细胞系，用质谱仪测定哺乳动物(小鼠、人)SLC4A11是否共转运Na和B。具体目的1B是利用非洲爪哇卵母细胞，在离子选择性微电极上，确定SLC4A11是否共转运Na和HCO3-。在特定目的2中，我们将进行免疫组织化学以确定SLC4a11是否位于小鼠角膜内皮的基底膜或顶膜。在具体目标3中，我们将产生并评估CHED2小鼠模型的表型，该模型是通过将已知导致人类CHED2的点突变(位于人R755Q的位置)引入小鼠SLC4a11而制备的。 与公众健康相关：保持角膜的透明度对良好视力至关重要。多种情况会导致角膜混浊，包括一种称为先天性遗传性内皮细胞营养不良(CHED)的遗传性疾病。某些形式的CHED是一种名为SLC4A11的运输蛋白突变的结果，其功能尚未很好地确定。本项目的目的是确定SLC4A11的功能，并建立CHED小鼠模型。

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.