Molecular and Functional characterization of the first known human mutation of the SLC12A2 gene

描述（由申请人提供）：NKCC 1是一种电中性阳离子-氯离子协同转运蛋白，属于进化古老基因家族SLC 12 A。这种转运蛋白从细菌到人类都有，因此已经进化到实现多种细胞功能。 NKCC 1表达于分泌Cl-的上皮细胞（如气道、肠、唾液腺、汗腺等）或分泌K+的上皮细胞（如 血管纹（内耳），分别参与Cl-或K+的跨上皮运动。在神经元中，NKCC 1参与调节细胞内Cl-，从而影响 GABA能和甘氨酸能神经传递。 协同转运蛋白也是细胞水合作用的关键因素，因为它通过细胞水分的损失而被激活，因此参与细胞体积的维持和调节。最近，在一名12岁患者的SLC 12 A2（编码NKCC 1的基因）外显子22中发现了11个碱基的从头缺失。 缺失截短了协同转运蛋白羧基末端尾部的40%。 我们假设突变型协同转运蛋白表达并对野生型协同转运蛋白产生显性负效应，或对细胞代谢产生毒性作用。在本申请中，我们提出1）检查异源表达系统中截短的协同转运蛋白的功能;和2）检查从患者分离和培养的细胞中突变协同转运蛋白的表达和功能;并创建重现该突变的小鼠模型。第一个目标将涉及在非洲爪蟾卵母细胞和HEK 293细胞中研究截短型和野生型协同转运蛋白的表达、运输、功能和相互作用。 这项研究将使我们能够详细描述突变的协同转运蛋白，并了解协同转运蛋白羧基末端尾部的分子作用。 将尝试评估是否有可能挽救截短协同转运蛋白功能。 第二个目标将检查突变转运蛋白对离子和体积稳态的影响，以及从患者分离的细胞（例如成纤维细胞和/或转化的淋巴细胞）的基本代谢特性。 利用CRISPR技术，我们还将创建和研究重现人类突变的小鼠模型。这些研究将使我们能够深入了解NKCC 1突变介导的细胞功能障碍，并解释患者的临床表现。

DESCRIPTION (provided by applicant): NKCC1 is an electroneutral cation‐chloride cotransporter which belongs to SLC12A, an evolutionary ancient gene family. The transporter is found from bacteria to humans and thus has evolved to fulfill a multitude of cellula functions. NKCC1 is expressed on the basolateral membrane of Cl‐ secreting epithelia such as airway, intestine, salivary gland, sweat gland, etc. Or K+ secreting epithelia, e.. stria vascularis (inner ear), where it participates to the transepithelial movement of Cl‐ or K+, respectively. In neurons, NKCC1 is involved in modulating intracellular Cl‐ thereby affecting GABAergic and glycinergic neurotransmission. The cotransporter is also a key factor for cell hydration as it is activated by loss of cell water and consequently participates in the maintenance and regulation of cell volume. Recently, a de novo deletion of 11 bases was found in exon 22 of SLC12A2, the gene encoding NKCC1, in a 12‐year old patient. The deletion truncates 40% of the carboxyl‐terminal tail of the cotransporter. We hypothesze that the mutant cotransporter is expressed and exerts dominant‐negative effects on wild‐type cotransporter, or exerts toxic effects on cell metabolism. In this application, we propose to 1) examine function of the truncated cotransporter in heterologous expression systems; and 2) examine expression and function of the mutant cotransporter in cells that are isolated and cultured from the patient; and create a mouse model recapitulating this mutation. The first aim will involve studies of expression, trafficking, function, interacton, of both truncated and wild‐type cotransporter in Xenopus laevis oocytes and HEK293 cells. This study will allow us to characterize in details the mutant cotransporter and learn about the molecular role of the carboxyl‐terminal tail of the cotransporter. Attempts wil be made to assess whether it is possible to rescue the truncation cotransporter functin. The second aim will examine the impact of the mutant transporter on ions and volume homeostasis, as well as basic metabolic properties of cells isolated from the patient (e.g. fibroblasts and/or transformed lymphocytes). Using CRISPR technology, we will also create and study a mouse model recapitulating the human mutation. These studies will allow us to gain insights into NKCC1 mutant‐mediated cellular dysfunction and explain the clinical presentations of the patient.