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

描述(申请人提供)：NKCC1是一个电子中性型阳离子转运蛋白，属于SLC12A，一个进化的古老基因家族。这种转运蛋白是从细菌到人类的，因此已经进化成履行多种细胞功能。NKCC1表达于呼吸道、肠、唾液腺、汗腺等氯离子分泌上皮细胞的基侧膜或K+分泌上皮细胞的基底膜上。血管纹(内耳)，分别参与Cl-或K+的跨上皮运动。在神经元中，NKCC1参与调节细胞内氯离子，从而影响 GABA能和甘氨酸能神经传递。共转运蛋白也是细胞水合的关键因素，因为它被细胞水分的丧失激活，从而参与细胞体积的维持和调节。最近，在一名12岁的患者中发现了编码NKCC1的基因SLc12a2外显子22的11个碱基的从头缺失。该缺失截断了辅转运体的40%的羧基末端尾巴。我们推测突变的共转运蛋白能够表达，并对野生型共转运蛋白产生显性-负性作用，或对细胞代谢产生毒性作用。在这个应用中，我们建议1)检测截短的共转运蛋白在异源表达系统中的功能；以及2)检测突变的共转运蛋白在从患者分离和培养的细胞中的表达和功能；并建立一个概括这种突变的小鼠模型。第一个目标将包括研究截短型和野生型共转运蛋白在非洲爪哇卵母细胞和HEK293细胞中的表达、运输、功能和相互作用。这项研究将使我们能够详细地表征突变的共转运体，并了解共转运体的羧基末端尾巴的分子作用。将尝试评估是否有可能挽救截断共转运体的功能。第二个目的将研究突变转运体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一个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NKCC1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.