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

DESCRIPTION（由申请人提供）：NKCC1是一种电中性阳离子-氯共转运蛋白，属于SLC12A，一个进化的古老基因家族。从细菌到人类都发现了这种转运蛋白，因此它已经进化到实现多种细胞功能。NKCC1表达于分泌Cl -上皮的基底外膜，如气道、肠、唾液腺、汗腺等。或K+分泌上皮，如…血管纹（内耳），在那里它分别参与Cl‐或K+的上皮运动。在神经元中，NKCC1参与调节细胞内Cl -从而影响

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.