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

第一个已知人类 SLC12A2 基因突变的分子和功能特征

• 批准号: 9095807
• 负责人: Eric J Delpire
• 金额: $ 23.7万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-06 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9095807
• 关键词: 12 year old; Address; Affect; Age; Alleles; Amino Acids; Antibodies; Apnea; Aspartic Acid; Bacteria; Base Pairing; Biology; CRISPR/Cas technology; Cations; Cell Volumes; Cell physiology; Cells; Cellular Metabolic Process; Childhood; Chloride Ion; Chlorides; Chronic; Clinical; Cyclic Neutropenia; Dehydration; Diagnosis; Dilated Cardiomyopathy; Dimerization; Disease; Dominant-Negative Mutation; Epithelium; Exons; Failure; Fatigue; Fibroblasts; Functional disorder; Gene Family; Generations; Genes; Genetic; Health; Homeostasis; Human; Hydration status; Hypoglycemia; Intestines; Introns; Ions; Knockout Mice; Laboratories; Labyrinth; Learning; Left ventricular structure; Lymphocyte; Maintenance; Measurement; Mediating; Membrane Transport Proteins; Metabolic; Methylation; Mitochondrial Diseases; Molecular; Molecular Cloning; Mothers; Movement; Mus; Muscle Fatigue; Mutation; NADH; Neurons; Obstruction; Oocytes; Oral Ulcer; Patients; Phenotype; Phenylalanine; Phosphotransferases; Production; Property; Protein Isoforms; Proteins; Recording of previous events; Regulation; Research; Role; Salivary Glands; Seizures; Stria Vascularis; Structure; Sweat Glands; Symptoms; System; Tail; Toxic effect; Translations; United States National Institutes of Health; Variant; Vomiting; Work; Xenopus laevis; base; basolateral membrane; cell water; exercise intolerance; insight; mitochondrial dysfunction; mouse model; mutant; neurotransmission; older patient; overexpression; premature; programs; protein expression; trafficking

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.

描述（由申请人提供）：NKCC1是一种电中性阳离子-氯化物协同转运蛋白，属于SLC12A，一个进化古老的基因家族。这种转运蛋白从细菌到人类都有发现，因此已经进化到能够履行多种细胞功能。 NKCC1表达于气道、肠、唾液腺、汗腺等Cl-分泌上皮细胞的基底外侧膜，或血管纹（内耳）等K+分泌上皮细胞的基底外侧膜上，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 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的关键因素，因为它会因细胞水分的流失而被激活，从而参与细胞体积的维持和调节。最近，在一名 12 岁患者的 NKCC1 编码基因 SLC12A2 的外显子 22 中发现了 11 个碱基的从头缺失。 该缺失截断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被表达并对野生型协同转运蛋白产生显性负效应，或者对细胞代谢产生毒性作用。在本申请中，我们建议1）检查截短的协同转运蛋白在异源表达系统中的功能； 2) 检查从患者体内分离和培养的细胞中突变型协同转运蛋白的表达和功能； 并创建一个重现这种突变的小鼠模型。第一个目标将涉及非洲爪蟾卵母细胞和 HEK293 细胞中截短型和野生型协同转运蛋白的表达、运输、功能和相互作用的研究。 这项研究将使我们能够详细描述突变型协同转运蛋白，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的协同转运蛋白功能。 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，以及从患者体内分离的细胞（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，我们还将创建并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 NKCC1 突变介导的细胞功能障碍并解释患者的临床表现。