NCC and ENaC form a Sodium Transporting Complex

NCC 和 ENaC 形成钠转运复合物

• 批准号: 9281535
• 负责人: Robert S Hoover
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-10-01 至 2019-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9281535
• 关键词: Address; Adult; Aldosterone; Animals; Binding; Biotinylation; Blood Pressure; Carrier Proteins; Cell model; Cells; Co-Immunoprecipitations; Complex; Confocal Microscopy; Data; Development; Diagnosis; Disease; Distal; Distal convoluted renal tubule structure; Diuretics; Electron Microscopy; Fluorescence Resonance Energy Transfer; Gene Silencing; Hyperactive behavior; Hypertension; Hypotension; Immunoblotting; Immunohistochemistry; In Vitro; Kidney; Lead; Lysine; Mammalian Cell; Mass Spectrum Analysis; Measurement; Mediating; Methodology; Methods; Modeling; Modernization; Mus; Nephrons; Operative Surgical Procedures; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; Play; Population; Property; Proteins; Regulation; Reporting; SLC12A3 gene; Sgk protein; Signal Transduction; Societies; Sodium; Sodium Chloride; Surface; Systemic blood pressure; Transfection; United States; Veterans; apical membrane; base; blood pressure regulation; epithelial Na+ channel; health administration; implantation; in vivo; inhibitor/antagonist; knock-down; osmotic minipump; particle; patch clamp; protein expression; protein transport; public health relevance; response; salt balance; small hairpin RNA; symporter; thiazide; uptake

 DESCRIPTION (provided by applicant): The sodium chloride co-transporter (NCC) and the epithelial sodium channel (ENaC) are two key sodium transporting proteins in the distal tubule of the mammalian kidney. Hyperactivity of either protein results in hypertension and hypoactivity results in hypotension. These two proteins are co-expressed in the second part of the distal convoluted tubule (DCT2) and are regulated by many of the same proteins. However, associations between the two have not been investigated. Preliminary data indicates that the proteins associate in a sodium transporting complex and co-localize in the apical membrane of the DCT2. Investigating the relationship between ENaC and NCC in this key nephron segment has the potential to significantly alter our understanding of salt transport in the mammalian kidney. We now propose to examine the interaction of NCC with each subunit of ENaC, examine the functional implications of this interaction and investigate association-dependent alterations in regulation of these proteins by aldosterone. Co-localization (in vivo and in vitro), native association, and proximity of NCC and each ENaC subunit will be examined. The ENaC subunit(s) that are critical for binding to NCC will also be determined. These studies will define the association between NCC and each ENaC subunit. Then the impact of this association on function, protein expression, surface expression and response to inhibitors of both sodium transporters will be investigated. These studies will determine the functional implications of this interaction. Then aldosterone-induced changes in function, protein expression and surface expression of NCC and ENaC will be examined in the absence or presence of the other. The aldosterone-induced phosphorylation of NCC with and without ENaC will also be investigated. Additionally the association of NEDD4-2 with NCC in the absence and presence of ENaC subunits will be determined. These studies will determine whether the NCC alters the regulation of ENaC by aldosterone and whether any of the ENaC subunits alter the regulation of NCC by aldosterone. A variety of methods will be utilized to address these issues. Function of NCC will be assessed by thiazide-sensitive 22Na+ uptakes in mouse distal convoluted tubule cells (mDCT15). For measurement of ENaC function, Dr. Doug Eaton's lab will use single channel whole-cell patch clamping to assess ENaC function. The proximity of these proteins will be assessed utilizing Fluorescence Resonance Energy Transfer (FRET). Signal will be detected with confocal microscopy. Native association will be examined through the use of Blue Native PAGE. Electron Microscopy will be used to examine association under aldosterone stimulation. Lentiviral shRNA transduction particles will be used to knockdown protein expression in mDCT15 cells. Small animal surgeries with implantation of osmotic minipumps will be used to examine the effects of aldosterone on the NCC-ENaC association on the organismal level. Additionally standard methodologies such as immunoblotting, SDS-PAGE, biotinylation, transfection, co- immunoprecipitation and immunohistochemistry will be used.

 描述（由申请人提供）： 氯化钠协同转运蛋白（NCC）和上皮钠通道（ENaC）是哺乳动物肾脏远端小管中两个关键的钠转运蛋白。任何一种蛋白质的过度活跃都会导致高血压，而过度活跃则会导致低血压。这两种蛋白质在远曲小管的第二部分 (DCT2) 中共表达，并受到许多相同蛋白质的调节。然而，两者之间的关联尚未得到调查。初步数据表明，这些蛋白质在钠转运复合物中结合，并共同定位在 DCT2 的顶膜中。研究这一关键肾单位部分中 ENaC 和 NCC 之间的关系有可能显着改变我们对哺乳动物肾脏中盐转运的理解。我们现在建议检查 NCC 与 ENaC 每个亚基的相互作用，检查这种相互作用的功能含义，并研究醛固酮调节这些蛋白质的关联依赖性改变。 将检查 NCC 和每个 ENaC 亚基的共定位（体内和体外）、天然关联和邻近性。还将确定对于与 NCC 结合至关重要的 ENaC 亚基。这些研究将定义 NCC 和每个 ENaC 亚基之间的关联。然后将研究这种关联对两种钠转运蛋白的功能、蛋白质表达、表面表达和对抑制剂的反应的影响。这些研究将确定这种相互作用的功能影响。然后，在另一种不存在或存在的情况下，将检查醛固酮诱导的 NCC 和 ENaC 的功能、蛋白质表达和表面表达的变化。还将研究使用和不使用 ENaC 时醛固酮诱导的 NCC 磷酸化。此外，将确定在 ENaC 亚基不存在和存在的情况下 NEDD4-2 与 NCC 的关联。这些研究将确定 NCC 是否会改变醛固酮对 ENaC 的调节，以及是否有任何 ENaC 亚基会改变醛固酮对 NCC 的调节。 将采用多种方法来解决这些问题。 NCC 的功能将通过小鼠远曲小管细胞 (mDCT15) 中噻嗪类敏感的 22Na+ 摄取来评估。为了测量 ENaC 功能，Doug Eaton 博士的实验室将使用单通道全细胞膜片钳来评估 ENaC 功能。这些蛋白质的接近度将利用荧光共振能量转移（FRET）进行评估。将用共焦显微镜检测信号。原生关联将通过使用 Blue Native PAGE 进行检查。电子显微镜将用于检查醛固酮刺激下的关联。慢病毒 shRNA 转导颗粒将用于敲低 mDCT15 细胞中的蛋白表达。植入微型渗透泵的小动物手术将用于在有机体水平上检查醛固酮对 NCC-ENaC 关联的影响。此外，还将使用免疫印迹、SDS-PAGE、生物素化、转染、免疫共沉淀和免疫组织化学等标准方法。