NCC and ENaC form a Sodium Transporting Complex

 描述(由申请人提供)： 氯化钠共转运蛋白(NCC)和上皮性钠通道(ENaC)是哺乳动物肾脏远端小管上皮细胞中两种关键的钠转运蛋白。任何一种蛋白质的过度活动都会导致高血压，而活动不足会导致低血压。这两个蛋白在远曲小管的第二部分(DCT2)共表达，并由许多相同的蛋白调控。然而，这两者之间的关联尚未得到调查。初步数据表明，这些蛋白结合在钠转运复合体中，并共同定位于DCT2的顶膜。研究ENaC和NCC在这一关键肾单位段的关系可能会显著改变我们对哺乳动物肾脏中盐运输的理解。我们现在建议研究NCC与ENaC每个亚基的相互作用，研究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相关性变化。将检查NCC和每个ENaC亚基的共定位(体内和体外)、天然关联和接近。还将确定与NCC结合至关重要的ENaC亚单位(S)。这些研究将确定NCC和每个ENaC亚单位之间的联系。然后，将研究这种关联对两种钠转运蛋白的功能、蛋白表达、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含义。然后，在没有或存在另一种细胞的情况下，研究醛固酮对NCC和ENaC功能、蛋白表达和表面表达的影响。此外，还将研究有无ENaC对醛固酮诱导的NCC的磷酸化作用。此外，将确定在没有和存在ENaC亚基的情况下NEDD4-2与NCC的关联。这些研究将确定NCC是否改变了醛固酮对ENaC的调节，以及是否有任何ENaC亚基改变了醛固酮对NCC的调节。将利用各种方法来解决这些问题。NCC的功能将通过小鼠远端曲管细胞(MDCT15)对噻嗪敏感的22Na+摄取来评估。对于ENaC功能的测量，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。这些蛋白质的接近程度将利用荧光共振能量转移(FRET)进行评估。信号将用共聚焦显微镜检测。本地关联将通过使用蓝色本地页面进行检查。电子显微镜将被用来检查在醛固酮刺激下的关联。慢病毒shRNA转导颗粒将用于敲除mDCT15细胞中的蛋白质表达。植入渗透性微泵的小动物手术将被用来检验醛固酮在生物水平上对NCC-ENaC协会的影响。此外，还将使用免疫印迹、SDS-PAGE、生物素化、转染法、免疫共沉淀法和免疫组织化学等标准方法。

 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.