NCC and ENaC form a Sodium Transporting Complex

 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的两个关键钠转运蛋白。任何一种蛋白质的过度活性都会导致高血压，而低活性则会导致低血压。这两种蛋白质在远曲小管的第二部分（DCT 2）中共表达，并且由许多相同的蛋白质调节。然而，两者之间的联系尚未调查。初步数据表明，蛋白质关联的钠转运复合物和共定位在顶端膜的DCT 2。研究ENaC和NCC在这个关键肾单位段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的每个亚基的相互作用，检查这种相互作用的功能意义，并研究醛固酮对这些蛋白质的调节中的关联依赖性改变。 将检查NCC和每个ENaC亚基的共定位（体内和体外）、天然缔合和邻近性。还将确定对结合NCC至关重要的ENaC亚基。这些研究将确定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，在存在或不存在另一种的情况下，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。还将研究在有和没有ENaC的情况下醛固酮诱导的NCC磷酸化。此外，将确定在不存在和存在ENaC亚基的情况下NEDD4 - 2与NCC的关联。这些研究将确定NCC是否改变醛固酮对ENaC的调节，以及是否有任何ENaC亚基改变醛固酮对NCC的调节。 将采用各种方法来解决这些问题。将通过小鼠远曲小管细胞（mDCT 15）中噻嗪敏感性22 Na+摄取来评估NCC的功能。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。将利用荧光共振能量转移（FRET）评估这些蛋白质的接近度。将用共聚焦显微镜检测信号。将通过使用蓝色非变性PAGE检查非变性结合。将使用电子显微镜检查醛固酮刺激下的相关性。慢病毒shRNA转导颗粒将用于敲低mDCT 15细胞中的蛋白质表达。将使用植入渗透微型泵的小动物手术来检查醛固酮在生物体水平上对NCC-ENaC缔合的影响。此外，将使用标准方法，如免疫印迹、SDS-PAGE、生物素化、转染、免疫共沉淀和免疫组织化学。

 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.