Gap Junction-mediated Regulation of the V-type H+-ATPase in a Renal Epithelium

描述（由申请人提供）：目前的R03重新提交是由PI （Piermarini）在康奈尔大学Klaus W. Beyenbach教授的指导下，在他获得K01奖的前两年取得的进展所推动的。该研究是PI正在进行的埃及伊蚊（Aedes aegypti）肾（malpiighian）小管分子和生理学研究的延伸，这是研究完全由液泡(V)型H+ atp酶驱动的上皮运输的有价值模型。拟议的R03研究将促进PI的职业发展，使他能够1)保持正在进行的马氏伊蚊小管K01研究的势头，2)为后续的R01申请收集初步数据。拟议的研究也将提供对哺乳动物细胞由v型H+ atp酶激活的见解。这些见解具有重要的生物医学意义，因为现在已知几种重要的病理与v型H+ atp酶的缺陷或功能障碍有关。最后，该研究有可能：(1)改变目前蚊子的肾功能模式，蚊子是人类疾病的重要媒介；(2)提供肾上皮细胞内pH （pHi）的间隙连接介导（细胞间）调节的第一个例子。亲本K01奖的研究目标是破译马氏伊蚊小管“主要”细胞中酸碱运输的分子机制。这些细胞类似于哺乳动物肾集合小管的分泌酸(a)插层细胞，它们富含线粒体，并表达顶端的v型H+ atp酶。基于这些相似性，PI假设主细胞表达基底侧Cl/HCO3阴离子交换器（AE），就像在哺乳动物a-插层细胞中发现的那样。PI现在已经表明，伊蚊AE （AeAE）在邻近的“星状”细胞的基底外侧膜上表达，这些细胞位于主细胞之间。此外，抑制马尔匹氏小管中的AeAE会破坏上皮转运，其方式与主细胞中v型H+ atp酶的抑制一致。因此，星状细胞中AeAE的活性似乎与主细胞中顶端v型H+ atp酶偶联。目前R03拨款申请的目的是确定间隙连接是否介导主细胞和星状细胞之间假定的功能耦合。在目标1中，我将使用药理学方法来验证以下假设：1)主细胞中v型H+ atp酶的活性，2)主细胞中pHi的调节，以及3)由主细胞介导的经上皮液分泌率，通过间隙连接在功能上与星状细胞中的AeAE偶联。在目标2中，我将使用一种功能遗传学方法（RNA干扰）来验证一种假设，即昆虫的缝隙连接蛋白是将主细胞与星状细胞偶联的分子实体。

DESCRIPTION (provided by applicant): The present R03 resubmission is motivated by progress made by the PI (Piermarini) during the first two years of his K01 award under the mentorship of Prof. Klaus W. Beyenbach at Cornell University. The proposed research is an extension of the PI's on-going molecular and physiological studies of the renal (Malpighian) tubules of the mosquito Aedes aegypti, which are a valuable model for studying transepithelial transport that is powered exclusively by the vacuolar (V)-type H+ATPase. The proposed R03 research will enhance the PI's career development by allowing him to 1) maintain the momentum of his on-going K01 research on Aedes Malpighian tubules, and 2) collect preliminary data for a subsequent R01 application. The proposed studies will also provide insights into mammalian cells that are energized by the V-type H+ATPase. Such insights are of significant biomedical interest, because several important pathologies are now known to be associated with defects or dysfunction of the V-type H+ATPase. Lastly, the proposed research has the potential to 1) shift the current paradigm of renal function in mosquitoes, which are important vectors of diseases that afflict humans, and 2) provide the first example of gap junction- mediated (intercellular) regulation of intracellular pH (pHi) in a renal epithelium. The research goal of the parent K01 award is to decipher the molecular mechanisms of acid-base transport in the 'principal' cells of Aedes Malpighian tubules. These cells resemble acid-secreting (a) intercalated cells of the mammalian renal collecting tubule in that they are mitochondrion-rich and express an apical V-type H+ ATPase. Based on these similarities, the PI hypothesized that principal cells express a basolateral Cl/HCO3 anion exchanger (AE), as is found in mammalian a-intercalated cells. The PI has now shown that the Aedes AE (AeAE) is expressed in the basolateral membrane of the neighboring 'stellate' cells, which intercalate between principal cells. Furthermore, inhibiting AeAE in Malpighian tubules disrupts transepithelial transport in a manner consistent with inhibition of the V-type H+ATPase in principal cells. Thus, the activity of AeAE in stellate cells appears to be coupled to the apical V-type H+ATPase in principal cells. The goal of the present R03 grant application is to determine if gap junctions mediate the putative functional coupling between principal and stellate cells. In aim 1, I will use a pharmacological approach to test the hypothesis that the 1) activity of the V-type H+ATPase in principal cells, 2) regulation of pHi in principal cells, and 3) rates of transepithelial fluid secretion mediated by principal cells, are functionally coupled to the AeAE in stellate cells via gap junctions. In aim 2, I will use a functional genetic approach (RNA interference) to test the hypothesis that innexins-the gap-junction proteins of insects-are the molecular entities that couple principal cells to stellate cells.