Mechanisms of Transport in Proximal and Distal Tubules

描述（由申请人提供）：常染色体显性多囊肾病由编码PC1或2的PKD1或PKD2基因突变引起。尽管越来越清楚PC1在质膜和初级纤毛上表达，但PC2的细胞定位一直存在争议。PC2定位于内质网、质膜和初级纤毛。一种流行的假设是PC1作为机械受体，PC2作为Ca2+内流通道在初级纤毛中感知肾血流。也有人认为PC2作为细胞内Ca2+通道起作用。此外，有证据表明，PC1突变可导致PC2从细胞表面到内质网的额外错误定位，这也可能进一步促进ADPKD的发病机制。因此，研究内质网定位的PC2在调节细胞内Ca2+信号传导中的作用是很重要的。我们认为PC1和pc2在内质网和质膜上与初级纤毛协同作用，调节细胞质内Ca2+。我们的研究表明，PC1和pc2都与IP3受体（IP3R）相互作用，以调节细胞内Ca2+信号传导。IP3介导的细胞内Ca2+信号通路是细胞内最重要的信号通路之一。我们还表明，PC1的一个片段，我们称之为PEC抑制Ca2+依赖性Cl-电流在卵母细胞中表明它抑制Ca2+进入。片段与储存Ca2+进入途径的一个组分STIM1共免疫沉淀。总体假设是PC1和pc2在设定细胞内Ca2+浓度范围方面具有精确的作用，在该范围内Ca2+从IP3R释放并同时抑制SOCE。我们推测PC1抑制储存操作的Ca2+进入是必要的，以平衡肾细胞的Ca2+内流通过初级纤毛作为Ca2+进入的主要途径。该提案有三个基本部分：第一部分：多囊素如何具体调节ER Ca2+释放？第二部分。多囊素是否调节储存操作的Ca2+进入？第三部分。IP3R和储存操作的钙进入在小管和囊肿形成中的作用是什么？过去25年的总体重点是近端和远端小管的运输机制。在本届奖项的任期内，我们在离子如何通过水通道蛋白水通道以及PC-1和2如何形成功能复合物方面取得了根本性的发现。我们将之前的范围扩展到小鼠模型领域。目前的应用为细胞内Ca2+信号传导领域提供了一个新的方向。

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease arises from mutations in either the PKD1 or the PKD2 gene which encode PC1 or 2. Although it is increasing clear that PC1 is expressed at the plasma membrane and the primary cilium, the cellular localization of PC2 has been controversial. PC2 localizes to the ER, the plasma membrane, and the primary cilium. A popular hypothesis is that PC1 acts as a mechanical receptor and PC2 as a Ca2+ influx channel in the primary cilium to sense renal flow. It has also been suggested that PC2 functions as an intracellular Ca2+ channel. In addition, evidence suggests that PC1 mutants can cause additional mis-localization of PC2 from the cell surface to the ER, which might also contribute even further to the pathogenesis of ADPKD. Therefore, it is important to investigate the role of the ER localized PC2 in regulating intracellular Ca2+ signaling. We propose that PC1 and 2 operate at the ER and at the plasma membrane in concert with the primary cilium to regulate cytosolic Ca2+. Our studies indicate that both PC1 & 2 functionally interact with the IP3 receptor (IP3R) to modulate intracellular Ca2+ signaling. IP3 mediated intracellular Ca2+ signaling is one of the most important intracellular signaling pathways. We also show that a fragment of PC1 which we call PEC inhibits Ca2+ dependent Cl- currents in oocytes suggesting that it inhibits Ca2+ entry. The fragment coimmunoprecipitates with a component of the store operate Ca2+ entry pathway, STIM1. The overall hypothesis is that PC1 & 2 have a precise role in setting the range of intracellular Ca2+ concentrations over which Ca2+ is released from IP3R and simultaneously inhibiting SOCE. We speculate that PC1 inhibition of store-operated Ca2+ entry is necessary to poise renal cells for Ca2+ influx via the primary cilium as a major route of Ca2+ entry. The proposal has three essential parts: Part I. How specifically do the polycystins modulate ER Ca2+ release? Part II. Do the polycystins regulate store-operated Ca2+ entry? Part III. What is the role of IP3R and store-operated calcium entry in tubule and cyst formation? The overall focus of the past 25 years has been on the mechanisms of transport in proximal and distal tubules. During the tenure of this current award, we made fundamental discoveries regarding how ions move through aquaporin water channels and how PC-1 & 2 form a functional complex. We expanded the previous scope into the area of mouse models. The current application takes a new direction into the area of intracellular Ca2+ signaling. PUBLIC HEALTH RELEVANCE Autosomal dominant polycystic kidney disease, a major cause of kidney failure, arises from mutations in more that one gene. When the polycystic kidney genes function abnormally, intracellular calcium is not regulated properly. Thus, understanding normal and abnormal calcium regulation in health and disease, respectively, will ultimately lead to new ways of treating this serious disease.