Polycystin-1, ATP-induced Ca2+ entry & C1 - conductance

描述（由申请人提供）： 常染色体显性多囊肾病（ADPKD）是最常见的遗传性疾病之一，导致肾功能丧失，每年约有1800名新患者接受透析治疗。这种疾病是由编码多囊蛋白-1的PKD 1基因突变引起的，其特征是存在多发性肾囊肿。这些囊肿的扩张与肾功能丧失密切相关，并由经上皮液体分泌驱动。本KO 8提案的目的是了解为什么多囊蛋白-1缺失会导致液体分泌和囊肿扩张。ATP是液体分泌的一种有效和普遍的刺激物，因此可能在ADPKD中发挥作用：ATP刺激的氯化物分泌所需的所有成分都存在于囊肿环境中。ATP通过异源三聚体G蛋白（Glphabetagamma）起作用，导致钙从内质网（ER）释放的初始爆发，这反过来又触发了细胞外钙进入的延长阶段（激动剂诱导的钙进入或ACE）。ACE也由与Galphabetagamma相关的ER储存独立途径触发。ATP刺激的氯分泌增加可能是由于多囊蛋白-1通过破坏其对ACE-型通道和Galphabetagamma的已知作用而丧失所致。 在初步的实验中，它被证明，分离的C-末端193个氨基酸的多囊蛋白-1（sIg-PKD 193融合蛋白）的表达增强ATP刺激的氯分泌通过在皮质集合管细胞系的存储独立的ACE的上调。过表达的多囊蛋白-1具有相反的作用，使细胞对ATP的钙响应减弱，表明slg-PKD 193融合蛋白的作用是内源性多囊蛋白-1的显性负抑制的结果。假设：Polyeystin-1通过减弱非钙库依赖性激动剂诱导的钙内流下调ATP刺激的氯分泌，并通过调节异源三聚体G蛋白偶联途径起作用。具体目标#1中的实验将确定多囊蛋白-1对ACE的ER储存依赖性和非依赖性组分的作用，并将这些作用与氯离子通道活性的调节相关联。这将通过多囊蛋白-1的过表达和抑制来完成。具体目标#2将确定多囊蛋白-1对ACE的作用机制，从而提供遗传病变和液体分泌之间的直接联系。Michael Sutters博士致力于成为临床医生和科学家。他将是KO 8应用程序的主要研究员。他工作的长期目标是通过描述ADPKD囊肿扩张的机制来促进新治疗策略的设计，以控制疾病进展

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary diseases, causing loss of renal function that leads to dialysis treatment in some 1800 new patients a year. The disease, arising from mutations in the PKD 1 gene encoding polycystin-1, is characterized by the presence of multiple renal cysts. Expansion of these cysts correlates closely with loss of renal function and is driven by transepithelial fluid secretion. The objective of this KO8 proposal is to understand why loss of polyeystin-1 results in fluid secretion and cyst expansion. ATP is a potent and universal stimulus for fluid secretion and might therefore play a role in ADPKD: All the components required for ATP-stimulated chloride secretion are resident in the cyst environment. ATP acts through heterotrimeric G proteins (Glphabetagamma) to cause an initial burst of calcium release from the endoplasmic reticulum (ER), which in turn triggers a prolonged phase of calcium entry from outside the cell (agonist induced calcium entry or ACE). ACE is also triggered by ER store-independent pathways linked to Galphabetagamma. Increased ATP-stimulated chloride secretion could arise from loss of polycystin-1 through disruption of its known effects upon ACE-type channels and Galphabetagamma. In preliminary experiments it was demonstrated that expression of the isolated C-terminal 193 amino acids of polycystin-1 (sIg-PKD193 fusion protein) augmented ATP-stimulated chloride secretion through up-regulation of store independent ACE in a cortical collecting duct cell line. Over-expressed polycystin-1 had the opposite effect, abbreviating the cell calcium response to ATP, suggesting that the slg-PKD193 fusion protein effect was the result of dominant negative inhibition of endogenous polycystin-1. It is hypothesized that: Polyeystin-1 down-regulates ATPstimulated chloride secretion through attenuation of store-independent agonist-induced calcium entry, and acts via modulation of heterotrimerie G protein coupled pathways. The experiments in Specific Aim #1 will define the effects of polycystin-1 upon the ER store dependent and independent components of ACE, and correlate these effects with the regulation of chloride channel activity. This will be done through over-expression and inhibition of polycystin-1. Specific aim #2 will identify the mechanism of the effect of polycystin-1 upon ACE, thereby providing a direct link between genetic lesion and fluid secretion. Dr. Michael Sutters has dedicated his career to becoming both a clinician and a scientist. He will be the principle investigator in this KO8 application. The long-term aim of his work is to facilitate the design of new therapeutic strategies to control disease progression through delineation of the mechanism of cyst expansion in ADPKD