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

描述(由申请人提供)： 常染色体显性遗传性多囊肾病(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，其特征是存在多个肾囊肿。这些囊肿的扩大与肾功能的丧失密切相关，并由跨上皮液分泌驱动。这项KO8提案的目的是了解为什么Polyeystin-1的丢失会导致液体分泌和囊性扩张。三磷酸腺苷是一种强大而普遍的液体分泌刺激物，因此可能在ADPKD中发挥作用：由三磷酸腺苷刺激的氯化物分泌所需的所有成分都驻留在囊环境中。ATP通过异三聚体G蛋白(Glphabetagamma)作用，引起内质网(ER)最初的钙释放，进而触发细胞外钙内流的延长阶段(激动剂诱导的钙内流或ACE)。ACE也是由与Galphabetagamma相连的ER商店独立通路触发的。通过阻断多囊蛋白-1对ACE型通道和Galphabetagma的已知作用，多囊蛋白-1的缺失可能导致ATP刺激的氯离子分泌增加。 初步实验证明，多囊蛋白-1的C端193个氨基酸(Sig-PKD193融合蛋白)的表达可通过上调皮质集合管细胞系中的血管紧张素转换酶(ACE)水平来增加ATP刺激的氯的分泌。过表达的多囊蛋白-1则有相反的作用，缩短了细胞对ATP的钙反应，提示sLG-PKD193融合蛋白的作用是内源性多囊蛋白-1显性负抑制的结果。推测：Polyeystin-1通过抑制非依赖性激动剂诱导的钙内流而下调ATP刺激的氯离子分泌，并通过调节异源三聚体G蛋白偶联通路起作用。具体目标1的实验将确定多囊蛋白-1对血管紧张素转换酶依赖和独立的内质网储存成分的影响，并将这些影响与氯通道活动的调节联系起来。这将通过过度表达和抑制多囊蛋白-1来实现。具体目标#2将确定多囊蛋白-1影响血管紧张素转换酶的机制，从而在遗传损伤和液体分泌之间提供直接联系。迈克尔·萨特斯博士致力于成为一名临床医生和一名科学家。他将是这份KO8申请的首席调查员。他的长期工作目标是帮助设计新的治疗策略，通过描绘ADPKD囊性扩张的机制来控制疾病的进展。

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