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

Polycystin-1，ATP 诱导 Ca2+ 进入

基本信息

批准号：
7117440
负责人：
MICHAEL SUTTERS
金额：
$ 13.44万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-01 至 2009-08-31
项目状态：
已结题

项目摘要

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

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，会导致肾功能丧失，导致每年约有 1800 名新患者接受透析治疗。该疾病由编码多囊蛋白-1 的 PKD 1 基因突变引起，其特征是存在多个肾囊肿。这些囊肿的扩张与肾功能丧失密切相关，并由跨上皮液体分泌驱动。该 KO8 提案的目的是了解为什么多囊蛋白-1 的缺失会导致液体分泌和囊肿扩张。 ATP 是液体分泌的有效且普遍的刺激物，因此可能在 ADPKD 中发挥作用：ATP 刺激的氯离子分泌所需的所有成分都驻留在囊肿环境中。 ATP 通过异三聚体 G 蛋白 (Glphabetagamma) 起作用，引起内质网 (ER) 中钙的初始爆发，进而触发钙从细胞外进入的延长阶段（激动剂诱导钙进入或 ACE）。 ACE 也由与 Galphabetagamma 相关的 ER 库独立通路触发。 ATP 刺激的氯离子分泌增加可能是由于多囊蛋白-1 的已知作用被破坏而导致的。多囊蛋白-1 对 ACE 型通道和 Galphabetagamma 的影响被破坏。初步实验表明，多囊蛋白-1（sIg-PKD193 融合蛋白）分离的 C 端 193 个氨基酸的表达通过上调皮质集合管细胞系中储存独立的 ACE 来增强 ATP 刺激的氯离子分泌。过表达的多囊蛋白-1具有相反的作用，缩短了细胞对ATP的钙反应，表明slg-PKD193融合蛋白效应是内源性多囊蛋白-1显性失活抑制的结果。假设：Polyeystin-1 通过减弱不依赖于储存的激动剂诱导的钙进入来下调 ATP 刺激的氯离子分泌，并通过调节异源三聚体 G 蛋白偶联途径发挥作用。具体目标 #1 中的实验将定义多囊蛋白-1 对 ACE 的 ER 储存依赖和独立成分的影响，并将这些影响与氯离子通道活性的调节相关联。这将通过多囊蛋白-1 的过度表达和抑制来完成。具体目标#2 将确定多囊蛋白-1 对 ACE 的影响机制，从而提供遗传病变和液体分泌之间的直接联系。迈克尔·萨特斯 (Michael Sutters) 博士致力于成为一名临床医生和科学家。他将担任本次 KO8 申请的首席研究员。他工作的长期目标是通过描述 ADPKD 囊肿扩张的机制，促进设计新的治疗策略来控制疾病进展