The Role of Polycystins in the Regulation of Intracellular Calcium and Vascular R

多囊蛋白在细胞内钙和血管 R 调节中的作用

• 批准号: 7144882
• 负责人: QI QIAN
• 金额: $ 7.4万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7144882
• 关键词: adrenergic receptor; calcium channel; calcium ion; cell line; gene expression; gene induction /repression; gene mutation; muscle cells; phosphorylation; polycystic kidney; protein structure function; protein transport; sarcoplasmic reticulum; small interfering RNA; vascular smooth muscle; vasoconstriction; voltage /patch clamp

DESCRIPTION (provided by applicant): The PI's long-term objective is to understand the pathogenesis of vascular manifestations associated with autosomal dominant polycystic kidney disease (ADPKD) by applying the fundamental concepts and broad technologies of cellular physiology and molecular biology. ADPKD is the most common life-threatening monogenic disease in the world; vascular manifestations, including hypertension and intracranial aneurysms, are its leading cause of death. Through her NIH (K08)-supported research, Dr. Qian has discovered that Pkd2+/- mutant vascular smooth muscle cells (VSMCs) manifest intracellular calcium ([Ca2+]i) dysregulation, including reductions in (1) the store-operated Ca2+ channel (SOC)-mediated Ca2+ entry, (2) the sarcoplasmic reticulum Ca2+ store, and (3) basal [Ca2+]i; and Pkd2+/- arteries develop hyperactive vasoconstriction in response to adrenergic receptor agonist (phenylephrine) stimulation. The current proposal is designed to further define the mechanisms underlying Pkd2 mutation-associated vascular abnormalities. The central hypothesis is that polycystin-2 (PC2, the protein product of the Pkd2 gene) regulates the activity of SOC channels. Pkd2+/- mutation reduces SOC-mediated Ca2+ entry. resulting VSMC [Ca2+]i dysregulation and abnormal adrenergic receptor trafficking, which in turn cause hyperactive vasoconstriction in response to stimulation. Specific Aim 1 examines whether and how PC2 regulates the cell-surface SOC channels in VSMCs. Specific Aim 2 examines whether and how Pkd2+/- mutation leads to an abnormal adrenergic receptor trafficking and hyperactive vasoconstriction. To complete these aims, the PI will employ established as well as new models, methods, and probes including: heterozygous Pkd1 and Pkd2 mutant mice, lymphoblastoid cell line, specific gene expression (PC1 - the protein products of Pkd1 gene, w/t PC2, mutant PC2- the protein product of D511, a disease causing PKD2 mutant gene, and TRPC1 - the protein product of trpc1 gene that is a SOC subunit) and gene silencing using small interfering RNA (siRNA), intracellular Ca2+ imaging, and patch clamp techniques including perforated whole-cell and cell-attached configurations. This proposal introduces novel concepts regarding the role of PC2 in the regulation of basal [Ca2+]i and alpha1-adrenergic receptor trafficking. The methodologies applied are direct, specific, and achievable. The information gained from the proposed study will greatly enhance our understanding in the pathogenesis of vascular complications in ADPKD and provide potential areas for therapeutic targets.

描述（由申请人提供）： PI 的长期目标是通过应用细胞生理学和分子生物学的基本概念和广泛技术，了解与常染色体显性多囊肾病 (ADPKD) 相关的血管表现的发病机制。 ADPKD 是世界上最常见的危及生命的单基因疾病；血管表现，包括高血压和颅内动脉瘤，是其死亡的主要原因。 通过 NIH (K08) 支持的研究，Qian 博士发现 Pkd2+/- 突变血管平滑肌细胞 (VSMC) 表现出细胞内钙 ([Ca2+]i) 失调，包括 (1) 钙库操纵通道 (SOC) 介导的 Ca2+ 内流减少，(2) 肌浆网 Ca2+ 储存减少，以及 (3)基础[Ca2+]i； Pkd2+/- 动脉因肾上腺素能受体激动剂（去氧肾上腺素）刺激而出现过度活跃的血管收缩。 目前的提案旨在进一步定义 Pkd2 突变相关血管异常的潜在机制。核心假设是多囊蛋白-2（PC2，Pkd2 基因的蛋白质产物）调节 SOC 通道的活性。 Pkd2+/- 突变减少 SOC 介导的 Ca2+ 进入。导致 VSMC [Ca2+]i 失调和肾上腺素能受体运输异常，进而导致对刺激的反应过度活跃的血管收缩。具体目标 1 检查 PC2 是否以及如何调节 VSMC 中的细胞表面 SOC 通道。具体目标 2 检查 Pkd2+/- 突变是否以及如何导致异常肾上腺素受体运输和过度活跃的血管收缩。 为了实现这些目标，PI 将采用已建立的以及新的模型、方法和探针，包括：杂合 Pkd1 和 Pkd2 突变小鼠、类淋巴母细胞系、特定基因表达（PC1 - Pkd1 基因的蛋白质产物，w/t PC2、突变体 PC2 - D511 的蛋白质产物（一种引起 PKD2 突变基因的疾病）和 TRPC1 - trpc1 基因（SOC 亚基）的蛋白质产物）和使用小干扰 RNA (siRNA) 的基因沉默、细胞内 Ca2+ 成像和膜片钳技术（包括穿孔全细胞和细胞贴壁配置）。 该提案引入了有关 PC2 在基础 [Ca2+]i 和 α1-肾上腺素受体运输调节中的作用的新概念。所采用的方法是直接、具体且可实现的。从拟议研究中获得的信息将极大地增强我们对 ADPKD 血管并发症发病机制的理解，并为治疗靶点提供潜在领域。