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

描述(由申请人提供)： PI的长期目标是应用细胞生理学和分子生物学的基本概念和广泛技术，了解常染色体显性遗传性多囊肾病(ADPKD)相关血管表现的发病机制。ADPKD是世界上最常见的威胁生命的单基因疾病，包括高血压和颅内动脉瘤在内的血管表现是其主要死亡原因。 通过NIH(K08)支持的研究，钱博士发现PKD2+/-突变的血管平滑肌细胞(VSMCs)表现出细胞内钙([Ca+]i)调节失调，包括：(1)钙库操作的钙通道(SOC)介导的钙内流，(2)肌浆网钙储存，(3)基础[钙]i；以及PKD2+/-动脉对肾上腺素能受体激动剂(苯肾上腺素)刺激产生高度活跃的血管收缩。 目前的建议旨在进一步确定PKD2突变相关血管异常的机制。中心假说是多囊蛋白-2(PC2，PKD2基因的蛋白产物)调节SOC通道的活动。PKD2+/-突变减少SOC介导的钙内流。导致VSMC[Ca~(2+)]i失调和肾上腺素能受体的异常转运，进而导致对刺激的过度兴奋的血管收缩。具体目的1研究PC2是否以及如何调节VSMC中的细胞表面SOC通道。特定目的2研究PKD2+/-突变是否以及如何导致肾上腺素能受体异常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、淋巴母细胞系、特定基因表达(PC1-PKD1基因的蛋白质产物、w/tPC2、突变PC2-导致PKD2突变基因的疾病D511的蛋白质产物和TRPC1-作为SOC亚单位的trpc1基因的蛋白质产物)和使用小干扰RNA(SiRNA)的基因沉默、细胞内钙成像和膜片钳技术，包括穿孔的全细胞和细胞连接配置。 这一建议引入了关于PC2在基础[Ca~(2+)]i和α1-肾上腺素能受体运输调节中的作用的新概念。所应用的方法是直接的、具体的和可实现的。从这项拟议的研究中获得的信息将极大地加深我们对ADPKD血管并发症发病机制的了解，并为治疗靶点提供潜在的领域。

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.