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

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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.