Role of TRP Channels on Collecting Duct Calcium Dynamics

TRP 通道对集合管钙动态的作用

• 批准号: 7046054
• 负责人: ROGER Gordon O'NEIL
• 金额: $ 28.71万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7046054
• 关键词: calcium channel; calcium flux; calcium ion; cell line; confocal scanning microscopy; immunofluorescence technique; immunoprecipitation; laboratory mouse; mechanical stress; membrane permeability; molecular /cellular imaging; northern blottings; phospholipase C; polymerase chain reaction; protein isoforms; protein kinase; protein localization; protein structure function; protein transport; renal tubular transport; renal tubule; small interfering RNA; tissue /cell culture; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant):The overall goal of this proposal is to elucidate the role of Ca-permeable TRPV channel isoforms in regulating mechanically-induced intracellular Ca, [Ca]i, dynamics and Ca reabsorption in renal cortical collecting duct. Mechanical stresses, such as fluid shear stress or hypoosmotic stress, vary widely in late distal tubule and cortical collecting duct (CCD) especially during pathophysiological states (e.g., hyponatremia, diabetes insipidus, diabetes mellitus, diuretic administration), which will contribute to hypocaliuric and/or hypercaliuric states that lead to altered Ca balance. We have implicated two TRPV channels as being mechanosensitive and central to regulating mechano-stimulated Ca entry and reabsorption in mouse CCD. The following four specific aims are proposed to achieve the overall goal: 1. To determine the relative role and pharmacological/ electrophysiological properties of mechanoregulated Ca entry pathways (Ca imaging, patch clamp) in control of [Ca]i dynamics in mouse CCD cells; 2. To identify TRPV channel isoforms expressed in mouse CCD cells (RT-PCR, Northern blot, immunofluorescence, immunoblots) and to determine which specific isoforms participate in mechanoregulated [Ca] dynamics; 3. To delineate the relative contribution of specific mechanoregulated TRPV channels in Ca reabsorption in mouse CCD cells (overexpression/siRNA knockdown) and to examine the mechanism of regulation of the channels (phospholipases, protein kinases); and 4. To evaluate the role of membrane localization/trafficking of TRPV isoforms (confocal microscopy/immunofluorescence, biotinylation-streptavidin purification) and the association with accessory proteins (immunoprecipitation/pull-downs), including other TRPs, on mechanoregulated [Ca]i dynamics and Ca reabsorption. The outcome of the study has important health relatedness as it will provide new insights into the poorly understood mechanisms of calcium signaling and reabsorption in renal tubules and provide a critical new understanding of the mechanism by which altered pathophysiological conditions regulate calcium reabsorption in renal cells.

描述（由申请人提供）：本提案的总体目标是阐明 Ca 渗透性 TRPV 通道亚型在调节肾皮质集合管中机械诱导的细胞内 Ca、[Ca]i、动力学和 Ca 重吸收中的作用。机械应力，例如流体剪切应力或低渗应力，在晚期远端小管和皮质集合管（CCD）中变化很大，尤其是在病理生理状态下（例如低钠血症、尿崩症、糖尿病、利尿剂给药），这将导致低尿钙和/或导致钙平衡​​改变的高尿钙状态。我们发现两个 TRPV 通道对机械敏感，并且是调节小鼠 CCD 中机械刺激的 Ca 进入和重吸收的核心。为了实现总体目标，提出了以下四个具体目标： 1. 确定机械调节 Ca 进入途径（Ca 成像、膜片钳）在控制小鼠 CCD 细胞中 [Ca]i 动力学中的相对作用和药理学/电生理学特性； 2. 鉴定小鼠 CCD 细胞中表达的 TRPV 通道异构体（RT-PCR、Northern blot、免疫荧光、免疫印迹），并确定哪些特定异构体参与机械调节 [Ca] 动力学； 3. 描述特定机械调节TRPV通道在小鼠CCD细胞Ca重吸收中的相对贡献（过表达/siRNA敲低）并检查通道调节机制（磷脂酶、蛋白激酶）； 4. 评估TRPV亚型的膜定位/运输（共焦显微镜/免疫荧光、生物素化-链霉亲和素纯化）的作用以及与辅助蛋白（免疫沉淀/下拉）（包括其他TRP）对机械调节[Ca]i的关联动力学和 Ca 重吸收。该研究的结果具有重要的健康相关性，因为它将为人们对肾小管中钙信号传导和重吸收的鲜为人知的机制提供新的见解，并为改变病理生理条件调节肾细胞中钙重吸收的机制提供重要的新认识。