Role of TRP Channels on Collecting Duct Calcium Dynamics

描述(申请人提供)：这项建议的总体目标是阐明钙通透性TRPV通道异构体在调节机械诱导的细胞内钙、[Ca]i、肾皮质集合管的动力学和钙重吸收中的作用。机械应力，如流体剪应力或低渗应力，在远端小管和皮质集合管的晚期变化很大，特别是在病理生理状态(如低钠血症、尿崩症、糖尿病、利尿剂给药)期间，这将导致低口径和/或高口径状态，导致钙平衡改变。我们推测有两个TRPV通道是机械敏感的，并且在调节机械刺激的钙进入和重吸收过程中起中心作用。为实现这一总体目标，提出了以下四个具体目标：1.确定机械调节的钙进入途径(钙成像、膜片钳)在控制小鼠CCD细胞[Ca]i动力学中的相对作用和药理/电生理特性；2.鉴定在小鼠CCD细胞中表达的TRPV通道异构体(RT-PCR、Northern印迹、免疫荧光、免疫印迹)，并确定哪些特定的异构体参与机械调节的[Ca]i动力学；3.描述特定的机械调节的TRPV通道在小鼠CCD细胞钙重吸收中的相对贡献(过表达/siRNA敲除)，并研究这些通道(磷脂酶、蛋白激酶)的调节机制；4.评价TRPV异构体的膜定位/转运(共聚焦显微镜/免疫荧光、生物素化-链霉亲和素纯化)以及与辅助蛋白(免疫沉淀/下拉)(包括其他TRP)在机械调节的[Ca]i动力学和钙重吸收中的作用。这项研究的结果具有重要的健康意义，因为它将为了解肾小管钙信号和重吸收的鲜为人知的机制提供新的见解，并为改变的病理生理条件调节肾细胞钙重吸收的机制提供关键的新理解。

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.