REGULATION OF KIDNEY MEMBRANE ION CHANNELS

肾膜离子通道的调节

基本信息

批准号：
6270421
负责人：
EMILE L BOULPAEP
金额：
$ 18.33万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-12-01 至 1998-11-30
项目状态：
已结题

项目摘要

The proposed studies plan to characterize channels in cell membranes of renal collecting duct and proximal tubule cells, from transgenic mouse, rat, rabbit, or salamander kidney, using a combination of preparations: cell cultures, isolated perfused and non-perfused renal tubules, and separated single cells which have preserved their epithelial polarity. Patch-clamp, optical and molecular biology techniques will be used. The objective are: 1. To study the physiological role and regulation of a cGMP-sensitive non-selective cation channel in the apical membrane of the M-1-cells line, derived from mouse cortical collecting duct. Incomplete cDNA sequences derived from M-1-cell line are extremely similar to that of the cGMP-gated non-selective channel of vertebrate photoreceptor. Full length kidney channel clones will be isolated and used to explore which genes are expressed in renal cells. Those genes will be studied further by expression in oocytes and their electrophysiology properties assessed. 2. To study the physiological role and regulation of single chloride channels in the basolateral membrane of mammalian proximal tubule cells using patch-clamp recordings, as well as of ensembles of channels through whole-patch and whole-cell current recording. In the basolateral membrane of single, isolated amphibian proximal tubule cells small-conductance chloride channels were detected, which are activated by forskolin and cAMP, and inhibited by the chloride channels will be explored by patch-clamping in unperfused mammalian proximal tubules, in controlled microperfusions of the lumen of cannulated proximal tubules, and during modulation of transepithelial chloride transport. The biophysics and kinetics of chloride channels in mammalian proximal tubule will be studied as well as the intracellular signal transduction pathways involved in regulation. In parallel, the effect of these regulator mechanisms will be tested on transepithelial chloride transport in perfused tubules. 3. To study a simple model of proximal tubule cell injury induced by angiotensin II, which is accompanied by elevation of intracellular calcium and plasma membrane blebbing. Using confocal vital microscopy and ion-sensitive fluorescent dyes on single isolated salamander proximal tubule cells, the mechanism of bleb formation will be studied as a direct response to increases in intracellular calcium, as a function of extracellular Na and of intracellular acidosis. Signal in order to develop possible interventions of cyto-protection. Cell membrane effects of angiotensin- induced cell injury will be monitored by conductance and capacitance determination in whole-cell patch-clamping. The overall scope of the project is the understand transepithelial solute movement by the kidney at the single cell membrane and single channel protein level and to contribute to the understanding of clinical disorders such as hypertension, metabolic alkalosis/acidosis, acute renal failure and potassium balance.

拟议的研究计划表征在细胞膜中的通道肾脏收集管和近端小管细胞，来自转基因小鼠，大鼠，兔子或sal骨肾脏，结合组合：细胞培养物，孤立的灌注和不被灌注的肾小管，以及分离的单细胞保留了其上皮极性。将使用斑块钳，光学和分子生物学技术。目的是：1。研究生理作用和调节 CGMP敏感的非选择性阳离子通道 M-1细胞系，源自小鼠皮质收集管道。来自M-1细胞系的不完整cDNA序列极为类似于脊椎动物的CGMP门控非选择通道感光器。全长肾通道克隆将被隔离，并且用于探索哪些基因在肾细胞中表达。那些基因将通过卵母细胞的表达进一步研究评估了电生理特性。 2。研究生理基底外侧单氯化物通道的作用和调节使用贴片钳记录，哺乳动物近端小管细胞的膜，以及通过全细胞和整个细胞的频道集合当前记录。在单一的基底外侧膜中两栖小管细胞小传导氯化物通道为检测到的，被福斯科林和cAMP激活，并被氯化物通道将通过贴片夹在未经灌注的情况下探索哺乳动物近端小管，在管腔的受控微型流体中插管近端小管，以及在旋转的调制期间氯化物运输。氯化物通道的生物物理学和动力学将研究哺乳动物近端小管以及细胞内涉及调节的信号转导途径。并行，这些调节器机制的效果将在transepithial上进行测试灌注小管中的氯化物运输。 3。研究一个简单的模型血管紧张素II诱导的近端小管细胞损伤，即伴随着细胞内钙和质膜的升高吹气。使用共聚焦重要显微镜和离子敏感的荧光单个孤立的sal端小管细胞上的染料，机理将研究BLEB形成的直接响应细胞内钙，作为细胞外Na的函数细胞内酸中毒。信号以发展可能细胞保护的干预措施。血管紧张素的细胞膜作用诱导的细胞损伤将通过电导和电容监测全细胞斑块夹的测定。该项目的总体范围是理解的跨度溶质肾脏在单细胞膜和单个通道上的运动蛋白质水平并有助于理解临床高血压，代谢碱/酸中毒，急性肾脏等疾病衰竭和钾平衡。