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细胞膜中的通道肾集合管和近端小管细胞，来自转基因小鼠，大鼠、兔子或蝾螈的肾脏，使用以下制剂的组合：细胞培养物、分离的灌注和非灌注肾小管，以及分离的单个细胞保留了其上皮极性。将使用膜片钳、光学和分子生物学技术。目的是： 1. 研究生理作用和调节顶膜中的 cGMP 敏感非选择性阳离子通道 M-1 细胞系，源自小鼠皮质集合管。来自 M-1 细胞系的不完整 cDNA 序列极其严重类似于脊椎动物的 cGMP 门控非选择性通道光感受器。将分离全长肾通道克隆并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及其表达进行进一步研究评估电生理学特性。 2. 生理学研究基底外侧单一氯离子通道的作用和调节使用膜片钳记录哺乳动物近端小管细胞的膜，以及通过全贴片和全细胞的通道集合当前录音。在单个、分离的基底外侧膜中两栖类近曲小管细胞小电导氯离子通道检测到，它们被毛喉素和 cAMP 激活，并被将通过膜片钳在未灌注的情况下探索氯离子通道哺乳动物近端小管，管腔的受控微灌注近端小管插管，以及跨上皮调节期间氯化物运输。氯通道的生物物理学和动力学将研究哺乳动物近曲小管以及细胞内参与调节的信号转导途径。与此同时，这些调节机制的效果将在跨上皮上进行测试氯化物在灌注小管中的转运。 3. 研究一个简单的模型血管紧张素II诱导的近曲小管细胞损伤伴有细胞内钙和质膜升高起泡。使用共焦活体显微镜和离子敏感荧光单个分离的蝾螈近端小管细胞的染料，其机制将研究气泡形成的直接反应，作为对增加的直接反应细胞内钙，作为细胞外Na和的函数细胞内酸中毒。信号以便开发可能细胞保护干预。血管紧张素对细胞膜的影响通过电导和电容监测诱导的细胞损伤全细胞膜片钳测定。该项目的总体范围是了解跨上皮溶质肾脏在单细胞膜和单通道上的运动蛋白质水平并有助于理解临床高血压、代谢性碱中毒/酸中毒、急性肾病等疾病失败和钾平衡。