REGULATION OF KIDNEY MEMBRANE ION CHANNELS

拟议的研究计划表征细胞膜中的通道 来自转基因小鼠的肾脏集合管和近端小管细胞， 大鼠肾、兔肾或火蜥蜴肾，使用多种制剂： 细胞培养，隔离的灌流和非灌流的肾小管，以及 分离的单个细胞保留了它们的上皮极性。 将使用膜片钳、光学和分子生物学技术。 本研究的目的是：1.研究血管紧张素转换酶的生理作用和调节 牙周炎根尖膜上cGMP敏感的非选择性阳离子通道 M-1细胞系，来源于小鼠皮质集合管。 从M-1细胞系中获得的不完整的cDNA序列非常 与脊椎动物cGMP门控非选择性通道相似 光感受器。全长肾通道克隆将被分离并 用于探索哪些基因在肾脏细胞中表达。这些基因 将通过在卵母细胞中的表达以及它们的 电生理学特性评估。2.生理学研究 单一氯离子通道在基底外侧区的作用和调节 使用膜片钳记录的哺乳动物近端小管细胞膜， 以及通过全斑块和全细胞的经络集合 当前录制。在单个、孤立的基底外侧膜上 两栖类近端肾小管细胞小电导氯通道 检测到的，它们被forskolin和cAMP激活，并被 氯离子通道将通过膜片钳技术在未灌流的 哺乳动物近端小管的管腔微灌流 在管状近端小管和跨上皮调节过程中 氯化物运输。氯离子通道的生物物理和动力学 将研究哺乳动物的近端小管以及细胞内 参与调控的信号转导通路。同时， 将测试这些调节机制对跨上皮细胞的影响 氯离子在灌流小管中的传输。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 伴随着细胞内钙和质膜的升高 流着泪。使用共聚焦生命显微镜和离子敏感荧光 染料对火蜥蜴单个离体近端小管细胞的作用机制 将研究滤过泡形成的直接反应 细胞内钙是细胞外钠和钙离子的函数 细胞内酸中毒。为了发展可能的信号 细胞保护的干预措施。血管紧张素的细胞膜效应- 将通过电导和电容来监测诱导的细胞损伤 全细胞膜片钳检测。 该项目的总体范围是了解跨上皮解决方案 肾在单细胞膜和单通道上的运动 蛋白质水平，有助于了解临床 高血压、代谢性碱中毒/酸中毒、急性肾脏等疾病 衰竭和钾平衡。

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.