REGULATION OF KIDNEY MEMBRANE ION CHANNELS

拟议的研究计划表征细胞膜中的通道， 肾集合管和近曲小管细胞，来自转基因小鼠， 大鼠、兔或蝾螈肾，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，其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。 目标是：1. 研究其生理作用和调节 一个cGMP敏感的非选择性阳离子通道在顶膜的 M-1-细胞系，来源于小鼠皮质集合管。 来源于M-1细胞系的不完整cDNA序列是非常重要的。 类似于脊椎动物cGMP门控非选择性通道 光感受器 将分离全长肾通道克隆， 用于探索哪些基因在肾细胞中表达。 这些基因 将通过在卵母细胞中的表达及其 评估电生理学特性。 2. 为了研究 基底外侧核单个氯通道的作用和调节 膜的哺乳动物近端小管细胞使用膜片钳记录， 以及通过整个膜片和整个细胞的通道集合 当前记录 在基底外侧膜的单个，孤立的 两栖类近端小管细胞小电导氯离子通道 检测到，这是由毛喉素和cAMP激活，并抑制了 氯离子通道将通过膜片钳在未灌注的 哺乳动物近端小管，在管腔的受控微灌注中 近端小管插管，并在调节跨上皮 氯离子转运 氯离子通道的生物物理学和动力学 哺乳动物近端小管将被研究，以及细胞内 参与调控的信号转导通路。 并行地将第 这些调节机制的作用将在跨上皮细胞上进行测试。 灌注小管中的氯离子转运。 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， 伴随着细胞内钙离子和质膜的升高 起泡 使用共聚焦活体显微镜和离子敏感荧光 染料对单个离体蝾螈近曲小管细胞的作用机制 将研究水泡形成的直接反应， 细胞内钙，作为细胞外钠和 细胞内酸中毒 信号，以便开发可能的 细胞保护干预。 血管紧张素Ⅱ的细胞膜效应 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经上皮溶质 肾在单细胞膜和单通道上的运动 蛋白质水平，并有助于了解临床 疾病，如高血压、代谢性肾功能衰竭/酸中毒、急性肾功能衰竭、 失败和钾平衡。

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.