REGULATION OF KIDNEY MEMBRANE ION CHANNELS

拟议的研究计划表征通道，特别是氯化物 通道，在近端小管细胞的细胞膜，从肾脏， 兔、蝾螈或CFTR敲除小鼠，使用以下组合： 分离的灌注肾小管、非灌注肾小管和分离的 保持上皮极性的单个细胞。膜片钳 将使用光学技术。具体目标是： 1.为了验证基底外侧的氯离子通道 近曲小管细胞的膜共享许多内在的 囊性纤维化跨膜传导调节因子CI的性质- 通道，我们将研究生物物理学和氯离子通道的动力学， 哺乳动物近端小管与细胞内信号转导 参与其调控的途径。 2.为了检测基底外侧氯离子通道的门控是否在脑缺血再灌注损伤中起作用， 近曲小管细胞通过ATP的水解和非水解相互作用 类似于CFTR CI通道。 3.为了验证基底外侧氯离子通道的假设， 近曲小管细胞通过膜调节跨细胞氯重吸收 近端小管和顶端氯间接摄取改变 调节基底外侧氯离子通道的功能。 4.探索氯离子跨细胞运动的替代途径 在CFTR敲除小鼠的近端小管中。虽然肾脏 表达CFTR，患者中似乎没有受损的离子转运 患有囊性纤维化我们将使用CFTR敲除小鼠， 为了确定氯离子通道如何有助于跨细胞 氯离子的运动，以及是否存在调节关系 CFTR和其他氯化物途径。 5.为了验证分离细胞中上皮极性是 不通过与紧密连接相关的内在蛋白质维持， 我们将研究一个近端小管细胞的模型， 为我们提供十天的上皮极性，并检查细胞骨架 这些相互作用对于保持极性至关重要。 该项目的总体范围是了解跨上皮溶质 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 如囊性纤维化、高血压、代谢性脂肪变性、酸中毒， 急性肾衰竭

The proposed studies plan to characterize channels, in particular chloride channels, in cell membranes of proximal tubule cells, from kidneys of rabbit, salamander, or CFTR knockout mouse, using a combination of isolated perfused tubules, non-perfused renal tubules, and separated single cells that have preserved their epithelial polarity. Patch-clamp and optical techniques will be used. The specific aims are: 1. To test the hypothesis that the chloride channel in the basolateral membrane of the proximal tubule cells share many of the intrinsic properties of the cystic fibrosis transmembrane conductance regulator CI- channel, we will study the biophysics and kinetics of chloride channels in mammalian proximal tubule and the intracellular signal transduction pathways involved in their regulation. 2. To test whether the gating of the basolateral chloride channel in proximal tubule cells by hydrolytic and non-hydrolytic interactions of ATP is analogous to that of the CFTR CI-channel. 3. To test the hypothesis that the basolateral chloride channel in proximal tubule cells regulates transcellular chloride reabsorption by the proximal tubule, and that alterations of apical chloride uptake indirect modulate the function of the basolateral chloride channel. 4. To explore the alternative pathways for transcellular chloride movement in proximal tubules of a CFTR-knockout mouse. Although the kidney expresses CFTR, there appears to be no impaired ion transport in patients suffering from cystic fibrosis. We will use a CFTR-knockout mouse, in order to establish how chloride channels contribute to transcellular chloride movement, and whether there is a regulatory relationship between CFTR and other chloride pathways. 5. To test the hypothesis that epithelial polarity in dissociated cells is not maintained by intrinsic proteins associated with the tight junction, we will study a model of a proximal tubule cells, that maintains epithelial polarity for us to ten days, and examine the cytoskeletal interactions that are essential for preserving polarity. The overall scope of the project is to 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 cystic fibrosis, hypertension, metabolic alkalosis, acidosis, and acute renal failure.