REGULATION OF KIDNEY MEMBRANE ION CHANNELS

拟议的研究计划表征通道，特别是氯化物。 近端肾小管细胞膜上的通道 兔子、火蜥蜴或CFTR基因敲除小鼠，使用 分离的灌流肾小管、非灌流肾小管 保持其上皮极性的单个细胞。膜片钳 并将使用光学技术。具体目标是： 1.检验基底外侧氯离子通道的假设 近端小管细胞的膜具有许多固有的 囊性纤维化跨膜电导调节剂CI-2的性质 通道，我们将研究氯通道的生物物理和动力学。 哺乳动物近端小管与细胞内信号转导 参与其调控的通路。 2.检测基底侧氯通道的门控是否在 近端小管细胞与ATP的水解性和非水解性相互作用 类似于CFTRCI通道。 3.为了检验假设，在 近端小管细胞通过调节跨细胞的氯离子重吸收 近端小管，以及间接摄取根尖氯变化 调节碱性侧氯离子通道的功能。 4.探索氯离子跨细胞移动的替代途径 在cftr基因敲除小鼠的近端小管中。虽说肾 表达CFTR，患者的离子转运似乎没有受损 患有囊性纤维症。我们将使用cftr基因敲除鼠标，在 以确定氯离子通道如何参与跨细胞 氯离子的移动与是否存在调控关系 Cftr和其他氯离子途径。 5.检验分离细胞中上皮极性是 不是由与紧密连接相关的内在蛋白质维持的， 我们将研究一个近端小管细胞的模型，它维持着 给我们上皮极性到十天，并检查细胞骨架 对保持极性至关重要的相互作用。 该项目的总体范围是了解跨皮细胞溶质 肾在单细胞膜和单通道上的运动 蛋白质水平并有助于理解临床疾病 如囊性纤维化、高血压、代谢性碱中毒、酸中毒以及 急性肾功能衰竭。

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.