REGULATION OF ION CHANNELS IN RENAL PROXIMAL TUBULE

近端小管维持体内平衡电解质和 面对饮食摄入量的急剧变化和水的再吸收 肾血流动力学显示跨细胞离子转运紧密 受监管的。过滤的钠进入细胞的一部分是顶端的Na-H 反向搬运工。阴离子转运蛋白与顶端Na-H逆向转运蛋白的偶联 结果导致细胞内氯的积累超过了预期 电化学平衡，为跨细胞提供动力 氯通过基底膜(BLM)从细胞移动到血液。 氯离子外流的确切性质仍然存在争议，主要是由于 直接观察发生在上的过程的内在困难 博洛姆。在该项目的第一阶段，假设氯化物存在 通过应用膜片钳来研究离子通道的发生 新鲜分离单个近端小管细胞的技术。这些细胞 值得注意的是，它们保留了上皮的极性，因此 BLM可通过贴片移液管访问，允许单次活动 氯离子通道可直接观察。使用全细胞钳位 配置时，将测量总氯电流，以便总体 跨细胞的氯离子重吸收可以被量化。一种钾 BLM上也存在渗透性，以允许K被泵入细胞 Na-K-ATPase循环。这种渗透性可能由几种类型的 通道，正如初步数据显示的，钾选择性通道和 一种非选择性阳离子通道。来自这些BLM的单通道录制 通道允许表征这种电导并研究 泵动力学和泵动力学之间存在功能联系的思想 阳离子通道活性。氯离子和钾离子通道的调节 细胞内第二信使和其他信号转导系统将 也可以在第一阶段使用单通道(电池连接和 切除补片)、常规全细胞和穿孔全细胞钳 特定激活剂和抑制剂的存在。附连的细胞和 穿孔贴片技术，其中细胞内环境是 保存下来的，将用于研究激素和细胞内的调节 第二个信使。该项目第二阶段将继续把重点放在 对跨细胞运输的调节，但将延伸到 这些BLM通道在细胞容量调节中起作用。这样做的最终目的是 该项目旨在加深我们对上皮细胞离子通道的了解 调节以及通道和其他膜运输过程是如何 结合起来协调整个细胞的运输。这可能已经 对高血压、代谢等临床疾病产生重大影响 碱中毒/酸中毒和钾平衡。

The ability of the proximal tubule to maintain homeostatic electrolyte and water reabsorption in the face of drastic changes in dietary intake and renal hemodynamics suggests that transcellular ion transport is tightly regulated. Filtered sodium enters the cell in part with apical Na-H antiporter. Coupling of anion transporters to the apical Na-H antiporter results in the intracellular accumulation of chloride above its predicted electrochemical equilibrium, providing a driving force for transcellular chloride movement from cell to blood across the basolateral membrane (BLM). The precise nature of chloride efflux remains controversial, primarily due to the intrinsic difficulties of directly observing processes occurring on the BLM. In Phase I of this project the hypothesis that chloride exit occurs through an ion channel will be studied by applying the patch-clamp technique to freshly isolated single proximal tubule cells. These cells are remarkable in that they retain their epithelial polarity so that the BLM is accessible to a patch pipette, allowing the activity of single chloride channels to be directly observed. Using the whole-cell clamp configuration, the total chloride current will be measured so that overall transcellular chloride reabsorption can be quantified. A potassium permeability also exists on the BLM to allow K pumped into the cell by the Na-K ATPase to recycle. This permeability may consist of several types of channels, as preliminary data show both a potassium selective channel and a non-selective cation channel. Single-channel recordings from these BLM channels allow characterization of this conductance and investigation of the functional linkage thought to exist between the pump kinetics and cation channel activity. Regulation of chloride and potassium channels by intracellular second messengers and other signal transduction systems will also be investigated during Phase I using single-channel (cell-attached and excised patch), conventional whole-cell, and perforated whole-cell clamp in the presence of specific activators and inhibitors. The cell-attached and perforated patch techniques, in which the intracellular milieu is preserved, will be used to study regulation by hormones and intracellular second messengers. Phase II of the project will continue to focus on regulation of transcellular transport but will extend into the role of these BLM channels in cell volume regulation. The ultimate goal of this project is to enhance our understanding of epithelial ion channel regulation and how channels and other membrane transport processes are coupled to coordinate overall cellular transport. This may have significant impact on clinical disorders such as hypertension, metabolic alkalosis/acidosis, and potassium balance.