REGULATION OF ION CHANNELS IN RENAL PROXIMAL TUBULE

近端小管维持体内电解质平衡的能力， 面对饮食摄入量的急剧变化时的水分重吸收， 肾血流动力学表明，跨细胞离子转运是紧密的， 监管. 过滤后的钠与顶端的Na-H一起部分进入细胞 反向转运蛋白。 阴离子转运体与顶端Na-H反向转运体的偶联 导致细胞内氯化物的积累超过其预测值 电化学平衡，为跨细胞的 氯离子从细胞穿过基底外侧膜（BLM）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 直接观察发生在地球上的过程的内在困难 BLM。 在该项目的第一阶段，假设氯化物存在 将通过应用膜片钳技术研究通过离子通道发生的 技术，以新鲜分离的单个近端小管细胞。 这些细胞 它们保持了上皮细胞的极性， BLM可通过贴片移液器进入，允许单个BLM的活性 可直接观察氯离子通道。 使用全细胞钳 配置时，将测量总氯化物电流， 可以量化跨细胞氯重吸收。 钾 渗透性也存在于BLM上，以允许K通过膜泵入细胞中。 Na-K ATP酶进行再循环。 这种渗透性可以由几种类型的 通道，因为初步数据显示钾选择性通道和 非选择性阳离子通道。 这些BLM的单通道记录 通道允许表征这种电导和调查 被认为存在于泵动力学和 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它信号转导系统将 也可以在第一阶段使用单通道（细胞附着和 切除的膜片）、常规的全细胞和穿孔的全细胞钳夹。 特定激活剂和抑制剂的存在。 细胞附着和 穿孔贴片技术，其中细胞内环境是 保存，将用于研究激素和细胞内的调节 第二信使 项目第二阶段将继续侧重于 调节跨细胞运输，但将扩展到的作用， 这些BLM通道在细胞体积调节中的作用。 这个项目的最终目标 该项目旨在提高我们对上皮细胞离子通道的理解 调节以及通道和其他膜转运过程是如何 耦合以协调整个蜂窝传输。 这可能 对高血压、代谢性疾病等临床疾病的显著影响 酸中毒/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.