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.
