Regulation of an Outwardly Rectifying Chloride Conductance in Renal Epithelial Cells by External and Internal Calcium

Abstract.

We have used perforated patch clamp and Fura-2 microfluorescence measurements to study Ca²⁺-activated Cl⁻ currents in cultured mouse renal inner medullary collecting duct cells (mIMCD-3). The conductance was spontaneously active under resting conditions and whole cell currents were time and voltage-independent with an outwardly rectifying current-voltage relationship. The channel blockers DIDS, niflumic acid, glybenclamide and NPPB reversibly decreased the basal currents, whereas the sulfhydryl agent, DTT produced an irreversible inhibition. Increasing or decreasing extracellular calcium produced parallel changes in the size of the basal currents. Variations in external Ca²⁺ were associated with corresponding changes in free cytosolic Ca²⁺ concentration. Increasing cytosolic Ca²⁺ by extracellular ATP or ionomycin, further enhanced Cl⁻ conductance, with whole cell currents displaying identical biophysical properties to the basal currents. However, the agonist-stimulated currents were now increased by DTT exposure, but still inhibited by the other channel blockers. Using RT-PCR, three distinct mRNA transcripts belonging to the CLCA family of Ca²⁺-activated Cl⁻ channel proteins were identified, two of which represent novel sequences. Whether different channels underlie the basal and agonist-stimulated currents in mIMCD-3 cells is unclear. Our findings establish a novel link between alterations in external and internal Ca²⁺ and the activity of Ca²⁺-activated Cl⁻ transport in these cells.