Regulation of an outwardly rectifying chloride conductance in renal epithelial cells by external and internal calcium

We have used perforated patch clamp and Fura-2 microfluorescence measuremen ts to study Ca2+-activated Cl- currents in cultured mouse renal inner medul lary collecting duct cells (mIMCD-3). The conductance was spontaneously act ive 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 dec reased the basal currents, whereas the sulfhydryl agent, DTT produced an ir reversible inhibition. Increasing or decreasing extracellular calcium produ ced parallel changes in the size of the basal currents. Variations in exter nal Ca2+ were associated with corresponding changes in free cytosolic Ca2concentration. Increasing cytosolic Ca2+ by extracellular ATP or ionomycin, further enhanced Cl- conductance, with whole cell currents displaying iden tical biophysical properties to the basal currents. However, the agonist-st imulated currents were now increased by DTT exposure, but still inhibited b y the other channel blockers. Using RT-PCR, three distinct mRNA transcripts belonging to the CLCA family of Ca2+-activated Cl- channel proteins were i dentified, two of which represent novel sequences. Whether different channe ls underlie the basal and agonist-stimulated currents in mIMCD-3 cells is u nclear. Our findings establish a novel link between alterations in external and internal Ca2+ and the activity of Ca2+-activated Cl- transport in thes e cells.