ClC-K2, a member of the ClC family of Cl − channels and transporters, forms the major basolateral Cl − conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl − absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl − , and Ca 2+ on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pH i ) and extracellular pH (pH o ) differentially modulate the voltage dependence curve: alkaline pH i flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pH o shifts the curve toward negative voltages. In addition, pH i , pH o , and extracellular Ca 2+ strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl − has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pH i and pH o on ClC-K2, might be a key regulator of NaCl absorption and Cl − /HCO 3 − exchange in type B intercalated cells.

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na + -Cl − cotransporter located at the apical membrane, and Na + , K + ATPase at the basolateral side. Cl − transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl − channels on microdissected mouse DCTs using the patch-clamp technique. A channel of ∼9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NP o in cell-attached patches was not modified when tubules were preincubated in the presence of 10 −5 M forskolin, but the channel was inhibited by phorbol ester (10 −6 M). In addition, NP o was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM ( NP o increased threefold), or pH increased from 6.4 to 8.0 ( NP o increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na + to Cl − relative permeability was 0.09, and the anion selectivity sequence Cl − ∼ I − > Br − ∼ NO 3 − > F − . Intracellular NPPB (10 −4 M) and DPC (10 −3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl − channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.