The ClC-3 Cl−/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl−/H+ antiport current (IClC-3) in HEK293 cells. The outward rectification and time dependence of IClC-3 closely resemble an endogenous HEK293 cell acid-activated Cl− current (IClacid) seen at extracellular pH ≤ 5.5. IClacid was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. IClacid was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (Erev) of IClC-3 between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl− and H+ transport become uncoupled at low pH. At pH 4.0 Erev was completely Cl− dependent (55.8 ± 2.3 mV/decade). Several findings linked ClC-3 with native IClacid; 1) RNA interference directed at ClC-3 message reduced native IClacid; 2) removal of the extracellular “fast gate” (E224A) produced large currents that were pH-insensitive; and 3) wild-type IClC-3 and IClacid were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10–500 μm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type IClC-3 and from native IClacid by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native IClacid. Low pH uncouples Cl−/H+ transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl−/H+ antiporters and perhaps for pH regulation in highly acidic intracellular compartments.