Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent

C1C-3 is a member of the C1C family of anion channels/transporters. Recently, the closely related proteins C1C-4 and C1C-5 were shown to be Cl-/H+ antiporters (39, 44). The function of C1C-3 has been controversial. We studied anion currents in HEK293T cells expressing wild-type or mutant C1C-3. The basic biophysical properties of C1C-3 currents were very similar to those of C1C-4 and C1C-5, and distinct from those of the swelling-activated anion channel. C1C-3 expression induced currents with time-dependent activation that rectified sharply in the outward direction. The reversal potential of the current shifted by -48.3 +/- 2.5 mV per 10-fold (decade) change in extracellular Cl-concentration, which did not conform to the behavior of an anion-selective channel based upon the Nernst equation, which predicts a -58.4 mV/decade shift at 22 degrees C. Manipulation of extracellular pH (6.35 - 8.2) altered reversal potential by 10.2 +/- 3.0 mV/decade, suggesting that C1C-3 currents were coupled to proton movement. Mutation of a specific glutamate residue (E224A) changed voltage dependence in a manner similar to that observed in other C1C C1(-)/H+ antiporters. Mutant currents exhibited Nernstian changes in reversal potential in response to altered extracellular Cl- concentration that averaged -60 +/- 3.4 mV/decade and were pH independent. Thus C1C-3 overexpression induced a pH-sensitive conductance in HEK293T cells that is biophysically similar to C1C-4 and C1C-5.