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

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Abstract
ClC-3 is a member of the ClC family of anion channels/transporters. Recently, the closely related proteins ClC-4 and ClC-5 were shown to be Cl−/H+antiporters ( 39 , 44 ). The function of ClC-3 has been controversial. We studied anion currents in HEK293T cells expressing wild-type or mutant ClC-3. The basic biophysical properties of ClC-3 currents were very similar to those of ClC-4 and ClC-5, and distinct from those of the swelling-activated anion channel. ClC-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°C. Manipulation of extracellular pH (6.35–8.2) altered reversal potential by 10.2 ± 3.0 mV/decade, suggesting that ClC-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 ClC Cl−/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 ClC-3 overexpression induced a pH-sensitive conductance in HEK293T cells that is biophysically similar to ClC-4 and ClC-5.