title: Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent.
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 degrees 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.
authors.affiliation: Department of Pediatrics, University of Iowa Children's Hospital, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Matsuda
authors.forename: Mohammed S
authors.initials: MS
authors.lastname: Filali
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Volk
authors.forename: Malia M
authors.initials: MM
authors.lastname: Collins
authors.forename: Jessica G
authors.initials: JG
authors.lastname: Moreland
authors.forename: Fred S
authors.initials: FS
authors.lastname: Lamb
mesh_terms: D002460:Cell Line; D018118:Chloride Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D009154:Mutation; D010693:Phloretin / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013629:Tamoxifen / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
chemical_list: D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; D018698:Glutamic Acid; D010693:Phloretin