The ClC-3 Cl-/H+ antiporter becomes uncoupled at low extracellular pH.
Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl(-)/H(+) antiport current (I(ClC-3)) in HEK293 cells. The outward rectification and time dependence of I(ClC-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH <or= 5.5. ICl(acid) 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. ICl(acid) was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E(rev)) of I(ClC-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 E(rev) was completely Cl(-) dependent (55.8 +/- 2.3 mV/decade). Several findings linked ClC-3 with native ICl(acid); 1) RNA interference directed at ClC-3 message reduced native ICl(acid); 2) removal of the extracellular "fast gate" (E224A) produced large currents that were pH-insensitive; and 3) wild-type I(ClC-3) and ICl(acid) were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10-500 microm)-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 I(ClC-3) and from native ICl(acid) by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl(acid). 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.
Matsuda
James J
JJ
Department of Pediatrics, University of Iowa Children's Hospital, Iowa City, Iowa 52242, USA.
Filali
Mohammed S
MS
Collins
Malia M
MM
Volk
Kenneth A
KA
Lamb
Fred S
FS
D000143:Acids / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000166:cytology; D002451:Cell Compartmentation / Q000502:physiology; D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008698:Mesylates / Q000494:pharmacology; D051379:Mice; D008817:Mice, Mutant Strains; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D016296:Mutagenesis; D018408:Patch-Clamp Techniques; D034741:RNA, Small Interfering; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000143:Acids; D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D008698:Mesylates; D034741:RNA, Small Interfering; D013439:Sulfhydryl Reagents; D049452:Green Fluorescent Proteins; C109153:(2-sulfonatoethyl)methanethiosulfonate; D018698:Glutamic Acid; D006859:Hydrogen