Comparison of amphibian and human ClC-5: similarity of functional properties and inhibition by external pH.
Loss of function mutations of the renal chloride channel, ClC-5, have been implicated in Dent's disease, a genetic disorder characterized by low weight proteinuria, hypercalciuria, nephrolithasis and, in some cases, eventual renal failure. Recently, our laboratory used an RT-PCR/RACE cloning strategy to isolate an amphibian cDNA from the renal epithelial cell line A6 that had high homology to human ClC-5. We now report a full-length native ClC-5 clone (xClC-5, containing 5' and 3' untranslated regions) isolated by screening a cDNA library from A6 cells that was successfully expressed in Xenopus oocytes. In addition, we compared the properties of xClC-5 and hClC-5 using isogenic constructs of xClC-5 and hClC-5 consisting of the open reading frame subcloned into an optimized Xenopus expression vector. Expression of the full-length "native" xClC-5 clone resulted in large, strongly rectifying, outward currents that were not significantly affected by the chloride channel blockers DIDS, DPC, and 9AC. The anion conductivity sequence was NO-3 > Cl- = I- > HCO-3 >> glutamate for xClC-5 and NO-3 > Cl- > HCO-3 > I- >> glutamate for hClC-5. Reduction of the extracellular pH (pHo) from 7.5 to 5.7 inhibited outward ClC-5 currents by 27 +/- 9% for xClC-5 and 39 +/- 7% for hClC-5. The results indicate that amphibian and mammalian ClC-5 have highly similar functional properties. Unlike hClC-5 and most other ClC channels, expression of xClC-5 in oocytes does not require the removal of its untranslated 5' and 3' regions. Acidic solutions inhibited both amphibian and human ClC-5 currents, opposite to the stimulatory effects of low external pH on other ClC channels, suggesting a possibly distinct regulatory mechanism for ClC-5 channels.
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Departments of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.
Hellmich
H L
HL
Fong
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P
Wood
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T
Embesi
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J
Wills
N K
NK
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions / Q000378:metabolism; D000873:Anthracenes / Q000494:pharmacology; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000187:drug effects*; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011522:Protons; D018075:RNA, Complementary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D014982:Xenopus laevis / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D000838:Anions; D000873:Anthracenes; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D002712:Chlorides; D018076:DNA, Complementary; D011522:Protons; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D062367:ortho-Aminobenzoates; C010030:9-anthroic acid; C001209:fenamic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid