Comparison of amphibian and human ClC-5: similarity of functional properties and inhibition by external pH

• Published in: The Journal of membrane biology Vol. 168; no. 3; pp. 253 - 264
• Main Authors: Mo, L, Hellmich, H L, Fong, P, Wood, T, Embesi, J, Wills, N K
• Format: Journal Article
• Language: English
• Published: United States 01-04-1999
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; Amino Acid Sequence; Animals; Anions - metabolism; Anthracenes - pharmacology; Chloride Channels - genetics; Chloride Channels - metabolism; Chlorides - metabolism; DNA, Complementary - genetics; Humans; Hydrogen-Ion Concentration; Ion Transport - drug effects; Microinjections; Molecular Sequence Data; Oocytes - drug effects; Oocytes - metabolism; ortho-Aminobenzoates - pharmacology; Protons; Recombinant Fusion Proteins - metabolism; RNA, Complementary - genetics; Species Specificity; Xenopus laevis - metabolism
• ISSN: 0022-2631; 1432-1424
• DOI: 10.1007/s002329900514

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.