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, agenetic disorder characterized by low weight proteinuria, hypercalciuria, nephrolithasis and, in some cases, eventual r enal failure. Recently, our laboratory used an RT-PCR/RACE cloning strategy to isolate an amphibian cDNA from the renal epithelial cell line A6 that h ad high homology to human ClC-5. We now report a full-length native ClC-5 c lone (xClC-5, containing 5' and 3' untranslated regions) isolated by screen ing 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 usin g isogenic constructs of xClC-5 and hClC-5 consisting of the open reading f rame subcloned into an optimized Xenopus expression vector. Expression of t he full-length "native" xClC-5 clone resulted in large, strongly rectifying , outward currents that were not significantly affected by the chloride cha nnel blockers DIDS, DPC, and 9AC. The anion conductivity sequence was NO3- > Cl- = I- > HCO3- >> glutamate for xClC-5 and NO3- > Cl- > HCO3- > I- >> g lutamate for hClC-5. Reduction of the extracellular pH (pH(o)) 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 hi ghly similar functional properties. Unlike hClC-5 and most other ClC channe ls, expression of xClC-5 in oocytes does not require the removal of its unt ranslated 5' and 3' regions. Acidic solutions inhibited both amphibian and human ClC-5 currents, opposite to the stimulatory effects of low external p H on other ClC channels, suggesting a possibly distinct regulatory mechanis m for ClC-5 channels.