Comparison of Amphibian and Human ClC-5: Similarity of Functional Properties and Inhibition by External pH |
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Authors: | L Mo H L Hellmich P Fong T Wood J Embesi NK Wills |
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Institution: | (1) Departments of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA, US;(2) Internal Medicine, Division of Gastroenterology, University of Texas Medical Branch, Galveston, TX 77555, USA, US;(3) Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555, USA, US;(4) Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510, USA, US |
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Abstract: | 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 (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 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.
Received: 28 August 1998/Revised: 13 January 1999 |
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Keywords: | : Chloride channel — ClC-5 — Human — Xenopus laevis oocytes — Amphibian renal cell line — Outward rectification — pH |
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