Functional characterization of recombinant human ClC-4 chloride channels in cultured mammalian cells.
Members of the ClC chloride channel family participate in several physiological processes and are linked to human genetic diseases. The physiological role of ClC-4 is unknown and previous detailed characterizations of recombinant human ClC-4 (hClC-4) have provided conflicting results. To re-examine the hClC-4 phenotype, recombinant hClC-4 was expressed in three distinct mammalian cell lines and characterized using patch-clamp techniques. In all cells, the expression of hClC-4 generated strongly outward-rectifying Cl(-) currents with the conductance sequence: SCN(-) >> NO(3)(-) >> Cl(-) > Br(-) approximate I(-) >> aspartate. Continuous activity of hClC-4 was sustained to different degrees by internal nucleotides: ATP approximately ATPgammaS >> AMP-PNP approximate GTP > ADP. Although non-hydrolysable nucleotides are sufficient for channel function, ATP hydrolysis is required for full activity. Changing the extracellular (2 mM or nominal Ca(2+)-free) or intracellular Ca(2+) (25 or 250 nM) concentration did not alter hClC-4 currents. Acidification of external pH (pH(o)) inhibited hClC-4 currents (half-maximal inhibition approximate 6.19), whereas neither external alkalinization to pH 8.4 nor internal acidification to pH 6.0 reduced current levels. Single-channel recordings demonstrated a Cl(-) channel active only at depolarizing potentials with a slope conductance of approximately 3 pS. Acidic pH(o) did not alter single-channel conductance. We conclude that recombinant hClC-4 encodes a small-conductance, nucleotide-dependent, Ca(2+)-independent outward-rectifying chloride channel that is inhibited by external acidification. This detailed characterization will be highly valuable in comparisons of hClC-4 function with native chloride channel activities and for future structure-function correlations.
Vanoye
Carlos G
CG
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
George
Alfred L
AL
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D018118:Chloride Channels / Q000096:biosynthesis* / Q000502:physiology; D004285:Dogs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009711:Nucleotides / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014162:Transfection
D018118:Chloride Channels; D009711:Nucleotides; D011994:Recombinant Proteins; D002118:Calcium