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Functional characterization of recombinant human ClC-4 chloride channels in cultured mammalian cells

Carlos G. Vanoye and Alfred L. George Jr

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^- >> NO₃^- >> Cl^- > Br^- I^- >> aspartate. Continuous activity of hClC-4 was sustained to different degrees by internal nucleotides: ATP ATPS >> AMP-PNP 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 Ca²⁺-free) or intracellular Ca²⁺ (25 or 250 nM) concentration did not alter hClC-4 currents. Acidification of external pH (pH_o) inhibited hClC-4 currents (half-maximal inhibition 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 ~3 pS. Acidic pH_o did not alter single-channel conductance. We conclude that recombinant hClC-4 encodes a small-conductance, nucleotide-dependent, Ca²⁺-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.

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