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Received August 8, 2001
Accepted after revision November 6, 2001
1 Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
2 Division of Genetic Medicine, 451 Preston Research Building, Vanderbilt University, Nashville, TN 37232-6304 USA
* To whom correspondence should be addressed. E-mail: al.george{at}vanderbilt.edu.
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 ATP
S >> 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 (pHo) 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 pHo 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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