ClC-4 and ClC-5 are members of the CLC gene family1, with ClC-5 mutated in Dent's disease2, a nephropathy associated with low-molecular-mass proteinuria and eventual renal failure. ClC-5 has been proposed to be an electrically shunting Cl- channel in early endosomes, facilitating intraluminal acidification3,4. Motivated by the discovery that certain bacterial CLC proteins are secondary active Cl-/H+ antiporters5, we hypothesized that mammalian CLC proteins might not be classical Cl- ion channels but might exhibit Cl--coupled proton transport activity. Here we report that ClC-4 and ClC-5 carry a substantial amount of protons across the plasma membrane when activated by positive voltages, as revealed by measurements of pH close to the cell surface. Both proteins are able to extrude protons against their electrochemical gradient, demonstrating secondary active transport. H+, but not Cl-, transport was abolished when a pore glutamate was mutated to alanine (E211A). ClC-0, ClC-2 and ClC-Ka proteins showed no significant proton transport. The muscle channel ClC-1 exhibited a small H+ transport that might be physiologically relevant. For ClC-5, we estimated that Cl- and H+ transport contribute about equally to the total charge movement, raising the possibility that the coupled Cl-/H+ transport of ClC-4 and ClC-5 is of significant magnitude in vivo.
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We thank L. Elia for technical assistance, T. Jentsch and A. Zdebik for providing the ClC-0, ClC-1, ClC-2(Δ16–61), ClC-3, ClC-4, ClC-Ka, barttin and ClC-5 cDNAs, E. Gaggero and G. Gaggero for constructing the high-impedance amplifier, and G. Gaggero for help in constructing the measuring chamber. Financial support by Telethon Italy and the Italian Research Ministry is gratefully acknowledged.
Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
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Picollo, A., Pusch, M. Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 436, 420–423 (2005) doi:10.1038/nature03720
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