- I. Neagoe
- T. Stauber
- P. Fidzinski
- E.Y. Bergsdorf
- T.J. Jentsch
- Journal of Biological Chemistry
- J Biol Chem 285 (28): 21689-21697
Members of the CLC protein family display the remarkable ability to function as either chloride channels or Cl-/H+ antiporters. Owed to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas indirect data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here we report that fusing the green fluorescent protein (GFP) to the N-terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl-/H+-exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. Like observed in other CLC exchangers, ClC-6-mediated H+-transport was abolished by mutations in either the gating or proton glutamate. Over-expression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl- > I- conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the anion-coordinating tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO3- conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl-/H+ antiporter.