The Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

Members of the CLC protein family of Cl⁻ channels and transporters display the remarkable ability to function as either chloride channels or Cl⁻/H⁺ antiporters. Due 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 recent 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. As observed in other CLC exchangers, ClC-6-mediated H⁺ transport was abolished by mutations in either the “gating” or “proton” glutamate. Overexpression 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.