The late endosomal ClC-6 mediates proton/chloride countertransport in heterologous plasma membrane expression

J Biol Chem. 2010 Jul 9;285(28):21689-97. doi: 10.1074/jbc.M110.125971. Epub 2010 May 13.

Abstract

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 NO(3)(-) conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl(-)/H(+) antiporter.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cell Membrane / metabolism*
  • Chloride Channels / metabolism*
  • Chloride Channels / physiology
  • Cricetinae
  • Cricetulus
  • Endosomes / metabolism*
  • Gene Expression Regulation*
  • Green Fluorescent Proteins / metabolism
  • Homeostasis
  • Ions
  • Models, Biological
  • Mutation
  • Oocytes / metabolism
  • Patch-Clamp Techniques
  • Protons
  • Xenopus laevis

Substances

  • CLCN6 protein, human
  • Chloride Channels
  • Ions
  • Protons
  • Green Fluorescent Proteins