title: The late endosomal ClC-6 mediates proton/chloride countertransport in heterologous plasma membrane expression.
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.
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie FMP and Max-Delbrück-Centrum für Molekulare Medizin, D-13125 Berlin, Germany.
authors.forename: Ioana
authors.initials: I
authors.lastname: Neagoe
authors.forename: Tobias
authors.initials: T
authors.lastname: Stauber
authors.forename: Pawel
authors.initials: P
authors.lastname: Fidzinski
authors.forename: Eun-Yeong
authors.initials: EY
authors.lastname: Bergsdorf
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Jentsch
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism* / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D011992:Endosomes / Q000378:metabolism*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006706:Homeostasis; D007477:Ions; D008954:Models, Biological; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011522:Protons; D014982:Xenopus laevis
chemical_list: C097539:CLCN6 protein, human; D018118:Chloride Channels; D007477:Ions; D011522:Protons; D049452:Green Fluorescent Proteins
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