title: Direct endosomal acidification by the outwardly rectifying CLC-5 Cl(-)/H(+) exchanger.
abstract: The voltage-gated Cl(-) channel (CLC) family comprises cell surface Cl(-) channels and intracellular Cl(-)/H(+) exchangers. CLCs in organelle membranes are thought to assist acidification by providing a passive chloride conductance that electrically counterbalances H(+) accumulation. Following recent descriptions of Cl(-)/H(+) exchange activity in endosomal CLCs we have re-evaluated their role. We expressed human CLC-5 in HEK293 cells, recorded currents under a range of Cl(-) and H(+) gradients by whole-cell patch clamp, and examined the contribution of CLC-5 to endosomal acidification using a targeted pH-sensitive fluorescent protein. We found that CLC-5 only conducted outward currents, corresponding to Cl(-) flux into the cytoplasm and H(+) from the cytoplasm. Inward currents were never observed, despite the range of intracellular and extracellular Cl(-) concentrations and pH used. Endosomal acidification in HEK293 cells was prevented by 25 microm bafilomycin-A1, an inhibitor of vacuolar-type H(+)-ATPase (v-ATPase), which actively pumps H(+) into the endosomal lumen. Overexpression of CLC-5 in HEK293 cells conferred an additional bafilomycin-insensitive component to endosomal acidification. This effect was abolished by making mutations in CLC-5 that remove H(+) transport, which result in either no current (E268A) or bidirectional Cl(-) flux (E211A). Endosomal acidification in a proximal tubule cell line was partially sensitive to inhibition of v-ATPase by bafilomycin-A1. Furthermore, in the presence of bafilomycin-A1, acidification was significantly reduced and nearly fully ablated by partial and near-complete knockdown of endogenous CLC-5 by siRNA. These data suggest that CLC-5 is directly involved in endosomal acidification by exchanging endosomal Cl(-) for H(+).
authors.affiliation: Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Smith
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Lippiat
mesh_terms: D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D009154:Mutation; D018408:Patch-Clamp Techniques; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C097503:CLC-5 chloride channel; D018118:Chloride Channels; D002712:Chlorides; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D018942:Macrolides; D011993:Recombinant Fusion Proteins; C040929:bafilomycin A1; D025262:Vacuolar Proton-Translocating ATPases