Direct endosomal acidification by the outwardly rectifying CLC-5 Cl-/H+ exchanger

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 mu m 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+.