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(+).
Pubmed ID: 20421284 RIS Download
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Software suite for electrophysiology data acquisition and analysis by Molecular Devices. Used for the control and recording of voltage clamp, current clamp, and patch clamp experiments. The software suite consists of Clampex 11 Software for data acquisition, AxoScope 11 Software for background recording, Clampfit 11 Software for data analysis, and optional Clampfit Advanced Analysis Module for sophisticated and streamlined analysis.
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