Alteration of Extracellular pH Dependence of CLC-5 By Modifications of Two Conserved Charged Residues

Abstract

The endosomal Cl(-) / H(+) antiporter, ClC-5, is inhibited by low extracellular pH. This could be caused by the saturation of a titratable residue which is directly involved in proton translocation towards the extracellular solution; alternatively, the reduction of currents at low pH could reflect an unspecific electrostatic effect of some titratable residue on proton and / or chloride movement within the external vestibule. To gain insight into the mechanism of the pH dependence we investigated the possible involvement of two highly conserved charged residues (D76 and K210) which are located in the extracellular vestibule. Mutants D76G and D76H drastically altered pH dependence. Preliminary experiments indicated that also K210C slightly altered the pH dependence. Interestingly, the K210C mutant could be modified by pCMBS (p-chloromercuriphenylsulfonic acid): application of 100 µM pCMBS led to a partial and irreversible inhibition of K210C mediated currents (but not WT ClC-5 currents). Our results favor the idea that neither D76 nor K210 are directly involved in proton translocation. However, further experiments, exploiting the possibility to modify K210C with various cysteine modifying reagents, are necessary to draw such a firm conclusion.