Determinants of anion-proton coupling in mammalian endosomal CLC proteins

Many proteins of the CLC gene family are Cl- channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl-/H- exchange. Mutating a gating glutamate (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular proton glutamate in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl- and H+ transport. Surface expression was unchanged by these mutations. Uncoupled Cl- transport could be restored in the ClC-4(E281A) and ClC-5(E268A) proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H (+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl-/H- exchange independently from the transport activity of the neighboring subunit. NO3- or SCN- transport was partially uncoupled from H+ countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl-/H+ exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl-/H+ exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211.