Determinants of anion-proton coupling in mammalian endosomal CLC proteins

Item Type:	Article
Title:	Determinants of anion-proton coupling in mammalian endosomal CLC proteins
Creators Name:	Zdebik, A.A. and Zifarelli, G. and Bergsdorf, E.Y. and Soliani, P. and Scheel, O. and Jentsch, T.J. and Pusch, M.
Abstract:	Many proteins of the CLC gene family are Cl(-) channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and ClC 5, mediate Cl(-)/H(+) exchange. Mutating a 'gating glutamate' (E224 in ClC-4; E211 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 (E281) and ClC-5 (E268), but not its replacement by 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- 4E281A and ClC-5E268A proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that WT 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. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) 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 pS. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and ClC-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 E268 to E211.
Keywords:	Amino Acid Sequence, Anions, Chloride Channels, Endosomes, Hydrogen-Ion Concentration, Molecular Sequence Data, Protons, Amino Acid Sequence Homology, Animals, Rats
Source:	Journal of Biological Chemistry
ISSN:	0021-9258
Publisher:	American Society for Biochemistry and Molecular Biology
Volume:	283
Number:	7
Page Range:	4219-4227
Date:	15 February 2008
Additional Information:	Copyright (c) 2008 by The American Society for Biochemistry and Molecular Biology
Official Publication:	https://doi.org/10.1074/jbc.M708368200
PubMed:	View item in PubMed

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