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J. Biol. Chem., Vol. 283, Issue 7, 4219-4227, February 15, 2008

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Determinants of Anion-Proton Coupling in Mammalian Endosomal CLC Proteins^*

Anselm A. Zdebik¹², Giovanni Zifarelli^¶1, Eun-Yeong Bergsdorf³, Paolo Soliani^¶, Olaf Scheel, Thomas J. Jentsch⁴, and Michael Pusch^¶5

From the Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), D-13125 Berlin, Germany, the Zentrum für Molekulare Neurobiologie, Universität Hamburg, D-20246 Hamburg, Germany, and the ^¶Istituto di Biofisica, Consiglio Nazionale delle Ricerche, I-16149 Genova, Italy

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. 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.

Received for publication, October 9, 2007 , and in revised form, November 16, 2007.

^* This work was supported in part by grants from the Deutsche Forschungs-gemeinschaft (to A. A. Z. and T. J. J.) and by Telethon Foundation Grant GGP04018 (to M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5 and Table 1.

¹ Both authors contributed equally to this work.

² Present address: London Epithelial Group, Royal Free Hospital, UCL, London NW 3 2PF, United Kingdom.

³ Fellow of the Leibniz Graduate School of Biophysics.

⁴ To whom correspondence may be addressed: MDC/FMP, Robert-Rössle-Str. 10, D-13125 Berlin, Germany. E-mail: jentsch{at}fmp-berlin.de.

⁵ To whom correspondence may be addressed: Istituto di Biofisica, CNR, Via de Marini 6, I-16149 Genova, Italy. E-mail: pusch{at}ge.ibf.cnr.it.

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