Conversion of the 2 Cl−/1 H+ antiporter ClC-5 in a NO3−/H+ antiporter by a single point mutation
Open Access
- 8 January 2009
- journal article
- research article
- Published by Springer Science and Business Media LLC in The EMBO Journal
- Vol. 28 (3), 175-182
- https://doi.org/10.1038/emboj.2008.284
Abstract
Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC‐5 protein that is mutated in Dent's disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl−/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3−/H+ antiporter atClCa, led to coupled NO3−/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3−/H+ exchange. We further found that ClC‐5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.Keywords
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