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Residues important for nitrate/proton coupling in plant and mammalian CLC transporters

Item Type:Article
Title:Residues important for nitrate/proton coupling in plant and mammalian CLC transporters
Creators Name:Bergsdorf, E.Y. and Zdebik, A.A. and Jentsch, T.J.
Abstract:Members of the CLC gene family either function as chloride channels or as anion/proton exchangers. The plant AtClC-a uses the pH gradient across the vacuolar membrane to accumulate the nutrient NO3- in this organelle. When Arabidopsis thaliana AtClC-a was expressed in Xenopus oocytes, it mediated NO3-/H+- and less efficiently Cl-/H+-exchange. Mutating the 'gating glutamate' E203 to alanine resulted in an uncoupled anion conductance that was larger for Cl- than NO3-. Replacing the 'proton glutamate' E270 by alanine abolished currents. These could be restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4 and ClC-5 mediate stoichiometrically coupled 2Cl-/H+-exchange, their NO3- transport is largely uncoupled from protons. By contrast, the AtClC-a-mediated NO3- accumulation in plant vacuoles requires tight NO3- to H+-coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this proline was mutated to serine (P160S), Cl-/H+ exchange of AtClC-a proceeded as efficiently as NO not3-/H+-exchange, suggesting a role of this residue in NO3-/H+-exchange. Indeed, when the corresponding serine of ClC-5 was replaced by proline, this Cl-/H+-exchanger gained efficient NO3-/H+-coupling. When inserted into the model Torpedo chloride channel ClC-0, the equivalent mutation increased nitrate relative to chloride conductance. Hence, proline in the CLC pore signature sequence is important for NO3-/H+-exchange and NO3- conductance both in plants and mammals. Gating and proton glutamates play similar roles in bacterial, plant, and mammalian CLC anion/proton exchangers.
Keywords:Amino Acid Sequence, Anions, Arabidopsis, Chloride Channels, Hydrogen-Ion Concentration, Mammals, Molecular Sequence Data, Nitrates, Oocytes, Patch-Clamp Techniques, Amino Acid Sequence Homology, Torpedo, Animals, Xenopus Laevis
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:284
Number:17
Page Range:11184-11193
Date:24 April 2009
Official Publication:https://doi.org/10.1074/jbc.M901170200
PubMed:View item in PubMed

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