Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

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 NO 3 in this organelle. When AtClC-a was expressed in Xenopus oocytes, it mediated NO 3 /H exchange and less efficiently mediated Cl /H exchange. Mutating the "gating glutamate" Glu-203 to alanine resulted in an uncoupled anion conductance that was larger for Cl than NO 3 . Replacing the "proton glutamate" Glu-270 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 NO 3 transport is largely uncoupled from protons. By contrast, the AtClCa-mediated NO 3 accumulation in plant vacuoles requires tight NO 3 /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 3 /H exchange, suggesting a role of this residue in NO 3 /H exchange. Indeed, when the corresponding serine of ClC-5 was replaced by proline, this Cl /H exchanger gained efficient NO 3