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

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 AtClC-a was expressed in Xenopus oocytes, it mediated NO3-/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 NO3-. 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 NO3-transport is largely uncoupled from protons. By contrast, the AtClC-amediated NO3- accumulation in plant vacuoles requires tight NO3-/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 NO3-/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.