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 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 AtClC-a-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)(-)/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 NO(3)(-)/H(+) exchange and NO(3)(-) conductance both in plants and mammals. Gating and proton glutamates play similar roles in bacterial, plant, and mammalian CLC anion/proton exchangers.
Bergsdorf
Eun-Yeong
EY
Department of Physiology and Pathology of Ion Transport, Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), D-13125 Berlin, Germany.
Zdebik
Anselm A
AA
Jentsch
Thomas J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D017360:Arabidopsis / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008322:Mammals / Q000378:metabolism; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017386:Sequence Homology, Amino Acid; D014101:Torpedo; D014982:Xenopus laevis / Q000378:metabolism
D000838:Anions; C086321:CLC-1 channel; D018118:Chloride Channels; D009566:Nitrates