Conversion of the 2 Cl(-)/1 H+ antiporter ClC-5 in a NO3(-)/H+ antiporter by a single point mutation

EMBO J. 2009 Feb 4;28(3):175-82. doi: 10.1038/emboj.2008.284. Epub 2009 Jan 8.


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 approximately 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Anion Transport Proteins / metabolism*
  • Antiporters / metabolism*
  • Chloride Channels / chemistry
  • Chloride Channels / metabolism*
  • Chlorides / metabolism*
  • Electric Conductivity
  • Fluorescence
  • Humans
  • Hydrogen-Ion Concentration
  • Intracellular Space / metabolism
  • Molecular Sequence Data
  • Mutant Proteins / chemistry
  • Nitrates / metabolism*
  • Point Mutation / genetics*
  • Protein Transport
  • Protons*
  • Reproducibility of Results
  • Sucrose / metabolism
  • Xenopus


  • Anion Transport Proteins
  • Antiporters
  • CLC-5 chloride channel
  • Chloride Channels
  • Chlorides
  • Mutant Proteins
  • Nitrates
  • Protons
  • nitrate transporters
  • Sucrose