Conversion of the 2 Cl(-)/1 H+ antiporter ClC-5 in a NO3(-)/H+ antiporter by a single point mutation.
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.
Zifarelli
Giovanni
G
Istituto di Biofisica, CNR, Via De Marini, Genova, Italy.
Pusch
Michael
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D027321:Anion Transport Proteins / Q000378:metabolism*; D017920:Antiporters / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004553:Electric Conductivity; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D000091285:Nitrate Transporters; D009566:Nitrates / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics*; D021381:Protein Transport; D011522:Protons; D015203:Reproducibility of Results; D013395:Sucrose / Q000378:metabolism; D014981:Xenopus
D027321:Anion Transport Proteins; D017920:Antiporters; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D002712:Chlorides; D050505:Mutant Proteins; D000091285:Nitrate Transporters; D009566:Nitrates; D011522:Protons; D013395:Sucrose