title: Extracellular determinants of anion discrimination of the Cl-/H+ antiporter protein CLC-5.
abstract: Mammalian CLC proteins comprise both Cl- channels and Cl-/H+ antiporters that carry out fundamental physiological tasks by transporting Cl- across plasma membrane and intracellular compartments. The NO3- over Cl- preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl-/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3- over Cl- preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl-. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext.
authors.affiliation: Istituto di Biofisica, CNR, Via De Marini 6, I-16149 Genova, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: De Stefano
authors.affiliation: Istituto di Biofisica, CNR, Via De Marini 6, I-16149 Genova, Italy.
authors.forename: Michael
authors.initials: M
authors.lastname: Pusch
authors.affiliation: Istituto di Biofisica, CNR, Via De Marini 6, I-16149 Genova, Italy. Electronic address: zifarelli@ge.ibf.cnr.it.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Zifarelli
mesh_terms: D000818:Animals; D000838:Anions; D001703:Biophysics / Q000379:methods; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000737:chemistry; D004252:DNA Mutational Analysis; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D009865:Oocytes / Q000166:cytology; D018408:Patch-Clamp Techniques; D011522:Protons; D014982:Xenopus laevis
chemical_list: D000838:Anions; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D002712:Chlorides; D011522:Protons; D008239:Lysine