New Insights into the Mechanism of NO₃^- Selectivity in the Human Kidney Chloride Channel ClC-Ka and the CLC Protein Family.

Abstract

The mechanism of anion selectivity in the human kidney chloride channels ClC-Ka and ClC-Kb is unknown. However, it has been thought to be very similar to that of other channels and antiporters of the CLC protein family, and to rely on anions interacting with a conserved Ser residue (Ser_cen) at the center of three anion binding sites in the permeation pathway S_cen. In both CLC channels and antiporters, mutations of Ser_cen alter the anion selectivity. Structurally, the side chain of Ser_cen of CLC channels and antiporters typically projects into the pore and coordinates the anion bound at S_cen. To investigate the role of several residues in anion selectivity of ClC-Ka, we created mutations that resulted in amino acid substitutions in these residues. We also used electrophysiologic techniques to assess the properties of the mutants. Mutations in ClC-Ka that change Ser_cen to Gly, Pro, or Thr have only minor effects on anion selectivity, whereas the mutations in residues Y425A, F519A, and Y520A increase the NO₃^-/Cl^- permeability ratio, with Y425A having a particularly strong effect. s ClC-Ka's mechanism of anion selectivity is largely independent of Ser_cen, and it is therefore unique in the CLC protein family. We identified the residue Y425 in ClC-Ka-and the corresponding residue (A417) in the chloride channel ClC-0-as residues that contribute to NO₃^- discrimination in these channels. This work provides important and timely insight into the relationship between structure and function for the kidney chloride channels ClC-Ka and ClC-Kb, and for CLC proteins in general.