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

Significance Statement The human chloride channels ClC-Ka and ClC-Kb, in complex with their accessory subunit barttin, are essential for NaCl reabsorption in the nephron. Mutations of ClC-Ka, ClC-Kb, or barttin cause type III and IV Bartter syndrome, characterized by renal salt wasting, hypokalemic metabolic alkalosis, and other effects. Using electrophysiologic techniques and mutational analysis, the authors show that the mechanism of discriminating Cl − versus NO 3 − anions in the human ClC-Ka channel is unique in the CLC protein family, and they identified previously unimplicated residues as responsible for anion selectivity. This represents significant progress in better understanding of the relationship between structure and function of kidney channels ClC-Ka and ClC-Kb and of CLC proteins in general. Background 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 . Methods 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. Results 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 NO 3 − /Cl − permeability ratio, with Y425A having a particularly strong effect. Conclusion 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 NO 3 − 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.