RT Journal Article
SR Electronic
T1 New Insights into the Mechanism of NO<sub>3</sub><sup>-</sup> Selectivity in the Human Kidney Chloride Channel ClC-Ka and the CLC Protein Family
JF Journal of the American Society of Nephrology
JO J. Am. Soc. Nephrol.
FD American Society of Nephrology
SP 293
OP 302
DO 10.1681/ASN.2018060593
VO 30
IS 2
A1 Lagostena, Laura
A1 Zifarelli, Giovanni
A1 Picollo, Alessandra
YR 2019
UL http://jasn.asnjournals.org/content/30/2/293.abstract
AB 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 NO3− 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 (Sercen) at the center of three anion binding sites in the permeation pathway Scen. In both CLC channels and antiporters, mutations of Sercen alter the anion selectivity. Structurally, the side chain of Sercen of CLC channels and antiporters typically projects into the pore and coordinates the anion bound at Scen.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 Sercen 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.Conclusion s ClC-Ka’s mechanism of anion selectivity is largely independent of Sercen, 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.