Molecular determinants of differential pore blocking of kidney CLC-K chloride channels

EMBO Rep. 2004 Jun;5(6):584-9. doi: 10.1038/sj.embor.7400169. Epub 2004 May 28.


The highly homologous Cl(-) channels CLC-Ka and CLC-Kb are important for water and salt conservation in the kidney and for the production of endolymph in the inner ear. Mutations in CLC-Kb lead to Bartter's syndrome and mutations in the small CLC-K subunit barttin lead to Bartter's syndrome and deafness. Here we show that CLC-Ka is blocked by the recently identified blocker 2-(p-chlorophenoxy)-3-phenylpropionic acid of the rat channel CLC-K1 with an apparent K(D) approximately 80 microM. We also found that DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), a generic Cl(-) channel blocker, inhibits CLC-Ka (K(D) approximately 90 microM). Surprisingly, the highly homologous channel CLC-Kb is fivefold to sixfold less sensitive to both compounds. Guided by the crystal structure of bacterial CLC proteins, we identify two amino acids, N68/D68 and G72/E72, in CLC-Ka and CLC-Kb, respectively, that are responsible for the differential drug sensitivity. Both residues expose their side chains in the extracellular pore mouth, delineating the probable drug binding site. These novel CLC-K channel blockers are promising lead compounds for the development of new diuretic drugs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2-Methyl-4-chlorophenoxyacetic Acid / analogs & derivatives*
  • 2-Methyl-4-chlorophenoxyacetic Acid / chemistry
  • 2-Methyl-4-chlorophenoxyacetic Acid / pharmacology*
  • 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / chemistry
  • 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / pharmacology*
  • Amino Acid Motifs / genetics
  • Anion Transport Proteins / antagonists & inhibitors*
  • Anion Transport Proteins / chemistry*
  • Anion Transport Proteins / genetics
  • Binding Sites / genetics
  • Chloride Channels / antagonists & inhibitors*
  • Chloride Channels / chemistry*
  • Chloride Channels / genetics
  • Dose-Response Relationship, Drug
  • Humans
  • Membrane Proteins / antagonists & inhibitors*
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Patch-Clamp Techniques
  • Point Mutation
  • Protein Structure, Tertiary


  • Anion Transport Proteins
  • CLCNKA protein, human
  • CLCNKB protein, human
  • Chloride Channels
  • Membrane Proteins
  • 2-(4-chlorophenoxy)propionic acid
  • 2-Methyl-4-chlorophenoxyacetic Acid
  • 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid