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

Abstract

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 KD∼80 μ M. We also found that DIDS (4,4′-diisothiocyanatostilbene-2,2′ -disulphonic acid), a generic Cl- channel blocker, inhibits CLC-Ka (KD∼90 μM). 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. © 2004 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION.