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

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 ∼80 μM. We also found that DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid), a generic Cl − channel blocker, inhibits CLC‐Ka ( K D ∼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.