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

Abstract

The highly homologous Cl À 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 B80 lM. We also found that DIDS (4,4 0 -diisothiocyanatostilbene-2,2 0 -disulphonic acid), a generic Cl À channel blocker, inhibits CLC-Ka (K D B90 lM). 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.

Molecular switch for CLC-K Cl − channel block/activation: Optimal pharmacophoric requirements towards high-affinity ligands

“…On the basis of the structural requirements and of the structure-activity relationship related to the principal classes of the studied compounds, namely flexible 3-phenyl-CPP derivatives (19), fenamates (12, 13), 3-phenyl-1-benzofuran-2-carboxylic acid derivatives, and 7-(trif luoromethyl)-9H-carbazole-1-carboxylic acid (GF-166), we hypothesize the ClCKa/Kb blockers/activators pharmacophore topography. The pharmacophore model (Fig.…”

“…Interestingly, GF-167 reversibly blocked ClC-Ka from the extracellular side with an apparent K d of 24 Ϯ 2 M at 60 mV ( Fig. 2A), a value that is Ϸ4-fold lower than that of 3-phenyl-CPP (12,19).…”

“…In the last few years, working with two structurally unrelated classes of compounds (CPP derivatives and fenamates), we identified CLC-K activators and inhibitors with moderate affinity (12,13,(17)(18)(19)(20). The only CLC-K opener identified was niflumic acid (NFA).…”

“…We have previously demonstrated that ClC-Ka possesses different blocking binding sites, the inhibitory binding site of NFA being different from that of 3-phenyl-CPP and FFA derivatives (12)(13)19). To start to explore the binding site of the benzofuran derivatives we used the ClC-Ka mutant N68D, that is significantly less sensitive to 3-phenyl-CPP and FFA induced block (12,19) but not to NFA-induced block (13). The mutant was Ϸ8-fold less sensitive to MT-189 compared with WT ClC-Ka (Fig.…”

“…3-Phenyl-CPP ( Fig. 2A, top left) is one of the most potent blockers of ClC-Ka described so far (12,19,20). To explore whether modification of the flexibility and the degree of planarity of the 3-phenyl-CPP structure could produce an alteration in drug blocking potency, we synthesized a molecule (GF-167; Fig.…”