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) 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.
Picollo
Alessandra
A
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via de Marini 6, 16149 Genova, Italy.
Liantonio
Antonella
A
Didonna
Maria Paola
MP
Elia
Laura
L
Camerino
Diana Conte
DC
Pusch
Michael
M
D008456:2-Methyl-4-chlorophenoxyacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000737:chemistry / Q000494:pharmacology*; D020816:Amino Acid Motifs / Q000235:genetics; D027321:Anion Transport Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D017434:Protein Structure, Tertiary
D027321:Anion Transport Proteins; C489932:CLCNKA protein, human; C412733:CLCNKB protein, human; D018118:Chloride Channels; D008565:Membrane Proteins; C048213:2-(4-chlorophenoxy)propionic acid; D008456:2-Methyl-4-chlorophenoxyacetic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid