Mechanism of interaction of niflumic acid with heterologously expressed kidney CLC-K chloride channels.
CLC-K Cl(-) channels belong to the CLC protein family. In kidney and inner ear, they are involved in transepithelial salt transport. Mutations in ClC-Kb lead to Bartter's syndrome, and mutations in the associated subunit barttin produce Bartter's syndrome and deafness. We have previously found that 3-phenyl-CPP blocks hClC-Ka and rClC-K1 from the extracellular side in the pore entrance. Recently, we have shown that niflumic acid (NFA), a nonsteroidal anti-inflammatory fenamate, produces biphasic behavior on human CLC-K channels that suggests the presence of two functionally different binding sites: an activating site and a blocking site. Here, we investigate in more detail the interaction of NFA on CLC-K channels. Mutants that altered block by 3-phenyl-2-(p-chlorophenoxy)propionic acid (CPP) had no effect on NFA block, indicating that the inhibition binding site of NFA is different from that of 3-phenyl-CPP and flufenamic acid. Moreover, NFA does not compete with extracellular Cl(-) ions, suggesting that the binding sites of NFA are not located deep in the pore. Differently from ClC-Ka, on the rat homologue ClC-K1, NFA has only an inhibitory effect. We developed a quantitative model to describe the complex action of NFA on ClC-Ka. The model predicts that ClC-Ka possesses two NFA binding sites: when only one site is occupied, NFA increases ClC-Ka currents, whereas the occupation of both binding sites leads to channel block.
Picollo
Alessandra
A
Istituto di Biofisica, CNR, Via De Marini, 6, I-16149 Genoa, Italy.
Liantonio
Antonella
A
Babini
Elena
E
Camerino
Diana Conte
DC
Pusch
Michael
M
D000818:Animals; D018118:Chloride Channels / Q000187:drug effects*; D004357:Drug Synergism; D005439:Flufenamic Acid / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D009544:Niflumic Acid / Q000494:pharmacology*; D010666:Phenylpropionates / Q000494:pharmacology; D051381:Rats; D014982:Xenopus laevis
C504521:2-(4-chlorophenoxy)-3-phenylpropanoic acid; C489932:CLCNKA protein, human; D018118:Chloride Channels; C082118:Clcnka protein, rat; D010666:Phenylpropionates; D009544:Niflumic Acid; D005439:Flufenamic Acid