Abstract
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.
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Acknowledgement
We thank T. Jentsch for providing all cDNA clones, M. De Bellis for help in molecular biology and G. Gaggero for help in constructing the recording chamber. The financial support of Telethon Italy (grant GGP04018) is gratefully acknowledged.
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Picollo, A., Liantonio, A., Babini, E. et al. Mechanism of Interaction of Niflumic Acid with Heterologously Expressed Kidney CLC-K Chloride Channels. J Membrane Biol 216, 73–82 (2007). https://doi.org/10.1007/s00232-007-9034-z
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DOI: https://doi.org/10.1007/s00232-007-9034-z