Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels.
OBJECTIVE
ClC-K kidney Cl(-) channels are important for renal and inner ear transepithelial Cl(-) transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non-steroidal anti-inflammatory drug, in a biphasic way: NFA activates ClC-Ka at low concentrations, but blocks the channel above approximately 1 mM. We attempted to identify the amino acids involved in the activation of ClC-Ka by NFA.


METHODS
We used site-directed mutagenesis and two-electrode voltage clamp analysis of wild-type and mutant channels expressed in Xenopus oocytes. Guided by the crystal structure of a bacterial CLC homolog, we screened 97 ClC-Ka mutations for alterations of NFA effects.


RESULTS
Mutations of five residues significantly reduced the potentiating effect of NFA. Two of these (G167A and F213A) drastically altered general gating properties and are unlikely to be involved in NFA binding. The three remaining mutants (L155A, G345S and A349E) severely impaired or abolished NFA potentiation.


CONCLUSIONS
The three key residues identified (L155, G345, A349) are localized in two different protein regions that, based on the crystal structure of bacterial CLC homologs, are expected to be exposed to the extracellular side of the channel, relatively close to each other, and are thus good candidates for being part of the potentiating NFA binding site. Alternatively, the protein region identified mediates conformational changes following NFA binding. Our results are an important step towards the development of ClC-Ka activators for treating Bartter syndrome types III and IV with residual channel activity.
Zifarelli
G
G
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, Italy.
Liantonio
A
A
Gradogna
A
A
Picollo
A
A
Gramegna
G
G
De Bellis
M
M
Murgia
A R
AR
Babini
E
E
Camerino
D Conte
DC
Pusch
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001665:Binding Sites; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009544:Niflumic Acid / Q000494:pharmacology*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D016415:Sequence Alignment; D014162:Transfection; D014981:Xenopus
D000894:Anti-Inflammatory Agents, Non-Steroidal; C489932:CLCNKA protein, human; D018118:Chloride Channels; D009544:Niflumic Acid