Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY 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 AND IMPLICATIONS: 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.

Channel or transporter? The CLC saga continues.

It was recently shown that the putative bacterial Cl- channel, ClC-ec1, is in reality a Cl--H+ antiporter. Our group has now shown that this is also the case for two human CLCs, ClC-4 and ClC-5. We found that the flux of Cl- in one direction is stoichiometrically coupled to the movement of protons in the opposite direction, unveiling a behaviour that is typical of a transporter rather than a channel. This discovery will surely stimulate further research to elucidate the molecular elements responsible for the behaviour as a transporter. On the physiological level, the antiport activity of ClC-4/ClC-5 must lead to a review of the role of CLC proteins in intracellular compartments. Small organic molecules have been extremely useful tools for studying ion channels and many commercial drugs target specific ion channel proteins. Several blockers have been found to inhibit the plasma membrane-localized CLC channels ClC-0, ClC-1 and ClC-Ka. These compounds include 9-anthracene-carboxylic acid (9-AC), p-chlorophenoxy-propionic acid (CPP) and its derivatives, and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Two different binding sites have been identified, one extracellular and one intracellular. However, high-affinity ligands for most CLC proteins are still missing. Apart from being useful biophysical tools, such drugs may provide a way to modulate protein function in vivo. With these tasks to be accomplished, it is definitely an exciting time in the chloride transport field.