Molecular Pharmacology of Kidney and Inner Ear CLC-K Chloride Channels

Abstract

CLC-K channels belong to the CLC gene family, which comprises both Cl(-) channels and Cl(-)/H(+) antiporters. They form homodimers which additionally co-assemble with the small protein barttin. In the kidney, they are involved in NaCl reabsorption; in the inner ear they are important for endolymph production. Mutations in CLC-Kb lead to renal salt loss (Bartter's syndrome); mutations in barttin lead additionally to deafness. CLC-K channels are interesting potential drug targets. CLC-K channel blockers have potential as alternative diuretics, whereas CLC-K activators could be used for the treatment of patients with Bartter's syndrome. Several small organic acids inhibit CLC-K channels from the outside by binding to a site in the external vestibule of the ion conducting pore. Benzofuran derivatives with affinities better than 10 μM have been discovered. Niflumic acid (NFA) exhibits a complex interaction with CLC-K channels. Below ∼1 mM, NFA activates CLC-Ka, whereas at higher concentrations NFA inhibits channel activity. The co-planarity of the rings of the NFA molecule is essential for its activating action. Mutagenesis has led to the identification of potential regions of the channel that interact with NFA. CLC-K channels are also modulated by pH and [Ca(2+)](ext). The inhibition at low pH has been shown to be mediated by a His-residue at the beginning of helix Q, the penultimate transmembrane helix. Two acidic residues from opposite subunits form two symmetrically related intersubunit Ca(2+) binding sites, whose occupation increases channel activity. The relatively high affinity CLC-K blockers may already serve as leads for the development of useful drugs. On the other hand, the CLC-K potentiator NFA has a quite low affinity, and, being a non-steroidal anti-inflammatory drug, can be expected to exert significant side effects. More specific and more potent activators will be needed and it will be important to understand the molecular mechanisms that underlie NFA activation.

Keywords: CLC; calcium; chloride channel; chloride transport; diuretic; fenamates; inner ear; kidney.

Figure 1

Transport models for NaCl reabsorption in the TAL (A) and for transepithelial K⁺ transport in the inner ear (B). (A) Schematic representation of an epithelial cell from the thick ascending limb of Henle's loop. All transporters involved in NaCl reabsorption are drawn in different colors. (B) Schematic representation of marginal cell from the stria vascularis (for simplicity other epithelial cell layers are not shown). See text for details.

Figure 2

Chemical structures of various substances that inhibit or activate CLC-K channels. (A) CPP (p-chlorophenoxy-propionic acid); (B) GF-100 (proper name); (C) 3-Ph-CPP (3-phenyl-CPP); (D) NFA (niflumic acid); (E) FFA (flufenamic acid); (F) DIDS (4,4′-Diisothiocyanato-2,2′-stilbenedisulfonic acid); (G) Triflocin; (H) GF-166 (proper name); (I) MT-189 (proper name); (J) RT-93 (proper name).

Figure 3

Binding sites on CLC-K channels. In (A) a view from the outside of the bacterial CLC-ec1 (pdb code 1OTS) homodimer is shown with one subunit in cartoon and the other in surface representation. In (B) a zoom of a selected region highlighting the residues involved in ligand binding. Cl⁻ ions in pink; gating glutamate in blue; N68 implicated in block by CPP in red (D54 in CLC-ec1); H497 mediating H⁺ block in yellow (L421 in CLC-ec1); E261 and D278 forming the Ca binding site in cyan (E235 and N250 in CLC-ec1); L155, G345, and A349 which are important for NFA potentiation in orange (L139, T312, and G316 in CLC-ec1).

Figure 4

Effect of 3-phenyl-CPP on CLC-Ka, CLC-Kb, and mutants. (A) The blocking effect of 3-phenyl-CPP is schematically indicated for CLC-Ka and CLC-Kb. (B) A sequence alignment of a short stretch of residues of helix B of CLC-Ka, CLC-Kb, CLC-K1, and StCLC, a bacterial homolog (Dutzler et al., 2002) is shown. Residues which are identical in CLC-Ka and CLC-K1, and different in CLC-Kb, are underlined. CLC-K residues at position 68 drastically affect block by 3-phenyl-CPP. DIDS block is additionally dependent on the residue at position 72. Sequence of the bacterial StCLC is shown because the mutagenesis work was guided by the crystal structure of this protein. Residues at positions 68 and 72 are mostly responsible for the difference between CLC-Ka and CLC-Kb and are shown in bold. (C) Schematically the blocking effect of 3-phenyl-CPP of mutants CLC-Ka-N68D and CLC-Kb-D68N. Plots were generated based on data from Picollo et al. (2004).

Figure 5

Blocking and potentiating effects of NFA and FFA on various CLC-K homologs (A: CLC-K1, B: CLC-Ka, C: CLC-Kb). The effect of NFA is schematically drawn in black, whereas the effect of FFA is shown in red. No measurements with FFA on CLC-K1 have been reported.

Figure 6

Effects of extracellular pH (A) and [Ca²⁺] (B) on WT CLC-Ka (black line) and the mutants (red lines) E261Q/D278N/H497M (A) and E261Q/D278N (B) schematically drawn according to the results of Gradogna et al. (2010).