Molecular determinants of differential pore blocking of kidney CLC-K chloride channels

Abstract

The highly homologous Cl(-) channels CLC-Ka and CLC-Kb are important for water and salt conservation in the kidney and for the production of endolymph in the inner ear. Mutations in CLC-Kb lead to Bartter's syndrome and mutations in the small CLC-K subunit barttin lead to Bartter's syndrome and deafness. Here we show that CLC-Ka is blocked by the recently identified blocker 2-(p-chlorophenoxy)-3-phenylpropionic acid of the rat channel CLC-K1 with an apparent K(D) approximately 80 microM. We also found that DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), a generic Cl(-) channel blocker, inhibits CLC-Ka (K(D) approximately 90 microM). Surprisingly, the highly homologous channel CLC-Kb is fivefold to sixfold less sensitive to both compounds. Guided by the crystal structure of bacterial CLC proteins, we identify two amino acids, N68/D68 and G72/E72, in CLC-Ka and CLC-Kb, respectively, that are responsible for the differential drug sensitivity. Both residues expose their side chains in the extracellular pore mouth, delineating the probable drug binding site. These novel CLC-K channel blockers are promising lead compounds for the development of new diuretic drugs.

Figure 1

Effects of 3-phenyl-CPP and DIDS on CLC-Ka. (A,B) Voltage-clamp traces of CLC-Ka currents before and during application of 200 μM 3-phenyl-CPP (A) and 200 μM DIDS (B), and immediately after wash-out. The pulse protocol is shown in the inset. Horizontal scale bars indicate 50 ms (A,B). Vertical scale bars indicate 5 μA (A,B). (C) Chemical structures of 3-phenyl-CPP and DIDS. (D) Slow recovery from DIDS block for a representative oocyte. Arrows indicate application of DIDS and wash-out. A pulse to 60 mV was applied every 2 s and the current is shown as a function of the elapsed time.

Figure 2

Effects of 3-phenyl-CPP and DIDS on CLC-Kb. Voltage-clamp traces of CLC-Kb currents before and during application of 200 μM 3-phenyl-CPP (A) and 200 μM DIDS (B), and immediately after wash-out. Horizontal scale bars indicate 100 ms. Vertical scale bars indicate 2 μA (A) and 1 μA (B).

Figure 3

Analysis of inhibition of wild-type channels. Dose–response relationship of inhibition by 3-phenyl-CPP (A) and DIDS (B) for CLC-Ka and CLC-Kb. The ratio of currents in the presence and absence of the drugs was determined as described in Methods and plotted versus concentration. Lines are drawn according to equation (1), with the apparent K_D values obtained as described in Methods and reported in Table 1.

Figure 4

Selection of candidate residues involved in drug binding. (A) View of the bacterial channel StCLC (Dutzler et al, 2002) from the extracellular side. One subunit of the dimer is shown in wireframe (right-hand side), whereas the other is shown in spacefill (left-hand side). Highlighted in dark grey are all amino acids that possibly expose their side chains in the extracellular pore entrance. Among these, we selected those residues that were different in CLC-Ka and CLC-Kb and that were identical in CLC-K1 and CLC-Ka. Four residues fulfilled this criterion, as shown in (B). The corresponding residues of StCLC are shown in colour in (A) (dark blue: V51, corresponding to CLC-Ka-Y65; red: E54, CLC-Ka-N68; orange: K55, CLC-Ka-F69; magenta: S58, CLC-Ka-G72). All four residues lie in helix B (yellow). E148 (green: V166 in CLC-Ka and CLC-Kb) indicates the location of the selectivity filter (Dutzler et al, 2002, 2003). (B) Alignment of the short stretch of helix B. Mutated amino acids are shown in bold. Corresponding residues in CLC-K1 are shown in bold and italic.

Figure 5

Phenotype of mutants. Typical voltage-clamp traces obtained with the pulse protocol described in Methods are shown for the indicated constructs. Horizontal scale bars indicate 50 ms (A–E) and 100 ms (F–J). Vertical scale bars indicate 5 μA (A,C), 1 μA (B,I), 3 μA (D,E), 2 μA (F), 4 μA (G), 0.5 μA (H) and 0.3 μA (J). Expression levels varied significantly between mutants. In particular, CLC-Kb and its mutants had lower expression levels than those of CLC-Ka. The reason for this difference is unknown. (K,L) Dose–response relationships for 3-phenyl-CPP (K) and DIDS (L) for the indicated constructs. Lines are drawn according to equation (1) with the apparent K_D values reported in Table 1.