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

Abstract

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.

Figure 1

Typical response of WT ClC-Ka to the application of 200 µM NFA. A 60 mV test pulse of 100 ms duration was applied about once per second, and the average current is plotted as a function of time. The current remaining after the application of 100 mM iodide was used as an estimate of endogenous/leak currents.

Figure 2

Location of mutants mapped on the structure of ClC-ec1. (A) A surface representation of the bacterial ClC-ec1 (pdb entry 1OTS) viewed from the extracellular side. The central glutamate residue (E148) is coloured in green indicating the pore. The residues corresponding to those selected for mutation are shown in colour. In the left subunit, the residues that emerged as the most interesting ones are coloured in pink (L139 corresponding to L155 of ClC-Ka), orange (T312 corresponding to G345 of ClC-Ka) and yellow (G316 corresponding to A349 of ClC-Ka) respectively. (B) A zoom of the region comprising the extracellular side of helix E and the extracellular loop between helices K and L is shown in cartoon representation with the four residues L139, E148, T312, G316 highlighted as stick with colouring as in (A). An NFA molecule is shown close to the putative binding site. In (C), alignments of sequence stretches comprising the five residues (shown in bold) that affected NFA potentiation are shown.

Figure 3

Mutational analysis of NFA-mediated potentiation. The ratio of currents in the presence of 200 µM NFA and in control solution at 60 mV is plotted for the mutations that resulted in significant functional expression (n ≥ 3; error bars indicate SEM). Mutants L155A, G167A, F213A, G345S and A349E were significantly different from WT (P < 0.01), whereas the remaining mutants were not significantly different (P > 0.05) (see Methods for statistical analysis). The red line indicates the value for WT; the black line indicates the value ‘1’ (i.e. implying no effect of NFA on current magnitude). The mutants E281D and E442D showed very small expression not allowing a quantitative assessment of NFA effects. The following mutants showed no expression above background: T55A, Y56C, C73R, R184G, V212A, A214W, A214T, E259Q, E281Q, D363N, G424A, M427A, E442Q.

Figure 4

Noise analysis revealed gating alterations. Typical results of non-stationary noise analysis of WT ClC-Ka (A), mutant F213A (B), mutant G167A (C) and mutant L155A (D) are shown. The mean (upper trace) and the variance (lower trace) are shown on the left, while the right shows the variance plotted versus the mean (symbols) and fitted with a parabola (line) as described in Methods. The parameters obtained by the fit are: (A): i = 2.1 pA, P_max < 0.1; (B): i = 1.4 pA, P_max = 0.61; (C): i = 1.2, P_max = 0.81; (D): i = 0.93, P_max < 0.1. Horizontal scale bars indicate 100 ms; vertical scale bars indicate: (A) 500 pA, 2000 pA²; (B) 200 pA, 100 pA²; (C) 50 pA, 20 pA²; (D) 20 pA, 20 pA². Mean values of P_max are shown in Table 1.

Figure 5

Representative current traces for WT and the indicated mutants in control solution and in 200 µM NFA. Horizontal scale bars correspond to 50 ms; vertical scale bars correspond to 5 µA for WT and 1 µA for the mutants. The pulse protocol comprised a prepulse to 60 mV followed by steps to values from –140 to 80 mV, and a tail pulse to –100 mV and is depicted in (A) as an inset.

Figure 6

Concentration dependence of the effects of NFA in WT and in the indicated mutants. Lines are fits to the equation I([NFA])/I(0) = 1/(1 + [NFA]/K) with the apparent dissociation constant K as the fitted parameter, resulting in values of 230 µM (L155A), 220 µM (G345S) and 460 µM (A349E) respectively.