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Figure 1. Multiple retigabine molecules modulate KCNQ2 and KCNQ3 channel subunits via an S5 Trp side chain.(a,b) Conductance–voltage relationships for (a) KCNQ2 (n=3) and KCNQ2[Trp236Phe] (n=6), and (b) KCNQ3* (n=5) and KCNQ3*[Trp265Phe] (n=3) homomeric channels along with indicated mutants (retigabine concentration of 100 μM). (c) Conductance–voltage relationships for heteromeric combinations of KCNQ2 and KCNQ3 (1:1 ratio of injected mRNA, with or without Trp→Phe mutations as indicated, n=5 for each combination), used to generate channels with reduced numbers of retigabine binding sites. (d) Summary of V1/2 shifts in saturating 100 μM retigabine for mutations of KCNQ2 Trp236 and KCNQ3 Trp265 as indicated (*P<0.05 in a paired Students t-test comparing control versus 100 μM retigabine in each experimental oocyte, n=3–6 per mutant). Only a Trp at either position is sufficient for retigabine sensitivity. (e) Exemplar currents of KCNQ3* and KCNQ3*[Trp265Phe] mutant coexpressed with CiVSP, illustrating that the Trp side chain responsible for retigabine sensitivity is not required for PIP2 sensitivity. (f) Summary data of tail current magnitude (−20 mV) after prepulses to a range of voltages, in oocytes expressing KCNQ3* (n=5) or KCNQ3*[Trp265Phe] (n=5) channels, along with CiVSP. In all panels, error bars represent s.e.m.
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Figure 2. Nonsense suppression for amino-acid incorporation in KCNQ3* channels.(a) Schematic diagram of the nonsense suppression method, in which mRNA (with a stop codon at Trp265) and amino-acylated tRNA are co-injected into Xenopus oocytes. Incorporation of the unnatural amino acid enables readthrough of the stop codon and expression of functional channels. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Trp (n=8, *P<0.05, Student's t-test). (c) Activation kinetics of KCNQ3* (n=5) and KCNQ3*[Trp265TAG] (n=8) channels rescued with Trp, in the presence or absence of 100 μM retigabine (*P<0.05, Student's t-test). (d–f) Exemplar currents from oocytes injected with KCNQ3*[Trp265TAG] mRNA, and indicated synthetic tRNAs. (g) Conductance–voltage relationships for Trp-rescued KCNQ3*[Trp265TAG] (n=8), with retigabine response, illustrating faithful incorporation of the desired side chain at position 265. For KCNQ3* channels V1/2=−44±1 mV, k=7.5±0.5 mV; for Trp-rescued KCNQ3*[Trp265TAG] V1/2=−43±2 mV, k=7.9±0.5 mV (± indicates s.e.). Error bars throughout represent s.e.m.
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Figure 3. The position of the Trp 265 indole nitrogen is essential for retigabine sensitivity.(a) Chemical structures of Trp and Ind side chains, illustrating the subtle change in the position of the indole nitrogen atom. (b) Exemplar currents elicited from a Xenopus oocyte with Ind-rescued KCNQ3*[Trp265TAG] channels illustrating retigabine insensitivity. (c) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Ind (n=7, *P<0.05, Student's t-test). (d) Conductance–voltage relationships for Ind-rescued KCNQ3*[Trp265TAG], in the presence and absence of retigabine, illustrating the importance of the correct positioning of the N–H group. For KCNQ3*[Trp265Trp], V1/2=−43±2 mV, k=7.9±0.5 mV; for KCNQ3*[Trp265Ind], V1/2=−48±2 mV, k=7.3±0.6 mV (no statistical significance, ±indicates s.e.). (e) Activation kinetics for KCNQ3*[Trp265Ind] measured at −20 mV in the presence and absence of 100 μM retigabine (n=7, no statistical significance). In all panels, error bars represent s.e.m.
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Figure 4. The polarity of the Trp265 indole nitrogen modulates retigabine sensitivity.(a) Chemical structures of Trp (with ring positions labelled) and F3-Trp, accompanied by colorimetric representations of electrostatic surface potentials. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino acylated with F3-Trp (n=12, *P<0.05, Student's t-test). (c) Exemplar currents from F3-Trp-rescued KCNQ3*[Trp265TAG] channels in the presence and absence of retigabine. (d) Conductance–voltage relationships illustrating the effects of 1 μM retigabine on Trp-rescued (n=12) and F3-Trp-rescued KCNQ3*[Trp265TAG] (n=12) channels. (e) Activation kinetics (−20 mV) for F3-Trp-rescued channels (n=3), in the presence and absence of 100 μM retigabine (*P<0.05, Student's t-test). (f) Concentration–response curves for retigabine effects on numerous fluoro-Trp analogues (n=9–12 per Trp analogue) substituted at position Trp265, illustrating enhanced retigabine potency with increased fluorination. (g) Conductance–voltage relationships with indicated retigabine concentrations on a KCNQ3 mutant (Asn220Cys, n=4) with an intrinsic hyperpolarizing shift in gating. Conductance–voltage relationships for F3-Trp substituted at Trp265TAG are shown for comparison. (h) Concentration–response curves for retigabine effects on KCNQ3* (n=5) and KCNQ3*[Asn220Cys] (n=4) channels. In all panels, error bars represent s.e.m.
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Figure 5. Detailed characterization of secondary retigabine binding residues and alternative binding site orientations.(a) Conductance–voltage relationships were gathered for the indicated KCNQ3* mutant channels (n=4–6 per mutant), in 0, 100 or 300 μM retigabine. (b) Maximal ΔV1/2 in 300 μM retigabine measured in each mutant channel. Error bars in a,b represent s.e.m. (c) Retigabine was docked into a molecular model of the pore-forming domain of KCNQ3 (see ref. 19). Two orientations are shown with the carbamate group in either the vicinity of Leu314 (‘original' model) or Trp265 (‘flip' model). The two binding models are superimposed in the ‘overlay', showing the similar space occupied by both drug orientations.
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Figure 6. ML-213 exhibits a stronger electrostatic surface potential and higher potency than retigabine for KCNQ3* activation.(a,b) Chemical structures and electrostatic surface potentials for retigabine and ML-213. Note the increased negative surface potential in the vicinity of the carbonyl oxygen atom in ML-213. The scale for electrostatic surface potential representation is red: −80 kcal, yellow: 0 kcal, blue: +80 kcal. (c) Concentration–response curves for retigabine and ML-213 (n=5) effects on KCNQ3* channels (n=5). Error bars represent s.e.m.
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Figure 7. Effects of retigabine analogues correlate with electrostatic surface potential.(a) Chemical structures and electrostatic surface potentials for a series of retigabine analogues. All structures and surface potential maps have been aligned on the basis of the location of the conserved amide-ester bond—note the gradient of the intensity of the negative surface potential around the carbonyl oxygen atom (scaling is the same as in Fig. 6). (b) Summary illustrating the EC50 of each drug on Trp-rescued, F3-Trp-rescued and Ind-rescued KCNQ3*[Trp265TAG] channels (n=4–9 per data point), and the maximal efficacy (ΔV1/2) of each drug in F3-Trp and Trp-rescued channels (effects on Ind-rescued channels are minimal and thus have been omitted). Error bars represent s.e.m.
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Figure 8. Diverse structures of KCNQ openers.Multiple structures of KCNQ channel openers are presented to highlight the overall features of an amide group flanked by various ring structures. Our findings highlight the importance of the amide carbonyl for interaction with KCNQ3 Trp 265 and likely equivalent positions in KCNQ2, 4 and 5. Drugs depicted are (a) retigabine, (b) ztz-240 (described in ref. 24), (c) acrylamide (s)-1, (d) BMS-204352 and (e) an unnamed experimental drug described in ref. 43.
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