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Graphical Abstract
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Figure 1 Hypothesised drug binding of QO-58 to the voltage sensing domain of KV7.2. (a) Top view of the KV7.2 structure (PDB ID: 7CR4). Drug-binding sites are marked by a green circle in the voltage sensing domain (VSD) of the assembled four sub-units. (b) Enlarged side view of ztz-240 highlighting D172 and F168, which have been reported to be critical for the binding of VSD-targeted drugs. (c) Chemical structures of QO-58, ICA-069673 and ztz-240; the chloropyridine region is indicated by a green circle. (d) Similar view as in Figure 1b with QO-58 (blue) overlaid with ztz-240 (yellow) in two proposed orientations (i and ii).
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Figiure 2 KV7.2 subtype specificity of QO-58. (a, c and e) Typical voltage clamp records of indicated channel types expressed in Xenopus oocytes. Oocytes were held at −80 mV and pulsed between −140 and +40 mV in 10 mV steps. (b, d, f) Conductance-voltage relationships for KV7.2 (b; n = 6–9), KV7.2/KV7.3 (d; n = 5–9), and KV7.3* (f; n = 6–9) after treatment with QO-58 at indicated concentrations. Conductance-voltage relationships were generated from tail currents normalized to maximum current amplitude within the same recording. Data shown here are means ± SEM. Values of V1/2 derived from these results are shown in Table 1.
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Figure 3 State-dependent binding of QO-58 to Kv7.2. (a) Typical recordings of wild-type KV7.2 channels, expressed in oocytes. QO-58 (10 μM) was applied for ∼2 min while holding at −80 (top) or −100 mV (bottom), as indicated, followed by two pulses to +20 mV spaced by 4 s. (b) Instantaneous current (Iinst., green lines shown in Figure 3a) of the first (white) and second pulse (green), as a percentage of the peak current, for holding potentials of −120, −100 and −80 mV (n = 6–9). # P < 0.05, first pulse Iinst significantly different from −120 mV Iinst); * P < .05 (second pulse Iinst significantly different from first pulse Iinst);. two-way ANOVA followed by post hoc Tukey's test. (c) Typical records obtained from oocytes incubated in 1 μM QO-58. Oocytes were pulsed to +20 mV with four sweeps of varying duration (250, 750, 1250 and 1750 ms), with an interpulse interval of 10 s at −120 mV. Inset, normalised tails at −120 mV after 250 ms (black) or 1750 ms (green) prepulses. d) One- (control) or two-component (QO-58) fits of deactivation were determined for each prepulse duration (n = 5–6). (e) The proportion of the slow component of the tail was quantified for each prepulse duration (n = 5). In (d, e),data shown are individual values with means ±SEM.
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Figure 4 Pore-targeted inhibition of KV7.2 by ML252 is not affected by QO-58. (a and b) Typical records of KV7.2 expressed in Xenopus oocytes, treated with QO-58 and ML252 as indicated. Oocytes were held at −80 mV and pulsed between −140 and +40 mV in 10 mV steps. (c and d) KV7.2 was expressed in HEK293 cells and patch-clamp recordings were obtained. (d) Normalised KV7.2 current peak at +20 mV upon application or washout with indicated sequence and combinations of 10 μM QO-58, 10 μM ML252 and QO-58 + ML252. Data shown are individual values with means ± SEM, (n = 5–10). * P < 0.05, significantly different from control and from QO-58 alone; Kruskal–Wallis test, with post hoc Dunn's test.
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Figure 5 QO-58 activates KV7.2 through the voltage-sensing domain. (a–c) Schematic view of swapped regions in each indicated KV7.2/KV7.3 chimera, expressed in HEK293 cells. Conductance–voltage relationships are presented for each chimera. (d–f) Typical traces for generation of conductance–voltage relationships. Cells were held at −80 mV and pulsed between −160 and +80 mV in 10 mV steps, with indicated QO-58 concentrations. Derived fit parameters are detailed in Table 2. Tail current amplitudes were normalised to the peak tail current in each experimental condition. In (a, b, c), data shown are means ±SEM.
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Figire 6. Rescue of QO-58 sensitivity in KV7.5 by the KV7.2 S2-S3 segments. Amino acid sequence alignment of (a) KV7.2 with KV7.3 or (d) KV7.5. Differences in residues between are highlighted in green. (b, e and g) Left, schematics of the KV7 chimeric channels expressed in oocytes. (centre) Typical sweeps of recordings obtained from oocytes in control or 1 μm QO-58 as indicated. Oocytes were held at −80 mV (b), or −100 mV (e and g), and pulsed for indicated durations between −140 and +40 mV in 10 mV steps. (c, f and h) Conductance–voltage relationships obtained from tail current amplitudes, normalised within each experimental condition. (b and c) KV7.3*–KV7.2[S2–S3] (n = 5), (e and f) KV7.5–KV7.2[S2–S3] (n = 7) and (g and h) wild-type (WT) KV7.5 (n = 5). In (c, f, h), data shown are means ±SEM.
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Figure 7 Differential effects of KV7.2 Phe168 position on VSD-targeted potentiators ICA-069673 and QO-58. (a, c, e, g) Typical records of wild-type (WT) KV7.2, KV7.2 [F168L], KV7.2 [F168W] and KV7.2 [F168Y] expressed in Xenopus oocytes with indicated QO-58 concentrations. (b, d, f, h) Corresponding normalised conductance–voltage relationships obtained from tail current amplitudes of WT KV7.2 (n = 6–15), KV7.2 [F168L] (n = 6–17), KV7.2 [F168W] (n = 5–6) and KV7.2 [F168Y] (n = 7–11) under indicated conditions, at 1 μM QO-58 and at 30 μM ICA-069673. (i–l) Tails at −120 mV elicited after opening channels with a prepulse to +20 mV under indicated conditions of control, QO-58 or ICA-069673. In (b, d, f, h), data shown are means ±SEM.
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Figure 8 Distinct current augmentation and drug unbinding kinetics of ICA-069673 and QO-58 in KV7.2. (a–c) Typical fast-perfusion patch-clamp recordings using HEK293 cells. Cells expressing KV7.2 were held at −100 mV, then pulsed to 0 mV followed by exposure to 10 μM ICA-069673 or QO-58. Next, the drug was washed off, and channels were simultaneously pulsed to 0, −50 or −100 mV, to observe drug unbinding at different voltages. (d) Comparison of augmentation of KV7.2 current after application of 10 μM ICA-069673 or 10 μM QO-58. * P < 0.05, significantly different from ICA-069673; Student's t-test. (e) Typical recordings of current decay, reflecting drug unbinding at −100 mV. (f) Current decay following drug wash off was measured at each indicated voltages. Current decay rate was quantified as the time (ms) to 50% closure. ICA-069673, QO-58, and control decay were compared at −50 and −100 mV. Data shown are means ±SEM, n = 6–15. * P < 0.05, significantly different from control; # P < 0.05, significantly different from QO-58; two-way ANOVA and post hoc Tukey's test.
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