Electro‐Mechanical Uncoupling of K V 7.1 Voltage Sensor and Pore by 1,4‐Benzodiazepines Is Modulated by Decoration of Position 1

Figures

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  Figure 1. Rationale and chemical structures of (R)‐L3 (2) and its analogues 1 and 3‐5. (A) Docking model of (R)‐L3 in KV7.1 [22]. Chemical modification strategy at 1‐position of the 1,4‐benzodiazepine scaffold. Residues in S4 that interact with the methyl group of (R)‐L3 (position coloured purple, (R)‐L3 coloured in magenta) in silico are shown in space fill. (B) Chemical structures of (R)‐L3 (2) and its analogues 1 and 3‐5. Modifications in 1‐position are coloured in red.

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  Figure 2. Representative TEVC KV7.1 current traces and current–voltage relation. (A) Example current traces recorded from Xenopus laevis oocytes expressing KV7.1 in control (upper traces) and compound‐treated conditions (lower traces, c = 10 µM). (B) Current amplitudes at the end of depolarizing pulses (indicated by red arrows) were determined and normalized mean results are plotted versus voltage of the depolarizing pulse. 1,4‐Benzodiazepines 1–3 exert clear activation of KV7.1 channel currents. Student's t‐test on currents at 40 mV revealed significant current increase upon application of compound 1: p = 0.0013 (indicated by **), compound 2: p = 0.0003 (indicated by ***) and compound 3: p = 0.0011 (indicated by **); n = 8‐11.

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  Figure 3. Activation kinetics of KV7.1 currents. (A) KV7.1 activates with two distinct kinetic components (biexponential activation kinetics). An example trace is shown at 40 mV. An exponential fit describing the fast (blue) and the slow (red) components is shown in dotted lines. In a simple linear gating scheme these components are accounted for by deep (C1) and intermediate (C2) closed states. (B, C) Voltage dependence of the slow (Ʈslow) and fast (Ʈfast) activation time constants. Time constants of the slow and fast components were plotted versus voltage. (D) Ratio of amplitudes (Afast/Aslow) as a function of voltage. Student's t‐test on values at a respective voltage revealed significant changes upon application. Significant changes are indicated by *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001 in the colour code of the respective compound; n = 3–14.

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  Figure 4. Deactivation kinetics and recovery from inactivation. (A) Pulse protocol to elicit currents. Channels were activated by depolarization to +40 mV (2 s) and stepping back to voltages of −120 to −40 mV in 20 mV increments (4.5 s). Representative tail currents are shown from before application (left traces) and after application of the indicated compounds (right traces). The tail currents are scaled to similar control size to allow for better vision on the compounds effect. Tail currents show two distinct kinetic components (biexponential kinetics). (B) An example trace is shown at −120 mV. An exponential fit describing the recovery from inactivation (blue) and the deactivation (red) components is shown in coloured lines. In a simple linear gating scheme, these components are accounted for with an inactivated state (I) and (O) open state(s). (C) Voltage dependence of deactivation time constants (Ʈdeactivation) versus voltage is represented. (D) Voltage dependence of recovery from inactivation (Ʈrecovery) plotted versus voltage is shown. (E) Ratio of recovery from inactivation to deactivation amplitudes. The relative amplitude of the inactivation component versus the amplitude of the…

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  Figure 5. Correlation of kinetic parameters with activating compound effects. Scatter plots of kinetic parameters (Ʈfast activation at 40 mV (blue) and Ʈrecovery from inactivation at −120 mV (red)) plotted versus activation (amplitude increase) of respective compound at 10 µM. Linear regression shows significant correlations of these parameters, whereas 5 does not follow the correlation of Ʈfast activation to activation.

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  Figure 6. Lack of correlation of kinetic parameters with other compound effects. Scatter plots of kinetic parameters plotted versus activation (amplitude increase) of respective compound at 10 µM concentration. Linear regression shows no significant correlations of these parameters to stimulation. Values of linear regressions shown underneath the respective graph.