Effect of (S)-2 on the deactivation kinetics of Kv7.2 and Kv7.4.

<p>Normalized tail current traces for Kv7.2 (A) and Kv7.4 (E) obtained at the indicated potentials in the absence and presence of (S)-2 illustrating the pronounced effect of the compound on the deactivation kinetics. Deactivation kinetics for Kv7.2 were determined by fitting the tail currents measured at potentials between –110 mV and –90 mV after an activating step to +40 mV to a double exponential function. The time constants τ_fast (B) and τ_slow (C) were plotted against the potential. (D) Relative contribution of the slow component of the deactivation kinetics for Kv7.2. Tail currents measured between −110 and −80 mV for Kv7.4 were fitted to a double exponential function and τ_fast (F) and τ_slow (G) were plotted against the potential. (H) Relative contribution of the slow component of the deactivation kinetics for Kv7.4. Asterisks indicate statistical significant difference between absence and presence of (S)-2 determined by two-way ANOVA followed by Bonferroni post-test. * P<0.05, ** P<0.01 and *** P<0.001. Bars represent S.E.M. and <i>n</i> = 5–14.</p

Sensitivity of heteromeric Kv7.4/Kv7.4-W242L channels to (S)-2.

<p>(A) Current-voltage (I-V) relationship of Kv7.4 and Kv7.4/Kv7.4-W242L in the absence and presence of 15 µM (S)-2. The steady state peak current measured at potentials between –100 and +40 mV were normalized against the current at +40 mV in control recordings and plotted against the test potential. (B) Effect of (S)-2 on the voltage-dependence of activation of Kv7.4 and Kv7.4/Kv7.4-W242L. Tail currents measured after stepping back to –120 mV from potentials between –100 and +60 mV in the absence and presence of 15 µM (S)-2 were normalized and plotted against the preceding potential. The tail current-voltage relationship was then fitted to the Boltzmann equation to yield half-activation potentials (V_0.5): Kv7.4 (5.1±1.3 mV); Kv7.4+(S)-2 (−30.5±1.6 mV); Kv7.4/Kv7.4-W242L (4.1±1.6 mV); Kv7.4/Kv7.4-W242L+(S)-2 (−15.0±1.0 mV). (C) Dose-response relationship of (S)-2 on Kv7.4 and Kv7.4-W242L. The steady state peak currents elicited by a 5 s step to 0 mV in response to increasing concentrations of (S)-2 were normalized to the current in the absence of compound and plotted as a function of the concentration of (S)-2. The values were then analyzed by non-linear regression to fit a sigmoidal curve. The EC₅₀ values were determined to 0.46 µM and 0.72 µM and the Hill coefficients to 1.32±0.17 and 1.24±0.25 for Kv7.4 and Kv7.4/Kv7.4-W242L, respectively. Bars represent S.E.M. and <i>n</i> = 6–10.</p

Activation of Kv7 channels by (S)-2.

<p>Representative two-electrode voltage-clamp current traces in the absence (left) and presence (middle) of 10 µM (S)-2 and effect of (S)-2 on current-voltage (I-V) relationship (right) of Kv7.1 (A), Kv7.2 (B), Kv7.2/Kv7.3 (C), Kv7.4 (D) and Kv7.5 (E) channels expressed in <i>Xenopus laevis</i> oocytes. The channels were activated by 5 s voltage steps from −80 mV to potentials ranging from –100 to +40 mV in 10 mV increments followed by a 2 S step to –120 mV. The steady state peak current amplitudes in the absence and presence of 10 µM (S)-2 were normalized against the current at +40 mV in control recordings and plotted against the test potential to obtain I-V curves (left). Bars represent S.E.M. and <i>n</i> = 6-17. Please note that in some instances the S.E.M. is so small that the error bars are not visible.</p

Effect of (S)-2 on the inactivation of Kv7.4.

<p>Representative two-electrode voltage-clamp recordings elicited by the voltage protocol shown in the inset in the absence (A) and presence (B) of 10 µM (S)-2. (C) The degree of inactivation in the absence and presence of (S)-2 is revealed by plotting the current amplitude measured at +40 mV normalized to the level measured after a prepulse potential of –120 mV as a function of the preceding prepulse potential. Bars represent S.E.M. and <i>n</i> = 11–14.</p

Chemical structure of (S)-2.

<p>Chemical structure of (S)-2.</p

Deactivation kinetics.

<p>Asterisks indicate statistical significant difference between absence and presence of (S)-2 determined by two-way ANOVA followed by Bonferroni post-test.</p>*<p>P<0.05,</p>**<p>P<0.01 and *** P<0.001.</p

Effect of (S)-2 on the voltage-dependence of activation.

<p>Tail currents measured for Kv7.1 (A), Kv7.2 (B), Kv7.2/Kv7.3 (C), Kv7.4 (D) and Kv7.5 (E) after stepping back to −120 mV from 5 s long potentials between –100 and +60 mV were normalized and plotted against the preceding potential in the absence and presence of 10 µM (S)-2. The tail current-voltage relationship was then fitted to the Boltzmann equation (Eq. 1) to yield half-activation potentials (V_0.5). Bars represent S.E.M. and <i>n</i> = 5–11.</p

Potency of (S)-2 on Kv7.2 and Kv7.4.

<p>Dose-response relationship for the effect of (S)-2 on (A) Kv7.2 (<i>n</i> = 6) and (B) Kv7.4 (<i>n</i> = 8) measured using an ⁸⁶Rb-flux assay.</p

Effects of gain-of-function <i>KCNA5</i> mutations.

<p><b>A</b> Effects of the mutations on the AP (i-iii) and APD-restitution (iv-vi) properties of human atrial myocytes elicited by updated <i>Colman et al</i>. model, <i>Courtemanche et al</i>. model and <i>Grandi et al</i>. model. <b>B</b> Effects of the mutations on the maximum sustained dominant frequency of excitation waves under the lone AF and chronic AF conditions using (i) <i>Colman et al</i>. model and (ii) <i>Courtemanche et al</i>. model. <b>C</b> Effects of the mutations on APD heterogeneity and tissue vulnerability window at the CT/PM junction in <i>Colman et al</i>. model. APD distribution among regional cells of whole atria and CT/PM for in (i) isolated single cells and (ii) in coupled tissue; the APD distribution in tissue is shown in boxplots with outlier limits of 1.5×IQR (interquartile range). (iii) Temporal vulnerability window to propagation wave break at the CT/PM junction.</p

<i>KCNA5</i> loss-of-function mutations induced EADs following the beta-adrenergic stimulation.

<p><b>A</b>(i) In the presence of ISO, EADs were produced by Y155C and P488S, but not in WT or D469E in RA; (ii) EADs were induced by D469E in CT but not in PM. <b>B</b> Under uniform application of ISO in a 1D strand model with D469E, EADs in the CT but not PM induced conduction block at an S2 of 689 ms (i) and success at 690 ms (ii). <b>C</b> 1D conduction patterns under mutations and application of ISO in various configurations. On the left of each panel is a breakdown of the regions in the 1D model and an illustration of the anatomical conduction pathway to which they correspond; on the right is the regions of tissue to which ISO was applied. (i) Regular conduction pattern under WT and uniform ISO application; (ii) Under uniform application of ISO, D469E led to alternating bidirectional conduction block due to EADs in the CT; (iii) D469E and non-uniform ISO can lead to unidirectional conduction block, resulting from EADs in the CT regions with ISO; (iv) Unidirectional block can also be attained through a different pathway to the PM, in which the CT on one side of the SAN is of insufficient extent to develop significant EADs. In the simulations effects of beta-adrenergic stimulation was modelled by simulated application of ISO (1 μM).</p