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

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

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

Effect of SMB-1 on K_V7.2-W236L.

<p>(<b>A</b>) Representative current traces for K_V7.2-W236L in the absence and presence of 10 µM SMB-1 (<b>B</b>) Effect of SMB-1 on current-voltage relationship. (<b>C</b>) Effect of SMB-1 on voltage-dependence of activation. (<b>D</b>) Effect of SMB-1 on deactivation kinetics. Statistical significance was determined by paired, two-tailed Student's <i>t</i>-test. Representative tail current traces in the absence and presence of 10 µM SMB-1 are shown in the inset. Effect of SMB-1 on the fast (<b>E</b>) and slow (<b>F</b>) component of the activation kinetics. Statistical significance was determined by two-way repeated measurements ANOVA followed by Bonferroni post-test. Y-values were log-transformed before the statistical analysis to meet the assumption of normality. * <i>p<</i>0.05, ** <i>p<</i>0.01, *** <i>p<</i>0.001. Bars represent S.E.M and <i>n = </i>4–6.</p

Plasma and brain exposure of SMB-1 in rats following subcutaneous administration of 20 mg/kg.

<p>Data shown as mean total concentrations ± S.E.M (n = 3).</p