Differential activation of vascular smooth muscle Kv7.4, Kv7.5, and Kv7.4/7.5 channels by ML213 and ICA-069673

Abstract

Recent research suggests that smooth muscle cells express Kv7.4 and Kv7.5 voltage-activated potassium channels, which contribute to maintenance of their resting membrane voltage. New pharmacologic activators of Kv7 channels, ML213 (N-mesitybicyclo[2.2.1]heptane-2-carboxamide) and ICA-069673 N-(6-chloropyridin-3-yl)-3,4-difluorobenzamide), have been reported to discriminate among channels formed from different Kv7 subtypes. We compared the effects of ML213 and ICA-069673 on homomeric human Kv7.4, Kv7.5, and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells. We found that, despite its previous description as a selective activator of Kv7.2 and Kv7.4, ML213 significantly increased the maximum conductance of homomeric Kv7.4 and Kv7.5, as well as heteromeric Kv7.4/7.5 channels, and induced a negative shift of their activation curves. Current deactivation rates decreased in the presence of the ML213 (10 μM) for all three channel combinations. Mutants of Kv7.4 (W242L) and Kv7.5 (W235L), previously found to be insensitive to another Kv7 channel activator, retigabine, were also insensitive to ML213 (10 μM). In contrast to ML213, ICA-069673 robustly activated Kv7.4 channels but was significantly less effective on homomeric Kv7.5 channels. Heteromeric Kv7.4/7.5 channels displayed intermediate responses to ICA-069673. In each case, ICA-069673 induced a negative shift of the activation curves without significantly increasing maximal conductance. Current deactivation rates decreased in the presence of ICA-069673 in a subunit-specific manner. Kv7.4 W242L responded to ICA-069673-like wild-type Kv7.4, but a Kv7.4 F143A mutant was much less sensitive to ICA-069673. Based on these results, ML213 and ICA-069673 likely bind to different sites and are differentially selective among Kv7.4, Kv7.5, and Kv7.4/7.5 channel subtypes.

Fig. 1.

ML213 induced a concentration-dependent enhancement of the Kv7.4 current accompanied by negative shift of the activation curve and prolonged Kv7.4 current deactivation. (A, B) Representative traces of Kv7.4 currents recorded with voltage-step protocol in the absence (A) and in the presence of 10 μM ML213 (B). (C) IV curves of normalized steady-state Kv currents recorded before (control, black circles) and after 10-minute treatment with increasing concentrations of ML213 as indicated (n = 5). (D) Averaged fractional conductance plots normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution (at increasing concentrations of ML213 as indicated in (C). (E) Representative traces of tail currents recorded in the absence (black, current scale at left) and in the presence of 10 μM ML213 (red, current scale at right) scaled to size of the currents recorded at 0 mV in the absence of ML213 for visual comparison. (F) The time constants (τ) of current deactivation, calculated from the single exponential fits of tail currents measured at voltages ranging from −130 mV to −90 mV in the absence (black circles) and in the presence of 10 μM ML213 (red circles) fitted to straight lines. *P < 0.05, paired Student’s t test, n = 5.

Fig. 2.

ML213 induced a concentration-dependent enhancement of Kv7.5 current accompanied by negative shift of the activation curve and decreased Kv7.5 current deactivation rate. (A, B) Representative traces of Kv7.5 currents recorded with a voltage step protocol in the absence (A) and in the presence of 10 μM ML213 (B). (C) IV curves of normalized steady-state Kv currents recorded before (control, black circles) and after 10-minute treatment with increasing concentrations of ML213 as indicated (n = 4 to 5). (D) Averaged fractional conductance plots normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution (at increasing concentrations of ML213 as indicated in (C). (E) Representative traces of tail currents recorded in the absence (black, current scale at left) and in the presence of 10 μM ML213 (red, current scale at right) scaled to size of the currents recorded at 0 mV in the absence of ML213 for visual comparison. (F) The time constants (τ) of current deactivation, calculated from the single exponential fits of tail currents measured at voltages ranging from −130 mV to −105 mV in the absence (black circles) and in the presence of 10 μM ML213 (red circles) fitted to straight lines. *P < 0.05, paired Student’s t test, n = 5.

Fig. 3.

ML213 induced a concentration-dependent enhancement of Kv7.4/7.5 current accompanied by negative shift of the activation curve and decreased Kv7.4/7.5 current deactivation rate. (A, B) Representative traces of Kv7.4/7.5 currents recorded with voltage step protocol in the absence (A) and in the presence of 10 μM ML213 (B). (C) IV curves of normalized steady-state Kv currents recorded before (control, black circles) and after 10-minute treatment with increasing concentrations of ML213 as indicated (n = 3–5). (D) Averaged fractional conductance plots normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution (at increasing concentrations of ML213 as indicated in C). (E) Representative traces of tail currents recorded in the absence (black, current scale at left) and in the presence of 10 μM ML213 (red, current scale at right) scaled to size of the currents recorded at 0 mV in the absence of ML213 for visual comparison. (F) The time constants (τ) of current deactivation, calculated from the single exponential fits of tail currents measured at voltages ranging from −130 mV to −90 mV in the absence (black circles) and in the presence of 10 μM ML213 (red circles) fitted to straight lines. *P < 0.05, paired Student’s t test, n = 5.

Fig. 4.

Summary of ML213-induced concentration-dependent negative shift of activation curves, increased maximal conductance, and decreased Kv7.4, Kv7.5, and Kv7.4/7.5 current deactivation rates. (A) Averaged ML213 dose dependence based on negative shift of V_0.5 estimated from Boltzmann fit of conductance plots for each experiment in A7r5 cells expressing Kv7.4 (black circles, n = 5), Kv7.5 (light gray circles, n = 5), and Kv7.4/7.5 (dark gray triangles, n = 5) fitted by the Hill equation. *Significant difference between Kv7.5 and Kv7.4 (P < 0.05, one-way ANOVA). (B) Averaged ML213 dose dependence based on increase in maximal conductance estimated from Boltzmann fit of conductance plots for each experiment in A7r5 cells expressing Kv7.4 (black circles, n = 5), Kv7.5 (light gray circles, n = 5) and Kv7.4/7.5 (dark gray triangles, n = 5) fitted by the Hill equation. (C) Time constant of current deactivation in the presence of 10 μM ML213, normalized to the control time constant of deactivation, was plotted against voltages to illustrate the voltage-dependent change in deactivation kinetics in the presence of 10 μM ML213 for Kv7.4 (black circles, n = 5), Kv7.5 (light gray circles, n = 5), and Kv7.4/7.5 (dark gray triangles, n = 5) fitted by straight lines. *Significant difference between Kv7.5 and Kv7.4 (P < 0.05, one-way ANOVA).

Fig. 5.

Comparison of effects of 10 µM ML213 on wild-type Kv7.5 and retigabine-insensitive mutant Kv7.5 W235L. (A) IV curves of normalized steady-state Kv7.5 W235L currents recorded before (control, filled circles) and after 10-minute treatment with 10 µM of ML213 (open circles, n = 6), followed by 10-minute treatment with 10 µM retigabine (open triangles, n = 5). **Significant difference between all groups (P < 0.01, repeated measures ANOVA). (B) Averaged fractional conductance plots for Kv7.5 W235L normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution (treatments as in A). (C) The time constants (τ) of current deactivation of Kv7.5 W235L, calculated from the single exponential fits of tail currents measured in control (filled circles) and in the presence of 10 μM ML213 (open circles) and 10 µM retigabine (open triangles). (D) Averaged V_0.5 estimated from Boltzmann fit of conductance plots in control (black bar for Kv7.5 wild-type and gray bar for Kv7.5 W235L) and in the presence of 10 µM ML213 (open bar for Kv7.5 wild-type and striped bar for Kv7.5 W235L). ***Significant difference from all groups (P < 0.001, one-way ANOVA, n = 5 or 6). (E) Averaged maximal conductance estimated from Boltzmann fit of conductance plots in the presence of 10 µM of ML213 normalized to control maximal conductance for wild-type Kv7.5 and Kv7.5 W235L (treatment indicated as in D). ***Significant difference from all groups (P < 0.001, one-way ANOVA, n = 5 or 6). (F) Time constant of current deactivation in the presence of 10 µM ML213, normalized to the deactivation time constant in control for wild type Kv7.5 (black circles, n = 5) and Kv7.5 W235L (gray circles, n = 5). **Significant difference between wild-type Kv7.5 and Kv7.5 W235L; P < 0.01, Mann-Whitney rank sum test.

Fig. 6.

Comparison of the effects of 10 µM ML213 on wild-type Kv7.4 and retigabine-insensitive mutant Kv7.4 W242L. (A) IV curves of normalized steady-state Kv7.4 W242L currents recorded before (control, filled circles) and after 10-minute treatment with 10 µM of ML213 (open circles, n = 4), followed by 10-minute treatment with 10 µM retigabine (open triangles, n = 4). (B) Averaged fractional conductance plots for Kv7.4 W242L normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution [treatments as in (A)]. (C). The time constants (τ) of current deactivation of Kv7.4 W242L, calculated from the single exponential fits of tail currents measured in control (filled circles) and in the presence of 10 μM ML213 (open circles) and 10 µM retigabine (open triangles). (D) Averaged V_0.5 estimated from Boltzmann fit of conductance plots in control (black bar for Kv7.4 wild-type and gray bar for Kv7.4 W242L) and in the presence of 10 µM ML213 (open bar for Kv7.4 wild-type and stripped bar for Kv7.4 W242L). **Significant difference from all groups (P < 0.01, one- way ANOVA, n = 4 or 5). (E) Averaged maximal conductance estimated from Boltzmann fit of conductance plots in the presence of 10 µM of ML213 normalized to control maximal conductance for wild-type Kv7.4 and Kv7.4 W242L [treatment indicated as in (D)]. ***Significant difference from all groups (P < 0.001, one-way ANOVA, n = 4 to 5). (F) Time constant of current deactivation in the presence of 10 µM ML213, normalized to the deactivation time constant in control for wild-type Kv7.4 (black circles, n = 5) and Kv7.4 W242L (gray circles, n = 4). ***Significant difference between wild-type Kv7.4 and Kv7.4 W242L, P < 0.001, Student’s t test.

Fig. 7.

ICA-069673 induced a profound negative shift of the Kv7.4 activation curve and drastically reduced Kv7.4 current deactivation rate. (A,B) Representative traces of Kv7.4 currents recorded with a voltage step protocol in the absence (A) and in the presence of 100 μM ICA-069673 (B). (C) IV curves of normalized steady-state Kv7.4 currents recorded before (control, black circles) and after 10-minute treatment with increasing concentrations of ICA-069673 as indicated (n = 4 to 5). (D) Averaged fractional conductance plots normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution (at increasing concentrations of ICA-069673 as indicated in C). (E) Representative traces of tail currents recorded in the absence (black; current scale at left) and in the presence of 100 μM ICA-069673 (red; current scale at right) scaled to size of currents recorded at 0 mV in the absence of ICA-069673 for visual comparison, 5 seconds of total 10-second tail duration shown for clarity. (F) The time constants (τ) of current deactivation, calculated from the single exponential fits of tail currents measured at voltages ranging from −130 mV to −90 mV in the absence (black circles) and in the presence of 100 μM ICA-069673 (red circles) fitted to straight lines. **P < 0.01, paired Student’s t test, n = 6.

Fig. 8.

ICA-069673 induced slight inhibition of Kv7.5 currents at 100 µM accompanied by negative shift of activation and reduction of current deactivation rate. (A,B) Representative traces of Kv7.5 currents recorded with voltage step protocol in the absence (A) and in the presence of 100 μM ICA-069673 (B). (C) IV curves of normalized steady-state Kv7.5 currents recorded before (control, black circles) and after 10-minute treatment with increasing concentrations of ICA-069673 as indicated (n = 4 or 5). **Significant difference from all groups (P < 0.01, repeated-measures ANOVA). (D) Averaged fractional conductance plots were normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution (at increasing concentrations of ICA-069673 as indicated in (C). **Significant difference from all groups (P < 0.01, repeated measures ANOVA). (E) Representative traces of tail currents recorded in the absence (black; current scale at left) and in the presence of 100 μM ICA-069673 (red; current scale at right) scaled to size of currents recorded at 0 mV in the absence of ICA-069673 for visual comparison. (F) The time constants (τ) of current deactivation, calculated from the single exponential fits of tail currents measured at voltages ranging from −130 mV to −90 mV in the absence (black circles) and in the presence of 100 μM ICA-069673 (red circles) fitted to straight lines. *P < 0.05, paired Student’s t test, n = 5.

Fig. 9.

ICA-069673 induces a negative shift of the Kv7.4/7.5 activation curve and reduced Kv7.4/7.5 current deactivation rate. (A,B) Representative traces of Kv7.4/7.5 currents recorded with voltage step protocol in the absence (A) and in the presence of 100 μM ICA-069673 (B). (C) IV curves of normalized steady-state Kv7.4/7.5 currents recorded before (control, black circles) and after 10-minute treatment with increasing concentrations of ICA-069673 as indicated (n = 4 or 5). (D) Averaged fractional conductance plots normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution (at increasing concentrations of ICA-069673 as indicated in C). (E) Representative traces of tail currents recorded in the absence (black; current scale at left) and in the presence of 100 μM ICA-069673 (red; current scale at right) scaled to size of currents recorded at 0 mV in the absence of ICA-069673 for visual comparison, 5 seconds of total 10-second tail duration shown for clarity. (F) The time constants (τ) of current deactivation, calculated from the single exponential fits of tail currents measured at voltages ranging from −130 mV to −90 mV in the absence (black circles) and in the presence of 100 μM ICA-069673 (red circles) fitted to straight lines. **P < 0.01, paired Student’s t test, n = 5.

Fig. 10.

ICA-069673 induced a concentration-dependent negative shift of activation curves and decreased Kv7.4, Kv7.5 and Kv7.4/7.5 current deactivation rates. (A) Averaged dose dependence of negative shift of V_0.5 estimated from Boltzmann fit of conductance plots for each experiment in A7r5 cells expressing Kv7.4 (black circles, n = 5), Kv7.5 (light gray circles, n = 5) and Kv7.4/7.5 (dark gray triangles, n = 5). ***Significant difference between all groups (P < 0.001 one-way ANOVA), ^###Significant difference of Kv7.5 from Kv7.4 and Kv7.4/7.5 (P < 0.001 one-way ANOVA). (B) Time constant of current deactivation in the presence of 100 μM ICA069673, normalized to the control time constant of deactivation, was plotted against voltages to illustrate the voltage-dependent change in deactivation kinetics in the presence of 100 μM ICA069673 for Kv7.4 (black circles, n = 5), Kv7.5 (light gray circles, n = 3) and Kv7.4.7.5 (dark gray triangles, n = 4). **Significant difference between all groups (P < 0.01 one-way ANOVA).

Fig. 11.

Comparison of effects of 100 µM ICA-069673 on wild-type Kv7.4, retigabine-insensitive mutant Kv7.4 W242L, and ztz240-insensitive mutant Kv7.4 F143A. (A) IV curves of normalized steady-state Kv7.4 W242L currents recorded before (control, filled circles) and after 10-minute treatment with 100 µM of ICA-069673 (open circles, n = 8). (B) Averaged fractional conductance plots of Kv7.4 W242L channels normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution [treatments as in (A)]. (C) The time constants (τ) of Kv7.4 W242L current deactivation, calculated from the single exponential fits of tail currents measured in control (filled circles) and in the presence of 100 μM ICA-069673 (open circles) fitted to straight lines. **P < 0.01, paired Student’s t test, n = 8. (D) IV curves of normalized steady-state Kv7.4 F143A currents recorded before (control, filled circles) and after 10-minute treatment with 100 µM of ICA-069673 (open circles, n = 5). (E) Averaged fractional conductance plots of Kv7.4 F143A channels normalized to maximal conductance in control for each experiment fitted to a Boltzmann distribution [treatments as in (A)]. (F) The time constants (τ) of Kv7.4 F143A current deactivation, calculated from the single exponential fits of tail currents measured in control (filled circles) and in the presence of 100 μM ICA-069673 (open circles) fitted to straight lines. **P < 0.01, paired Student’s t test, n = 5. (G) Averaged V_0.5 estimated from Boltzmann fit of conductance plots in control (black bar for Kv7.4 wild-type, light gray bar for Kv7.4 W242L and dark gray bar for Kv7.4 F143A) and in the presence of 100 µM ICA-069673 (open bar for Kv7.4 wild type, striped bar for Kv7.4 W242L and dotted bar for Kv7.4 F143A). ***Significant difference from Kv7.4 wild type, and Kv7.4 W242L, Kv7.4F143A in control, and Kv7.4 F143A in the presence of 100 μM ICA-069673, (P < 0.001, one-way ANOVA, n = 5–8), ^###Significant difference from Kv7.4 wild-type and Kv7.4 W242L in control (P < 0.001, one-way ANOVA, n = 5–8), ^^^Significant difference from all groups (P < 0.001, one- way ANOVA, n = 5–8). H. Time constant of current deactivation in the presence of 100 µM ICA-069673, normalized to the deactivation time constant in control for wild-type Kv7.4 (open circles, n = 6), hKv7.4 W242L (light gray circles, n = 8), and Kv7.4 F143A (dark gray circles, n = 5). Dotted line (at y-axis value = 1) represents the level of no changes in deactivation kinetic. **Significant difference from wild-type Kv7.4 and Kv7.4 W242L, P < 0.01, ANOVA on rank sum test).