Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

Abstract

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4.

Fig. 1.

Enhancement of endogenous Kv7.5 current in A7r5 cells by isoproterenol depends on activation of endogenous βARs and PKA. (A) Representative traces of endogenous Kv7.5 currents recorded in an A7r5 cell before (control, left panel), after 5 minutes in the presence of 1 μM isoproterenol (middle panel), and following addition of 1 μM XE991 (right panel) (C = 151 pF, representative of 11 similar experiments). (B) Current-voltage relationships of endogenous Kv7.5 current densities recorded in A7r5 cells before (control, solid circles), after 5 minutes of treatment with 1 μM isoproterenol (open circles), and after 10 minutes of treatment with 1 μM isoproterenol in combination with 1 μM XE991 (solid triangles). The asterisks (*) indicate significant difference from control, P < 0.05, one-way repeated-measures analysis of variance (ANOVA), n = 5–11. (C) Conductance-voltage relationships of endogenous Kv7.5 channels normalized to maximal conductance (G_max) before (solid circles, n = 5) and after addition of 1 μM isoproterenol (open circles, n = 5) fitted to the Boltzmann equation (solid lines). Absolute values of conductance densities are shown in the inset. (D) Deactivation tail currents recorded at −120 mV before (black) and after addition of 1 μM isoproterenol (red), fitted by a single exponential function (solid lines). Endogenous Kv7.5 channels were activated by 5-second steps to −20 mV, followed by a 1-second step to −120 mV; five traces were averaged for each condition. The inset shows tail currents normalized to peak amplitude in order to highlight the differences in deactivation kinetics. (E) Normalized currents recorded at −20 mV in control (black bar) and in the presence of the following: 1 μM isoproterenol (ISO, open bar, n = 5); 10 μM propranolol (P, dark gray bar, n = 4); 10 μM propranolol plus 1 μM isoproterenol (P + ISO, dark gray striped bar, n = 4); 1 μM KT5720 (KT, light gray bar, n = 4); and 1 μM KT5720 plus 1 μM isoproterenol (KT + ISO, light gray striped bar, n = 4). The asterisks (*) indicate significant difference from control (P < 0.05, one-way ANOVA on ranks).

Fig. 2.

Enhancement of endogenous Kv7.5 current in A7r5 cells by elevation of intracellular [cAMP]. (A) Current-voltage (I-V) relationships of endogenous Kv7.5 currents recorded in A7r5 cells before (control, solid circles), after 15 minutes of treatment with 1 mM 8-bromo cAMP (8Br-cAMP) (open circles), and after 10 minutes of washout (solid triangles). Currents were normalized to currents recorded at −20 mV before application of 8Br-cAMP [n = 4, * indicates significant difference from control, P < 0.05, one-way repeated-measures analysis of variance (ANOVA)]. (B) I-V relationships of endogenous Kv7.5 currents recorded in A7r5 cells before (control, solid circles), after 5 minutes of treatment with 1 μM forskolin (open circles), and after 10 minutes of washout (solid triangles). Currents were normalized to currents recorded at −20 mV before application of forskolin (n = 6, * indicates significant difference from control, P < 0.05, one-way repeated-measures ANOVA). (C) I-V relationships of endogenous Kv7.5 currents recorded in A7r5 cells before (control, solid circles), after 5 minutes of treatment with 1 μM forskolin in the presence of 500 μM IBMX (open circles), and after 10 minutes of washout (solid triangles). Currents were normalized to currents recorded at −20 mV before application of forskolin/IBMX (n = 4, * indicates significant difference from control, P < 0.05, one-way repeated-measures ANOVA). (D) I-V relationships of endogenous Kv7.5 currents recorded in A7r5 cells before (control, solid circles) and after treatment with papaverine (10 μM open circles, 100 μM open triangles). Currents were normalized to currents recorded at −20 mV before application of papaverine (n = 4, * indicates significant difference from control, P < 0.01, one-way repeated-measures ANOVA).

Fig. 3.

Inhibition of PDE4 with rolipram enhances endogenous Kv7.5 current in A7r5 cells in a PKA-dependent manner. (A) Current-voltage (I-V) relationships of endogenous Kv7.5 currents recorded in A7r5 cells before (control, solid circles) and during treatment with increasing concentrations of rolipram (open symbols: circle, 1 nM; triangle, 10 nM; reversed triangle, 30 nM; square, 100 nM; and diamond, 1 μM). (B) Cumulative rolipram dose-response curve for Kv7.5 current enhancement, presented as fold increase in amplitude of current recorded at −20 mV, fitted to the Hill equation [n = 4–5, * indicates significant difference from control, P < 0.001, one-way repeated-measures analysis of variance (ANOVA)]. (C and D) I-V relationships of Kv7.5 currents recorded in A7r5 cells before (control, solid circles), after 30 minutes of pretreatment with either vehicle for KT5720 (0.01% dimethylsulfoxide, solid triangles) (C) or 1 μM KT5720 (D), and after 5 minutes of treatment with 100 nM rolipram (open circles). Currents were normalized to currents recorded at −20 mV before application of rolipram (n = 4, * indicates significant difference from control, P < 0.05, one-way repeated-measures ANOVA).

Fig. 4.

Regulation of endogenous Kv7 currents in mesenteric artery myocytes by isoproterenol, rolipram, and forskolin/IBMX. (A) Current-voltage (I-V) relationships of endogenous Kv7 currents recorded in MASMCs before (control, solid circles) and after 5 minute treatment with 1 μM isoproterenol (open circles, n = 6). (B) I-V relationships of endogenous Kv7 currents recorded in MASMCs before (control, solid circles) and after 5 minutes of treatment with 100 nM rolipram (open circles, n = 5), followed by application of ML213 (10 μM, open triangles, n = 5) and 10 μM XE991 in the presence of 10 μM ML213 (solid triangles, n = 4). The asterisks (*) indicate significant difference from control, P < 0.05, one-way repeated-measures analysis of variance. (C) I-V relationships of endogenous Kv7 currents recorded in MASMCs before (control, solid circles) and after 5 minutes of treatment with 1 μM forskolin in the presence of 500 μM IBMX (open circles, n = 5, * indicates significant difference from control, P < 0.05, paired Student’s t test) and after 10 minutes of washout (solid upward triangles), followed by application of 1 μM XE991 (solid downward triangles, n = 4).

Fig. 5.

Differential regulation of hKv7.5, hKv7.4, and hKv7.4/7.5 by forskolin/IBMX and rolipram. (A and D) Current-voltage (I-V) curves of steady-state Kv currents recorded in A7r5 cells overexpressing hKv7.5 before (control, solid circles) and after 5 minutes of treatment with 10 μM forskolin in the presence of 500 μM IBMX (A, open circles, n = 7) or 100 nM rolipram (D, open circles, n = 7). (B and E) I-V curves of steady-state Kv currents recorded in A7r5 cells overexpressing hKv7.4 before (control, solid circles) and after 5 minutes of treatment with 10 μM forskolin in the presence of 500 μM IBMX (B, open circles, n = 3) or 100 nM rolipram (E, open circles, n = 5). (C and F) I-V curves of steady-state Kv currents recorded in A7r5 cells overexpressing hKv7.4/7.5 before (control, solid circles) and after 5 minutes of treatment with 10 μM forskolin in the presence of 500 μM IBMX (C, open circles, n = 6) or 100 nM rolipram (F, open circles, n = 5). The asterisks (*) indicate significant difference from control, P < 0.05, paired Student’s t test.

Fig. 6.

Activation of endogenous βARs in A7r5 cells enhances exogenous hKv7.5, hKv7.4 and hKv7.4/7.5 to varying degrees. (A–C) Current-voltage curves of steady-state Kv currents recorded in A7r5 cells overexpressing hKv7.5 (A, n = 6), hKv7.4 (B, n = 6), or hKv7.4/7.5 (C, n = 5) before (control, solid circles) and after 5 minutes of treatment with 1 μM isoproterenol (open circles). The asterisks (*) indicate significant difference from control, P < 0.05, paired Student’s t test. (D–F) Conductance-voltage relationships of exogenous hKv7.5 (D), Kv7.4 (E), and Kv7.4/7.5 (F) channels normalized to maximal conductance (G_max) before (solid circles, n = 5-6) and after addition of 1 μM isoproterenol (open circles, n = 5–6) fitted to the Boltzmann equation (solid lines); conductances normalized to the maximal control conductance (G_{max_c}) are shown in the insets.

Fig. 7.

Activation of endogenous βARs in A7r5 cells enhances PKA-dependent phosphorylation of exogenous hKv7.5 but not hKv7.4 channels. (A) Representative images of A7r5 cells exogenously expressing hKv7.5 channels (top panels) and hKv7.4 channels (bottom panels); untreated (control), treated with 1 μM isoproterenol (ISO) for 5 minutes, and treated with 1 μM ISO for 5 minutes after pretreatment with 1 μM KT5720 for 30 minutes (ISO + KT). PLAs were conducted on hKv7.5-expressing cells labeled with a combination of mouse anti-FLAG antibody and rabbit anti-phospho-Ser/Thr PKA substrate antibody or on hKv7.4-expressing cells labeled with a combination of mouse anti-KCNQ4 antibody and rabbit anti-phospho-Ser/Thr PKA substrate antibody. (B and C) Bar graphs summarizing the number of PLA signals/cell in A7r5 cells exogenously expressing hKv7.5 (B) and hKv7.4 (C) under control conditions (c), after 30 minutes of treatment with 1 μM KT5720 (c + KT), after 5 minutes of treatment with 1 μM ISO in the absence (ISO) or presence of pretreatment with 1 μM KT5720 (ISO + KT), and in cells where the primary antibodies were omitted (−1°ab); * and #, P < 0.001 from all group analyses of variance on ranks, n = 12–21.