Fundamental role for the KCNE4 ancillary subunit in Kv7.4 regulation of arterial tone

Abstract

Key points: KCNE4 alters the biophysical properties and cellular localization of voltage-gated potassium channel Kv7.4. KCNE4 is expressed in a variety of arteries and, in mesenteric arteries, co-localizes with Kv7.4, which is important in the control of vascular contractility. Knockdown of KCNE4 leads to reduced Kv7.4 membrane abundance, a depolarized membrane potential and an augmented response to vasoconstrictors. KCNE4 is a key regulator of the function and expression of Kv7.4 in vascular smooth muscle.

Abstract: The KCNE ancillary subunits (KCNE1-5) significantly alter the expression and function of voltage-gated potassium channels; however, their role in the vasculature has yet to be determined. The present study aimed to investigate the expression and function of the KCNE4 subunit in rat mesenteric arteries and to determine whether it has a functional impact on the regulation of arterial tone by Kv7 channels. In HEK cells expressing Kv7.4, co-expression of KCNE4 increased the membrane expression of Kv7.4 and significantly altered Kv7.4 current properties. Quantitative PCR analysis of different rat arteries found that the KCNE4 isoform predominated and proximity ligation experiments showed that KCNE4 co-localized with Kv7.4 in mesenteric artery myocytes. Morpholino-induced knockdown of KCNE4 depolarized mesenteric artery smooth muscle cells and resulted in their increased sensitivity to methoxamine being attenuated (mean ± SEM EC50 decreased from 5.7 ± 0.63 μm to 1.6 ± 0.23 μm), which coincided with impaired effects of Kv7 modulators. When KCNE4 expression was reduced, less Kv7.4 expression was found in the membrane of the mesenteric artery myocytes. These data show that KCNE4 is consistently expressed in a variety of arteries, and knockdown of the expression product leads to reduced Kv7.4 membrane abundance, a depolarized membrane potential and an augmented response to vasoconstrictors. The present study is the first to demonstrate an integral role of KCNE4 in regulating the function and expression of Kv7.4 in vascular smooth muscle.

Figure 1. Whole‐cell patch‐clamp recordings on HEK cells stably expressing Kv7.4, transfected with either an empty vector or KCNE4

A, representative recordings of the Kv7.4 and Kv7.4/KCNE4 currents. Currents were elicited by the voltage protocol shown. The current elicited by a −30 mV test potential is indicated in red. B, current–voltage relationship of Kv7.4 (black; n = 19) and Kv7.4/KCNE4 (red; n = 9). Normalized current values are plotted against test the potentials and were obtained from steady‐state peak current amplitudes in response to depolarizing pulses from a holding potential of −80 mV. Values are normalized to the cell capacitance. A two‐way ANOVA followed by a Bonferroni post hoc test was performed. *P < 0.05; ***P < 0.001. C, comparison of the voltage dependence of activation in Kv7.4 (black; n = 7) and Kv7.4/KCNE4 (red; n = 13) expressing HEK cells. The current–voltage relation was obtained by plotting the normalized tail current amplitude at −30 mV against the preceding step potential. The tail currents were normalized against the maximal tail current measured in each experiment and the half‐activation potential was calculated using a Boltzmann fit. The mean half‐activation potential was determined for Kv7.4 and Kv7.4/KCNE4 currents and compared using an unpaired t test. ***P < 0.001. D, channel activation kinetics comparing Kv7.4 and Kv7.4/KCNE4 currents. The activation kinetics were fitted to a single exponential function. The time constants are shown as a function of the step potential. A two‐way ANOVA followed by a Bonferroni post hoc test was performed. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2. Expression of KCNE4 in the rat mesenteric artery

A, QPCR analysis of the relative abundance of KCNE mRNA in the rat mesenteric artery. The relative abundance of each gene was calculated using the 2^−ΔCq method (n = 3). B, representative western blot of KCNE4 bands (indicated by arrows) in HEK cell lysate with and without KCNE4 protein expression, and native rat mesenteric artery protein lysate. C, representative fluorescence and transmitted light (insert) confocal mid‐cell xy‐sections of HEK cells transfected with (a) KCNE4 or (b) an empty vector and both stained for KCNE4 (green), as well as freshly isolated mesenteric artery myocytes probed with (c) a primary antibody for KCNE4 (green) or (d) a no primary control. The nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (blue). Scale bars = 10 μm.

Figure 3. In situ PLA detection of Kv7.4, Kv7.5 and KCNE4 protein interactions in rat mesenteric artery myocytes

Aa–Af, representative fluorescence and transmitted light (insert) confocal mid‐cell xy‐sections of mesenteric artery myocytes probed with primary antibody combinations for KCNE4, Kv7.4 or Kv7.5 together with appropriate PLA probes. Red punctae indicate target proteins are in close proximity (<40 nm). Nuclei are shown in blue as defined by 4′,6‐diamidino‐2‐phenylindole. B, quantification of the mean ± SEM number of PLA signals per mid‐cell xy‐section for each antibody combination and a no primary control (NPC). **P < 0.01 for Kv7.4 + KCNE4 vs. NPC with anti‐rabbit (R) and anti‐mouse (M) PLA probes and ***P < 0.001 for Kv7.5 + KCNE4 or Kv7.4 + Kv7.5 vs. NPC with anti‐rabbit (R) and anti‐goat (G) PLA probes, according to a one‐way ANOVA, Tukey's multiple comparisons test. Scale bar = 10 μm.

Figure 4. KCNE4 knockdown depolarises rat mesenteric artery myocytes

Rat mesenteric artery arteries were transfected with either a KCNE4‐targeted or the respective control morpholino. Aa, representative western blot, with KCNE4 bands indicated by arrows. Ab, mean data comparing the KCNE4 band intensity in the lysates from mesenteric arteries transfected with the targeted or control morpholino. *P < 0.05 according to a paired t test. The number of cells analysed is indicated in each bar. Representative traces (Ba) and scatter plot (Bb) showing the membrane potential from current‐clamp experiments on myocytes isolated from mesenteric arteries after transfection with the KCNE4 control morpholino (n = 21 cells from six transfections and animals), which was significantly more hyperpolarized than the membrane potential in myocytes derived from the arteries transfected with the KCNE4‐targeted morpholino (n = 17 cells from six transfections and animals; P = 0.0004 according to a Mann–Whitney test). C, current–voltage relationship comparing isolated mesenteric artery myocytes transfected with either a KCNE4‐targeted (red) or control (black) morpholino (n = 5 cells from two transfections for each group). Current values are plotted against the test potentials elicited by depolarizing steps from −80 mV to + 50 mV (10 mV increments, 750 ms duration) from a holding potential of −80 mV. A Bonferroni post hoc test was performed following a two‐way ANOVA. **P < 0.01; ***P < 0.001.

Figure 5. Knockdown of KCNE4 increases the contractile reactivity of rat mesenteric arteries through reduced Kv7 channel function

Representative traces (Aa) and mean concentration–effect curves (Ab) for methoxamine in isometric tension recordings performed on vessels transfected with either the control (black) or KCNE4‐targeted (red) morpholino. *P < 0.05 and ***P < 0.0001, respectively, according to a Bonferroni post hoc test following a two‐way ANOVA. Ac, scatter plot of the methoxamine EC₅₀ values of each experiment. ***P < 0.0001 according to an unpaired t test with Welch's correction. The effect of 3 μmol L⁻¹ linopirdine in vessels transfected with either the (Ba) KCNE4 control or (Bb) KCNE4‐targeted morpholino. *P < 0.05 and ***P < 0.001 according to a two‐way ANOVA followed by a Bonferroni post hoc test, respectively. C, comparison of the relaxation effect of S‐1 (1 μmol L⁻¹) in control and targeted KCNE4 morpholino transfected vessels. *P < 0.05 according to a Mann–Whitney test. D, effect of 1 μmol L⁻¹ HMR1556 in vessels transfected with either the (Ba) KCNE4 control or (Bb) KCNE4‐targeted morpholino. Data are the mean ± SEM.

Figure 6. Immunostaining of mesenteric artery smooth cells transfected with either the control or KCNE4‐targeted morpholino

A, representative smooth muscle cells isolated and stained for KCNE4 (green) and Kv7.4 (red). 4′,6‐diamidino‐2‐phenylindole is shown in blue. Scale bars = 20 μm. Mean ± SEM intensity  of (Ba) KCNE4 and (Bb) Kv7.4 staining in the smooth muscle cells comparing vessels transfected with the KCNE4 control (n = 5 from three transfections) or KCNE4‐targeted morpholino (n = 7 from three transfections). Bc and Bd, percentage of KCNE4 and Kv7.4 staining in or near the membrane of these cells relative to the total expression. Data are the mean ± SEM (n ≥ 5 cells from three transfections). *P < 0.05, according to a Mann–Whitney test.

Figure 7. KCNE4 expression increases Kv7.4 membrane abundance in HEK cells

A, representative HEK cells expressing Kv7.4 (red) alone or Kv7.4 with KCNE4 (green). The white boxes in the merged panel are expanded to the right to show membrane fluorescence. B, quantification of Kv7.4 membrane‐like expression as a percentage of the total Kv7.4 expression. Data are the mean ± SEM. The number of cells analysed is indicated in each bar as taken from three separate experiments. ***P < 0.001, according to a Mann–Whitney test.