TASK1 (K(2P)3.1) K(+) channel inhibition by endothelin-1 is mediated through Rho kinase-dependent phosphorylation

Abstract

Background and purpose: TASK1 (K(2P)3.1) two-pore-domain K(+) channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin-1 (ET-1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET-1-mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET-1.

Experimental approach: Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record TASK1 currents from hPASMC and Xenopus oocytes.

Key results: ET-1 inhibited TASK1-mediated I(KN) currents in hPASMC, an effect attenuated by Rho kinase inhibition with Y-27632. In Xenopus oocytes, TASK1 current reduction by ET-1 was mediated by endothelin receptors ET(A) (IC(50) = 0.08 nM) and ET(B) (IC(50) = 0.23 nM) via Rho kinase signalling. TASK1 channels contain two putative Rho kinase phosphorylation sites, Ser(336) and Ser(393) . Mutation of Ser(393) rendered TASK1 channels insensitive to ET(A) - or ET(B)-mediated current inhibition. In contrast, removal of Ser(336) selectively attenuated ET(A) -dependent TASK1 regulation without affecting the ET(B) pathway.

Conclusions and implications: ET-1 regulated vascular TASK1 currents through ET(A) and ET(B) receptors mediated by downstream activation of Rho kinase and direct channel phosphorylation. The Rho kinase pathway in PASMC may provide a more specific therapeutic target in pulmonary arterial hypertension treatment.

Figure 1

Endothelin-1 (ET-1) inhibition of TASK1/I_KN currents in human pulmonary artery smooth muscle cells. Representative experiments before (A) and after exposure to 10 nM ET-1 (B) are displayed. The corresponding current voltage relationship is shown in (C). (D) the time course of inhibition upon ET-1 exposure (10 nM) was monitored over a period of 20 min and is shown together with the respective current measurements under control conditions, after co-application of ET-1 (10 nM) and Y-27632 (10 µM) and following additional application of anandamide (10 µM) at the end of each experiment. (E) Summary of current changes after 20 min of drug exposure. Control values after incubation in bath solution (20 min) are not significantly different from results after application of ET-1 (10 nM) and Y-27632 (10 µM). In contrast, the effect of ET-1 (10 nM) was significantly different from control values; ***P < 0.001 vs. untreated controls.

Figure 2

Endothelin-1 (ET-1) inhibits TASK1 channels in Xenopus oocytes upon co-expression with endothelin receptors. Representative current recordings before and after exposure to 20 nM ET-1 are displayed for ET_A (A, B) and ET_B (E, F) receptors. (C, G) corresponding current voltage (I/V) relationships. (D, H) time course of TASK1 current blockade by 20 nM endothelin in the presence of ET_A (D; n = 7) and ET_B receptors (H; n = 7).

Figure 3

Dose-dependent inhibition of TASK1 channels in oocytes. The IC₅₀ values of the inhibitory effect of endothelin-1 on TASK1 currents were 0.08 ± 0.04 nM for ET_A receptor activation (A) and 0.23 ± 0.05 nM for ET_B receptor stimulation (B). Five to nine cells were studied at each concentration.

Figure 4

Intracellular signalling kinases associated with ET-1 regulation of TASK1 in oocytes, co-expressing TASK1 channels and ET_A or ET_B receptors. In order to investigate the signalling pathways, a range of low MW protein kinase inhibitors were co-applied together with 20 nM endothelin-1. Results for ET_A receptors (A) and ET_B receptors (B) are displayed. For comparison, the ET-1 effect in the absence of inhibitors is shown separated by a dashed line. The following kinase inhibitors were used: Y-27632 (10 µM) to inhibit Rho kinase; staurosporine (1 µM), chelerythrine (10 µM) or RO-32–0432 (3 µM) to inhibit protein kinase C; U73122 (10 µM) to inhibit phospholipase C; KT5720 (2.5 µM) to inhibit protein kinase A; ODQ (10 µM) to inhibit cGMP-dependent signalling; LY294002 (30 µM) and wortmannin (10 µM) to inhibit PI3kinase and myosin light chain kinase; and KN-93 (10 µM) to inhibit CamKII. Data are given as mean ± SEM; *P < 0.05; ***P < 0.001 versus ET-1 alone.

Figure 5

Upstream Rho kinase signalling in oocytes with TASK1 channels and ET_A or ET_B receptors. The small molecule Rac1 inhibitor (50 µM) was applied together with 20 nM ET-1. In addition, C3 toxin, an inhibitor of RhoA, was injected 3 h prior to the experiments (1 ng per oocyte) with ET-1 20 nM. Panel (A) shows the results for ET_A receptors, and data obtained from ET_B receptors are displayed in (B). Data are shown as mean ± SEM; *P < 0.05 versus ET-1 alone.

Figure 6

Downstream Rho kinase signalling in oocytes with mutant TASK1 channels and ET_A or ET_B receptors. Mutant TASK1 channels lacking the consensus sites for Rho kinase phosphorylation (TASK1-S336A; TASK1-S393A) were generated. Effects of ET-1 (20 nM) on mutant TASK1 channels in the presence of ET_A receptors (A) or ET_B receptors (B) are displayed. In each panel, control recordings (30 min) and the effect of ET-1 (20 nM) on TASK1 wild type channels are shown for comparison. Data are given as mean ± SEM; ***P < 0.001 versus wild type TASK1.

Figure 7

Overview of ET-dependent regulation of TASK1 channels. See text for details.