Impact of TASK-1 in Human Pulmonary Artery Smooth Muscle Cells

Abstract

Abstract-The excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K ϩ ) conductances. Although studies suggest that background K ϩ currents carried by 2-pore domain K ϩ channels are important regulators of resting membrane potential in PASMC, their role in human PASMC is unknown. Our study tested the hypothesis that TASK-1 leak K ϩ channels contribute to the K ϩ current and resting membrane potential in human PASMC. We used the whole-cell patch-clamp technique and TASK-1 small interfering RNA (siRNA). Noninactivating K ϩ current performed by TASK-1 K ϩ channels were identified by current characteristics and inhibition by anandamide and acidosis (pH 6.3), each resulting in significant membrane depolarization. Moreover, we showed that TASK-1 is blocked by moderate hypoxia and activated by treprostinil at clinically relevant concentrations. This is mediated via protein kinase A (PKA)-dependent phosphorylation of TASK-1. To further confirm the role of TASK-1 channels in regulation of resting membrane potential, we knocked down TASK-1 expression using TASK-1 siRNA. The knockdown of TASK-1 was reflected by a significant depolarization of resting membrane potential. Treatment of human PASMC with TASK-1 siRNA resulted in loss of sensitivity to anandamide, acidosis, alkalosis, hypoxia, and treprostinil. These results suggest that (1) TASK-1 is expressed in human PASMC; (2) TASK-1 is hypoxia-sensitive and controls the resting membrane potential, thus implicating an important role for TASK-1 K ϩ channels in the regulation of pulmonary vascular tone; and (3) treprostinil activates TASK-1 at clinically relevant concentrations via PKA, which might represent an important mechanism underlying the vasorelaxing properties of prostanoids and their beneficial effect in vivo. Key Words: pulmonary circulation Ⅲ potassium channels Ⅲ TASK-1 Ⅲ treprostinil Ⅲ hypoxic pulmonary vasoconstriction T he membrane potential of pulmonary artery smooth muscle cells (PASMC) is an important regulator of arterial tone. These cells have a resting membrane potential of approximately Ϫ65 to Ϫ50 mV in vitro, close to the predicted equilibrium potential for potassium (K ϩ ) ions. The opening of K ϩ channels in the PASMC membrane increases K ϩ efflux, which causes membrane hyperpolarization. This closes voltage-dependent Ca 2ϩ channels, decreasing Ca 2ϩ entry and leading to vasodilatation. Conversely, inhibition of K ϩ channels causes membrane depolarization, Ca 2ϩ entry, cell contraction, and vasoconstriction.Background or leak K ϩ -selective channels, as defined by a lack of time and voltage dependency, play an essential role in setting the resting membrane potential and input resistance in excitable cells. Two-pore domain K ϩ (2-PK) channels have been shown to conduct several leak K ϩ currents. The activity of 2-PK channels is strongly regulated by protons, protein kinases, and hypoxia. Alteration of K ϩ conductance can influence cellular activity via membrane potential changes.Both RT-PCR and Northern blot analyses p...

The Impact of Heterozygous KCNK3 Mutations Associated With Pulmonary Arterial Hypertension on Channel Function and Pharmacological Recovery

“…KCNK3 currents contribute to the resting potential of native PASMCs, and the downregulation or inhibition of KCNK3 causes PASMC depolarization, excessive proliferation, and pulmonary arterial constriction 5, 7, 8, 9. Cultured PASMCs have been used previously to study overexpressed potassium channel activity, regulation, and pharmacology in the context of PAH 19, 20.…”

“…KCNK3 is expressed in hPASMCs, while KCNK9 has been reported absent from hPASMCs 5. We performed gene expression analysis on whole human lung tissue samples from healthy (control) subjects, and from patients with familial PAH.…”

“…Bath (extracellular) solution (in mmol/L) contained: 150 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES adjusted to pH 6.4, 7.4, or 8.4 with NaOH. In hPASMCs, solutions were adapted from a prior study 5. Pipette (internal) solution (in mmol/L) contained: 135 K‐methanesulphonate, 20 KCl, 2 Na2ATP, 1 MgCl2, 1 EGTA, 20 HEPES, adjusted to pH 7.2 with KOH.…”

“…KCNK3 currents contribute to the PASMC resting potential and impact pulmonary arterial tone and smooth muscle cell growth; KCNK3 downregulation or inhibition leads to PASMC depolarization, proliferation, and pulmonary arterial constriction 5, 7, 8, 9. Notably, KCNK3 downregulation was recently identified as a pathogenic hallmark of PAH in humans and in a monocrotaline‐induced pulmonary hypertension rat model, and administration of ONO alleviated signs of pulmonary hypertension in the animal model, further implicating KCNK3 as a therapeutic target in PAH 3, 8…”

Potassium channels in pulmonary arterial hypertension

“…KCNK3 (also called TASK1) encodes a pH-sensitive K 2P channel, which has been shown to be expressed by PASMCs and to contribute significantly to the resting membrane potential [79]. Indeed, TASK1 knockdown in human PASMCs caused a depolarisation of the resting membrane potential [80]. In direct connection with PAH, TASK1 was documented as being inhibited in human PASMCs by endothelin-1 through an endothelin receptor type A-PLC-PIP 2 -DAG-PKC-dependent signalling cascade [81].…”