Two-pore domain K+ channel TASK-1 in human pulmonary artery smooth muscle cells and its regulation by the vasoconstrictor endothelin-1

Abstract

The characteristics of the two pore-domain potassium channel and its modulation by endothelin-1 (ET-1) were tested by means of the whole-cell patch-clamp technique combined with TASK-1 small interfering RNA (siRNA) in primary human pulmonary arterial smooth muscle cells. We have found that the TASK-1 protein is present in primary hPASMC via immunofluorescence and immunoprecipitation methods. PCR studies detect only TASK-1 mRNA in primary and cultured hPASMC.Under whole cell voltage-clamp conditions, noninactivating K+ current is clearly but reversibly inhibited by anandamide, which is a direct and selective TASK-1 channel blocker. Its inhibition induces a significant membrane depolarization. As a consequence of the inhibition of the outward current, anandamide also influences the reversal potential of the current. The anandamide sensitive current is reversed close to -84 mV, the calculated Nernst equilibrium potential for K+ under these conditions. Moreover, under symmetrical K+ conditions in the same cell, anandamide causes a clear inhibition of the K+ current. The anandamide sensitive current is linear and reversed at or near 0 mV, the calculated Nernst equilibrium potential for K+ under these conditions. We have found that the TASK-1 channels are their extreme sensitive to variations in the extracellular pH. It behaves in a pH dependent manner in primary hPASMC across the full voltage range. A pH of 8.3 significantly hyperpolarizes the cells, whereas acidification causes a membrane depolarization in primary hPASMC (-10±1 mV versus 13±2 mV; n=5).By assessing the O2 sensitivity of this channel in primary hPASMC, under current-clamp conditions, we have found that hypoxia causes a marked cell depolarization (by 10±1 mV; n=9) and that the hypoxia-sensitive current reverses close to -84 mV.Further investigations, using the TASK-1 small interfering RNA (siRNA) technique show that TASK-1 knockdown causes a depolarization of the resting membrane potential compared to control cells (-26±1 mV versus -40±2 mV; P<0.05). Pretreatment of hPASMC with TASK-1 siRNA results in a lack of significant further suppression of the IKN by anandamide, acidosis, hypoxia, or ET-1.In order to detect the modulation by ET-1, we have found that ET-1 markedly reduces the TASK-1 current in primary hPASMCs and significantly and dose-dependently depolarizes hPASMCs. The concentration-response curve calculation shows that IC50 is 1.6 ± 0.3 nM (n = 5). The effect of ET-1 on the TASK-1 current is completely abolished by a pre-application of 10 uM anandamide or a pH of 6.3. The ET-1-sensitive current is reversed close to -84 mV, the calculated Nernst equilibrium potential for K+ under these conditions. Application of isoflurane (1mM) enhances the TASK-1 current, and this facilitation is blocked by ET-1.In order to investigate the possible physiological relevance of TASK-1 for the ET-1 induced pulmonary vasoconstriction, the ET-1-induced pressor response in the isolated perfused mouse lungs was carried out. ET-1 induces a dose-dependent increase in the PA pressure. The PA vasoconstriction is more pronounced when anandamide is given prior to ET-1. The application of 8 nM ET-1 increases PAP with 15.6 ± 2.3 mmHg. When anandamide is given prior to ET-1, the increase is 23.8 ± 1.0 mmHg (n =5).To investigate the possible involvement of the ET-1-mediated signal transduction in human PASMCs, we have employed different signaling molecular blockers and demonstrated that the endothelin-sensitivity of TASK-1 requires ETA receptors, phospholipase C (PLC), phosphatidylinositol 4,5-biphosphate (PIP2), diacylglycerol (DAG) and protein kinase C (PKC) in hPASMCs. However, caveolins are not involved in this process. Additionally, ET-1 stimulates the threonine phosphorylation of TASK-1, but not its tyrosine phosphorylation.Our results suggest that TASK-1 is expressed in human PASMC and is hypoxia-sensitive. Furthermore, TASK-1 is regulated by ET-1 involved in ETA-PLC-PIP2-DAG-PKC pathway at clinically relevant concentrations, which might represent a novel pathological mechanism related to pulmonary arterial hypertension. In dieser Arbeit wurden die Effekte von Endothelin-1 (ET-1) am two-pore domain Kaliumkanal TASK-1 von humanen primären pulmonalarteriellen glatten Muskelzellen (PASMCs) geprüft. Für die Untersuchungen wurde die Patch-Clamp Methode mit der small interfering RNA Methode kombiniert.Mittels pharmakologischen Tools wurde der TASK-1 Kaliumkanal, der auch als Hintergrundkaliumkanal bezeichnet wird, in der Ganzzell-Konfiguration der Patch-Clamp Methode untersucht. Der nicht-inaktivierende Kaliumstrom des TASK-1 Kanals wurde durch Anandamide (spezifischer Blocker von TASK-1), durch extrazelluläre Azidose und durch Hypoxie inhibiert. Die Anwendung der small interfering RNA gegen TASK-1 führte zu einer Inhibierung des TASK-1 Stromes und zu einem Verlust der Effekte von Anandamide, Azidose und Hypoxie sowie zu einer signifikanten Depolarisation des Membranpotentials der humanen PASMCs (-26±1 mV vs. -40±2 mV; P<0.05). Die Existenz von TASK-1 wurde auch mittels PCR und Immunhistochemie bestätigt.Der sehr effektive Vasokonstriktor Endothelin-1 blockierte den TASK-1 Kanal in klinisch relevanten Konzentrationen signifikant. Der Effekt war dosisabhängig mit einer halb-maximalen Konzentration (IC50) von 1.6 ± 0.3 nM. Die Effekte von ET-1 am TASK-1 Kanal wurden durch Vorapplikation von Anadamide oder Azidose aufgehoben. Die Anwendung von ET-1 führte zu einer signifikanten Vasokonstriktion in den Pulmonalarterien in der isoliert-perfundierten Mauslunge.Weitere Untersuchungen zeigten, dass für die Effekte von ET-1 der Endothelinrezeptor A, die Aktivierung von Phospholipase C (PLC), Phosphatidylinositol 4,5-biphosphate (PIP2), Diacylglycerol (DAG) und Proteinkinase C (PKC) in humanen PASMCs essentiell sind.Diese Ergebnisse zeigen, dass TASK-1 für die Effekte von ET-1 eine zentrale Bedeutung hat und daher einen neuen Signalweg repräsentiert, der für die Entstehung der pulmonalen Hypertonie, aber auch für mögliche therapeutische Ansätze eine wichtige Rolle spielen könnte.