Publikationsserver der Universitätsbibliothek Marburg

Titel:Regulation of TASK potassium channels by G-protein coupled receptors
Autor:Wilke, Bettina
Weitere Beteiligte: Oliver, Dominik (Prof. Dr.)
Veröffentlicht:2017
URI:https://archiv.ub.uni-marburg.de/diss/z2017/0257
URN: urn:nbn:de:hebis:04-z2017-02573
DOI: https://doi.org/10.17192/z2017.0257
DDC: Medizin, Gesundheit
Titel(trans.):Regulation von TASK Kalium Kanälen durch G-Protein gekoppelte Rezeptoren
Publikationsdatum:2017-03-21
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
zerebelläre Körnerzellen, Phospholipase C, Potassium channel, GPCR, Kaliumkanal, Phospholipase C, Diacylglycerol, Diacylglycerol, Cerebellar granule neurons, G-Protein gekoppelte Rezeptoren

Summary:
TASK potassium channels control the membrane potential in many cell types and thus affect a plethora of cellular functions such as excitability of neurons and cardiac muscle, and secretion of aldosterone in the adrenal gland. Although commonly termed ‘leak channels’, TASK channels are highly regulated. Most importantly, they are strongly inhibited by a variety of hormones and neurotransmitters activating Gq-protein coupled receptors (GqPCRs). Despite extensive studies of TASK inhibition by the GqPCR-induced signaling cascade, the underlying mechanism of channel regulation has not been elucidated. Thus I aimed to unravel the second messenger responsible for GqPCR-mediated TASK channel inhibition and validate my findings from the heterologous expression system in cerebellar granule neurons. The signaling cascade induced by GqPCRs is initiated by activation of Gαq, which in turn stimulates phospholipase Cβ to hydrolyze the membrane phospholipid phosphatidylinositol( 4,5)bisphosphate producing the second messengers 1,2-diacylglycerol (DAG) and inostol( 1,4,5)trisphosphate. Using different approaches, I first established that phospholipase C is critical for GqPCR-mediated TASK channel inhibition. Next, I found that direct application of a DAG analog was sufficient to inhibit TASK channels. Accordingly, experimental attenuation of the DAG transients evoked by GqPCR stimulation diminished TASK channel inhibition, indicating that DAG is responsible for the current reduction following receptor activation. Because it had been previously established that a six amino acid motif within the proximal C-terminus is important for TASK channel regulation by GqPCRs, I compared the effects of GqPCR stimulation and DAG application on TASK channel proteins either truncated or mutated within this motif. A correlation of the sensitivities towards DAG and GqPCR activation further supported the hypothesis of DAG production as the underlying mechanism for the GqPCR-mediated effect. Lastly, to test whether native TASK-mediated currents were also inhibited by DAG, I probed application of this lipid on dissociated cerebellar granule neurons that express the TASK-mediated standing outward potassium current (IKSO). IKSO was inhibited by muscarinic receptor agonist as well as by direct application of DAG, producing a significant membrane depolarization. In conclusion, my findings demonstrate that DAG mediates the GqPCR-induced inhibition of TASK channels in an expression system as well as native, TASK-mediated currents. Thus, my data expand the view on the signaling effects of the small membrane lipid DAG and establish a link between DAG and cell excitability. Additionally, they may pave the way towards understanding the mechanism of DAG action on ion channels as atypical DAG effector proteins.

Zusammenfassung:
TASK Kaliumkanäle tragen in vielen Zelltypen zur Generierung des Membranpotentials bei, wodurch sie zahlreiche zelluläre Funktionen beeinflussen. Obwohl TASK-vermittelte Ströme häufig als „Hintergrundleitfähigkeit“ bezeichnet werden, sind sie vielfach reguliert; unter anderem werden TASK Kanäle durch jene Hormone und Neurotransmitter gehemmt, welche Gαq/11-Protein-gekoppelte Rezeptoren (GqPCR) aktivieren. Diese Kanalregulation spielt zum Beispiel für die Anpassung der Erregbarkeit von Nerven- und Herzmuskelzellen, wie auch für die Aldosteronsekretion in der Nebenniere eine Rolle. Trotz der physiologischen Bedeutung von TASK Kanälen und deren Inhibition konnte der zugrunde liegende Mechanismus für die GqPCRvermittelte Regulation noch nicht aufgeklärt werden. Die vorliegende Arbeit hat zum Ziel, das verantwortliche Signalmolekül zu identifizieren und seinen Effekt auf zerebelläre Körnerzellen zu untersuchen. Die klassische GqPCRs Signalkaskade beginnt mit der Aktivierung von Gαq, welches die Phospholipase Cβ aktiviert. Diese spaltet daraufhin das Membranlipid Phosphatidylinositol( 4,5)bisphosphat zu Diacylglycerol (DAG) und Inositol(1,4,5)trisphosphat. Mit verschiedenen experimentellen Ansätzen habe ich zuerst die Bedeutung der Phospholipase C für die GqPCR-vermittelte TASK Regulation herausgearbeitet. In weiterer Folge konnte ich zeigen, dass die direkte Applikation eines DAG-Analogons ausreicht, um den Kanal zu inhibieren. Die Abschwächung des durch Stimulation von GqPCRs erreichten DAG-Transienten durch Überexpression DAG-metabolisierender Enzyme reduzierte gleichermaßen die Inhibition der TASK Kanäle. Da bereits gezeigt wurde, dass ein sechs Aminosäuren langes Motiv im TASK C-Terminus essentiell für die GqPCR-vermittelte Inhibition ist, habe ich den Kanal in diesem Motiv mutiert und die Mutanten auf ihre DAG-Sensitivität untersucht. Die Ergebnisse zeigen eine Korrelation zwischen der Inhibition durch Aktivierung eines GqPCRs und der Applikation des DAG-Analogons und untermauern die vorangegangenen Resultate, dass die DAG Produktion der GqPCR-vermittelten TASK Inhibition zugrunde liegt. Um die Ergebnisse aus den heterolog exprimierenden Zellen im nativen System zu validieren, untersuchte ich den TASK-vermittelten Strom IKSO in dissoziierten zerebellären Körnerzellen. Sowohl die Aktivierung muskarinischer Rezeptoren, als auch die Applikation des DAG-Analogons führten zu einer deutlichen Reduktion des IKSO, welche mit einer Depolarisation der Zellmembran einher ging. Zusammengefasst zeigen meine Ergebnisse, dass DAG der verantwortliche second messenger in der GqPCR Signalkaskade ist, welcher zur Schließung der TASK Kanäle führt. Das erweitert die Sicht auf die Signalwirkung des kleinen Membranlipids DAG und betont den Zusammenhang zwischen DAG und zellulärer Erregbarkeit.

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