Hämoxygenase-2 reguliert die Öffnungswahrscheinlichkeit des Kaliumkanals TREK-1durch die Produktion von Kohlenstoffmonoxid

Der mechanosensitive K2P-Kanal TREK-1 wird durch eine Vielzahl von physikalischen und chemischen Stimuli, sowie durch eine Reihe von Interaktionspartnern reguliert. Das Ziel dieser Arbeit bestand darin, die Interaktion des TREK-1 Kanals mit dem Enzym Hämoxygenase 2 (HO-2), welches den stereospez...

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Bibliographische Detailangaben
1. Verfasser: Koschinsky, Kristin
Beteiligte: Preisig-Müller, Regina (PD Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Deutsch
Veröffentlicht: Philipps-Universität Marburg 2015
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The mechanosensitive K2P-channel TREK-1 is regulated by various physical and chemical stimuli, as well as interaction partners. The aim of this study was to characterize the interaction between TREK-1 and the enzyme HO-2, which catalyzes the breakdown of heme into biliverdin, Fe2+ and CO, and to clarify the functional consequences. Furthermore, the influence of CO on the TREK-1 channel was examined. The interaction between TREK-1 and HO-2 was confirmed by coimmunoprecipitation in HeLa cell lysates. In addition, yeast-two-hybrid experiments with HO-2 deletion mutants showed that the distal carboxyterminus of HO-2 is crucial for the interaction with the TREK-1 channel. The functional consequences of this interaction were examined by using two-electrodevoltage- clamp (TEVC) measurements in Xenopus oocytes. Coexpression of human HO-2 and TREK-1 led to a significant increase of TREK-1 current amplitude, whereas coexpression of the catalytically inactive enzyme HO-2H45N, which still interacts with TREK-1, evoked no significant change in the TREK-1 current amplitude. The catalytic activity of HO-2 in Xenopus oocytes was modulated by application of HO-2 activators and inhibitors. Xenopus oocytes constitutively express HO-2. Application of Hemin, a HO-2 activator, led to a significant increase in TREK-1 currents. Additional coexpression of human HO-2 led to a significantly higher activation of TREK-1 currents. Application of zinc protoporphyrin, an HO-2 inhibitor, led to significant reduction of TREK-1 currents, which was higher after additional coexpression of human HO-2. Application of carbon monoxide (CO) via CO-releasing molecule CORM-2 produced a significant increase of TREK-1 currents in Xenopus oocytes and in HEK293 cells. Further studies with the PhotoCORM-S1 and CO gas also increased TREK-1 currents significantly. Furthermore, the related mechanosensitive K2P-channels TREK-2 and TRAAK could also be activated by CO by a comparable degree as TREK-1. CO is an important cellular signaling molecule which interacts with various proteins and modulates a number of signaling pathways. Investigations of a possible CO-dependent signaling pathway showed that the different phosphorylation sites S362 (PKG-phosphorylation site) and S344 (PKAphosphorylation site) in the carboxyterminus of the channel were not involved in CO-mediated activation of the channel. Furthermore, deletion of the TREK-1 amino- or carboxyterminus showed that the shortened channels could still be activated by CO. Activation of TREK-1 currents by the gaseous signaling molecule NO was abolished in measurements with the carboxyterminal deletion mutant. Thus, the gaseous signaling molecules CO and NO appear to influence the open probability of TREK-1 via different signaling pathways. A direct COeffect on TREK-1 channels was proven by using inside-out measurements (giant-patches) in Xenopus oocytes. My results support the hypothesis that HO-2 binds to TREK-1 channels and regulates the open probability of the channel via local production of CO.