Hämoxygenase-2 reguliert die Öffnungswahrscheinlichkeit des Kaliumkanals TREK-1durch die Produktion von Kohlenstoffmonoxid
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Philipps-Universität Marburg
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Abstract
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.
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Dates
Created: 2015Issued: 2016-04-14Updated: 2018-06-20
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
nitric oxide (NO)CO-releasing molecules (CORMs)carbon monoxide (CO)TREK-1K2P-channelheme oxygenase 2 (HO-2)Protein- Protein Interaktion, CO-freisetzende Moleküle (CORMs), Stickstoffmonoxid (NO)protein-protein interactionK2P-Kanäle
DFG-subjects
KohlenstoffmonoxidTREK-1Hämoxygenase-2
DDC-Numbers
610
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Koschinsky, Kristin: Hämoxygenase-2 reguliert die Öffnungswahrscheinlichkeit des Kaliumkanals TREK-1durch die Produktion von Kohlenstoffmonoxid. : Philipps-Universität Marburg 2016-04-14. DOI: https://doi.org/10.17192/z2015.0526.
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This item has been published with the following license: In Copyright