Untersuchungen zur Interaktion zwischen dem Kaliumkanal TREK-1 und Hämoxygenasen
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Philipps-Universität Marburg
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Abstract
Introduction
The mechanosensitive K2P-channel TREK-1 is expressed at high levels in the nervous system and is regulated by diverse physical and chemical stimuli and interaction partners. Several in vivo studies have demonstrated that TREK-1 is involved in pathophysiological processes like neuroprotection, pain and depression. TREK-1 channels stabilize the resting membrane potential of neurons and consequently have a key role in the regulation of membrane excitability. The aim of this study was to compare the direct interaction of the inducible enzyme heme oxygenase 1 (HO-1) and the constitutively expressed heme oxygenase 2 (HO-2) with TREK-1 and to identify amino acid regions responsible for the interaction.
Material and Methods
To characterize the direct interaction of TREK-1 with HO-1 und HO-2, the split-ubiquitin yeast two-hybrid system was used, which is specialized for membrane proteins. This method utilizes complementation between separable domains of ubiquitin to study protein interactions. The C-terminal half of ubiquitin (Cub), along with an artificial transcription factor complex was fused to TREK-1 (bait) and the N-terminal half of ubiquitin (NubG) was fused to HO-1 or HO-2 (preys). Interaction between bait and prey proteins brings Cub and NubG together which activates the transcription factor complex. This complex enters the nucleus and activates the expression of several reporter genes which in turn enable the yeast cells to grow on a selective medium lacking histidine and turn blue in a -galactosidase assay.
To investigate the physiological relevance of the interaction of TREK-1 with HO-1 and HO-2 the channel and the two heme oxygenase isoforms were co-expressed in Xenopus laevis oocytes and the TREK-1 currents were measured with the two-electrode voltage-clamp method.
In order to produce TREK-1 constructs with specific antigenic epitopes the HA-epitope (N-terminally) and the EGFP (C-terminally) were fused to the TREK-1 by using the site-directed mutagenesis method. Subsequently the fusion constructs were characterized with the Westernblot technique.
Results
Specific and strong interactions between several K2P-channels (TREK-1, TREK-2c and TASK-1) and HO-2 but not with its closely related isoform HO-1 could be demonstrated using a yeast two-hybrid direct interaction study. Additionally, it could also been shown that neither HO-2 nor HO-1 interacted with the inwardly rectifying potassium channel Kir2.1. Furthermore, physiological significance of the interaction between TREK-1 and HO-2 was demonstrated by two-electrode voltage-clamp measurements showing an increase in channel currents following the co-expression of TREK-1 and HO-2 in Xenopus laevis oocytes. On the contrary, co-expression of TREK-1 with HO-1 did not yield any changes in the channel currents.
Discussion
From the two studied heme oxygenases only HO-2 could be identified as a specific interaction partner of TREK-1 and other K2P-channels. Functionally, the interaction between TREK-1 and HO-2 leads to an increase of the channel currents, which in turn stabilizes the membrane potential of the cells in which both proteins are expressed. Although HO-1 and HO-2 have a high similarity (76 %) in their amino acid sequences, they exhibit several structural differences especially in the distal C-termini and the heme regulatory motifs of HO-2. These structural differences could be responsible for the absence of interaction between HO-1 and TREK-1 and in contrary be important for the specific interaction between HO-2 and TREK-1. Further analysis of defined HO-2 deletion mutants and the exchange of the distal C-termini between HO-2 and HO-1 could give more information about the importance of these regions for their interaction with TREK-1. Designing more experiments with the TREK-1 fusion constructs could be helpful for carrying out further immunochemical studies. Hilfe der ortsgerichteten Mutagenese mit TREK-1 fusioniert. Anschließend wurden die Fusionskonstrukte durch Westernblot-Analysen immunochemisch charakterisiert.
Ergebnisse
Es konnten spezifische und starke Interaktionen zwischen verschiedenen K2P-Kanälen (TREK-1, TREK-2c und TASK-1) und HO-2 mit Hilfe der Hefe-Zwei-Hybrid-Methode gezeigt werden, die für den nahen Verwandten HO-1 nicht nachweisbar waren. Zudem konnte gezeigt werden, dass weder HO-2 noch HO-1 mit dem einwärtsgleichrichtenden Kaliumkanal Kir2.1 interagieren. Die physiologische Relevanz der Interaktion zwischen TREK-1 und HO-2 konnte durch Messungen mit der Zwei-Elektroden-Spannungsklemme gezeigt werden, bei denen es nach Coexpression beider Proteine in Xenopus laevis Oozyten zu einer signifikanten Erhöhung des Ionenkanalstroms kam. Im Gegensatz dazu führte die Coexpression von TREK-1 und HO-1 zu keinen signifikanten Änderungen der Ionenkanalströme.
Diskussion
Von beiden untersuchten Hämoxygenasen konnte nur HO-2 als ein spe-zifischer Interaktionspartner für TREK-1 und andere K2P-Kanäle nachge-wiesen werden. Funktionell wirkt sich die Interaktion zwischen TREK-1 und HO-2 in einer Zunahme des Ionenkanalstroms aus, wodurch das Membran-potential von Zellen, in denen beide Proteinpartner exprimiert werden, stabilisiert werden könnte. Obwohl HO-1 und HO-2 auf Ebene der Amino-säuresequenzen eine hohe Similarität (76 %) haben, zeigen sich einige strukturelle Unterschiede, vor allem in den distalen C-Termini und bei den hämregulatorischen Motiven der HO-2. Diese strukturellen Unterschiede könnten zum einen für die fehlende Interaktion von HO-1 und TREK-1 verant-wortlich sein und zum anderen für die spezifische Interaktion von HO-2 und TREK-1 wichtig sein. Weitere Untersuchungen von speziellen HO-2 Deletions-mutanten und der Austausch der distalen C-Termini zwischen HO-2 und HO-1 könnten Aufschluss über die Wichtigkeit dieser Regionen bei der Interaktion mit TREK-1 geben. Experimente mit den TREK-1 Fusionskonstrukten könnten bei weiteren immunochemischen Charakterisierungen hilfreich sein.
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Dates
Created: 2012Issued: 2012-08-29Updated: 2012-08-29
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
potassium channel TREK-1 hemeoxygenase Yeast-two hybrid TEVC
DFG-subjects
HämoxgenaseTEVCTREK-1Hefe-zwei-Hybrid-SystemKaliumkanal
DDC-Numbers
610
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Kocher , Vivien-Isabell (geb. Lauer): Untersuchungen zur Interaktion zwischen dem Kaliumkanal TREK-1 und Hämoxygenasen. : Philipps-Universität Marburg 2012-08-29. DOI: https://doi.org/10.17192/z2012.0840.
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This item has been published with the following license: In Copyright