Charakterisierung der 2',3'-zyklischen Nukleotid 3'-Phosphodiesterase als neuen Interaktionspartner des Hyperpolarisations-aktivierten und durch zyklische Nukleotide modulierten HCN2 Kanals
Loading...
Files
Date
Authors
Publisher
Philipps-Universität Marburg
Supervisors
Abstract
In this dissertation, I investigated the modulation of the HCN (hyperpolarization-activated cyclic nucleotide-gated) channel family on a cellular, electrophysiological and molecular biological level by a potential new interaction partner, the preferentially glially expressed enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). The HCN channel family contains four channel subtypes (HCN1-4) that mediate a depolarizing cation inward current upon hyperpolarization. This current has a crucial role in the spontaneous generation of neuronal and cardiac rhythmic activities. The so-called cyclic nucleotide-binding domain (CNBD) in the C-terminus of the HCN protein is conserved within the channel family and enables cAMP (adenosine-3',5'-monophosphate) dependent regulation of the electrophysiological channel properties. In addition to cAMP, other important modulators of the HCN channels, such as phosphatidylinositol-4,5-bisphosphate (PIP2), TPR-containing Rab8b-interacting protein (TRIP8b) and vesicle-associated membrane protein-associated protein B (VAPB) are known to modulate HCN channel trafficking or gating. In this thesis a further interaction partner of the HCN2 channel has now been characterized, that had been identified using a split-ubiquitin yeast-two hybrid (Y2H) screen of a human brain cDNA library. This interaction partner is the membrane-associated enzyme CNP. CNP catalyzes the hydrolysis of the cyclic nucleotide 2',3'-cAMP to 2'-AMP. CNP represents a novel, previously undescribed modulator of the HCN2 channel. Therefore, the aim of this dissertation was to characterize CNP as an interaction partner of the HCN2 channel and to functionally elucidate HCN channel regulation by CNP. Initially, the interaction of CNP with HCN2 was investigated. For this purpose, the results of the Y2H screen were verified biochemically by co-immunoprecipitation and a protein-protein interaction of the HCN2-CNP channel complex was detected. Furthermore, the interaction was investigated electrophysiologically using the two-electrode voltage-clamp (TEVC) technique. The TEVC-experiments show, for the first time, that CNP leads to a concentration-dependent reduction of HCN2-encoded currents. In addition, TEVC measurements confirm HCN channel subtype-dependent modulation. While HCN4 currents, comparable to those of the HCN2 channel, show a decrease in current amplitude due to CNP coexpression, HCN1 currents show a reduction to a lesser extent, and HCN3 currents show no modulation. TEVC measurements of classical representatives of different potassium channel families also show no modulation of current amplitude by CNP. To investigate the mechanism underlying the reduced HCN2 current densities by CNP, fluorescence microscopic analyses were performed, indicating reduced localization of HCN2 channels at the plasma membrane under coexpression with CNP. Furthermore, the modulatory effect of CNP on HCN2 channels was investigated using ELISA-based quantification of HCN2 surface expression. This analysis confirms the results of the fluorescence microscopy experiments by showing a significant decrease in HCN2 surface expression due to CNP. To identify the interacting HCN2 domains with CNP, functional TEVC measurements were performed with C- and N-terminally truncated HCN2 channels. The analysis of the data reveal, that the N- as well as the C-terminus are important for the interaction between CNP and HCN2, but not exclusively, because the truncations do not completely prevent modulation. Furthermore, the endogenous expression of the HCN2-CNP channel complex was investigated. For this purpose, reverse transcriptase (RT)-PCR was performed to confirm endogenous expression of CNP, as well as HCN1, HCN2 and HCN4 in primary murine oligodendrocyte progenitor cells (OPC) and differen¬tiated oligodendrocytes (OLG). In addition, immunofluorescence staining for coexpression of HCN2 and CNP was performed. The images show colocalization of both proteins in OPC and OLG, however, the expression of HCN2 seems to be restricted to the OLG soma. This suggests a maturation-dependent regulation of HCN2 localization, possibly affected by CNP. Although the cyclic nucleotid-regulation is characteristic for HCN channels, no data exist on the effect of 2',3' cAMP on HCN2 channel regulation. However, in this dissertation, using inside-out macropatch clamp measurements, a 2',3'-cAMP-regulation of HCN2 channels was observed, which in turn is dependent on the coexpression of the channels with CNP.
In summary, CNP was confirmed to be a subtype-selective HCN channel modulator based on the experiments and analyses performed in this dissertation. Moreover, CNP was shown to modulate not only surface expression and, in this context, current amplitude, but also the 2',3' cAMP sensitivity of the HCN2 channel. To date, HCN2 current modulation by an interaction of CNP with the HCN2 channel protein and the function of this modulation have not been described. Strikingly, the characterization of the newly identified HCN2-CNP interaction performed in this work provides first evidences on the regulation of HCN2 channels in glial cells and thus may contribute to the understanding of electrical activity in oligodendrocytes and of demyelinating processes.
Review
Metadata
Contributors
Supervisor:
Dates
Created: 2022Issued: 2025-12-01Updated: 2022-12-12
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
CNPHCN2ion channelhyperpolarization-activated and cyclic nucleotide-gated HCN2 channelIonenkanal2',3'-cyclic nucleotide 3'-phosphodiesteraseOligodendrozyten
DFG-subjects
CNPaseHCN22',3'-zyklischen Nukleotid 3'-PhosphodiesteraseCNP
DDC-Numbers
500
show more
Komadowski, Marlene: Charakterisierung der 2',3'-zyklischen Nukleotid 3'-Phosphodiesterase als neuen Interaktionspartner des Hyperpolarisations-aktivierten und durch zyklische Nukleotide modulierten HCN2 Kanals. : Philipps-Universität Marburg 2025-12-01. DOI: https://doi.org/10.17192/z2023.0023.
License
This item has been published with the following license: In Copyright