Untersuchungen zu molekularen Mechanismen der Spannungsabhängigkeit des µ-Opioid Rezeptors

In jüngerer Zeit haben eine Reihe von Studien gezeigt, dass die membranständigen G-Protein-gekoppelten Rezeptoren (GPCR) in ihrer Funktion durch das elektrische Membranpotenzial beeinträchtigt werden. Diese spannungsabhängige Modulation kann je nach betrachtetem Rezeptor und je nach Liganden zu eine...

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Bibliographic Details
Main Author: Ruland, Julia
Contributors: Bünemann, Moritz (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2020
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Table of Contents: Recently, in a series of studies it has been proven, that the function of membranous G-protein coupled receptors (GPCR) is modulated by the membrane potential Depending on the type of receptor or ligand, the observed voltage-dependent changes may lead to an enhanced or decreased receptor activity which may be due to a change in affinity and also efficacy. Despite of an increasing number of receptors being characterized, the underlying mechanism of voltage sensitivity remains rather obscure. Also, there is little knowledge about pharmacological implications of voltage sensitivity in native tissue. In the present study the μ-opioid receptor (MOR) has been characterized with regard to voltage sensitivity. As activation of the MOR leads to a decreased propagation of pronociceptive stimuli, the receptor is the most important target for analgesics in treatment of moderate to severe pain. Being expressed in neuronal tissue, the MOR is exposed to robust and high-frequency changes in membrane potential. In the present study voltage sensitivity was elucidated at different levels: in G-protein activation, G-protein coupled receptor kinase 2 (GRK2)- and Arrestin3-recruitment, the voltage sensitivity of the MOR was elucidated on a molecular level by analyzing protein - protein interactions of effectors. To this aim, a setup which allows for the determination of ratiometric Foerster Resonance Energy Transfer (FRET) measurements between fluorescently labelled fusion proteins upon manipulation of the membrane potential by whole cell voltage clamp was used. A further set of experiments, which measured alterations of G-protein activated inwardly rectifying K+ (GIRK) currents, allowed for investigation of the pharmacological consequences of voltage sensitivity. To this aim, MOR-evoked GIRK currents were characterized upon depolarization both in inward and outward direction in transfected HEK 293T cells. Moreover, these currents were also determined in neurons that contained preparations of Locus Coeruleus (LC) neurons2. On a molecular basis, a strong voltage dependent increase in morphine-mediated receptor activation was observed on all investigated levels of signaling. The most striking impacts of voltage on signaling were found in the GRK2- and Arrestin3-recruitment to the morphine-activated MOR. According to previous publications, morphine has been characterized to be a ligand with low intrinsic efficacy, causing only minor recruitment of GRK2 and Arrestin3 in non-depolarized cells to the receptor, when compared with the peptidergic ligands DAMGO or Met-enkephaline. However, upon depolarization to different membrane potentials within the physiological range, a remarkable increase of the initial recruitment was observed and the occurring changes could be identified to demonstrate a change in efficacy. In contrast, investigation of voltage sensitive modulations of the DAMGO-activated receptor showed only minor changes in all observed interactions and the Met-enkephaline activated receptor even exhibited no detectable voltage sensitivity. In a subset of experiments, voltage sensitivity of fentanyl and buprenorphine, two further opioids of therapeutic relevance were tested. While the morphine-like substance buprenorphine showed – similar to morphine – a strong increase in efficacy upon depolarization, for fentanyl which is a structurally different type of ligand, a decrease of MOR-activity was observed upon depolarization. Characterization of changes in morphine-induced MOR-mediated GIRK currents due to depolarization in transfected HEK 293T cells showed an increase of currents both in outward and inward direction, although in these measurements only a small range of potentials within the physiological range of membrane potentials was suited for characterization, due to the physiologic properties of these channels. In a further set of experiments, the investigation of voltage sensitive effects on morphine-mediated GIRK currents in native tissue was performed. In these experiments, slices containing LC neurons were used for measurements. Although measurements in this delicate tissue allow for measurements in an even smaller range of membrane potentials, a significant increase in morphine-mediated GIRK currents, as compared to DAMGO- or Met-enkephaline-mediated currents was observed upon depolarization. In summary, this work demonstrates the strong voltage dependent modulation of MOR-signaling, which occurs to different extents and qualities dependent on the ligand applied. In case of the strong voltage sensitivity of the morphine-activated receptor, these changes become evident even on the level of GIRK currents in physiological tissue suggesting pharmacological relevance of voltage induced alterations of MOR activity.