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Voltage modulates the effect of ?-receptor activation in a ligand-dependent manner.

ABSTRACT: BACKGROUND AND PURPOSE:Various GPCRs have been described as being modulated in a voltage-dependent manner. Opioid analgesics act via activation of ? receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on ? receptor signalling. EXPERIMENTAL APPROACH:We investigated potential voltage sensitivity of ? receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer-based assays. Depolarization-induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials. KEY RESULTS:Studying ? receptor function and signalling in cells, we discovered that morphine-induced signalling was strongly dependent on the membrane potential (VM ). This became apparent at the level of G-protein activation, G-protein coupled inwardly rectifying potassium channel (Kir 3.X) currents and binding of GPCR kinases and arrestin3 to ? receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine-induced ? receptor activation was also observed at the level of Kir 3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate ? receptors was not (Met-enkephalin) or only moderately ([D-Ala2 , N-Me-Phe4 , Gly5 -ol]-enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate ? receptors. CONCLUSION AND IMPLICATIONS:Our results indicate a strong ligand-dependent modulation of ? receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity.

SUBMITTER: Ruland JG

PROVIDER: S-EPMC7348086 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Voltage modulates the effect of μ-receptor activation in a ligand-dependent manner.

Ruland Julia G JG Kirchhofer Sina B SB Klindert Sebastian S Bailey Chris P CP Bünemann Moritz M

British journal of pharmacology 20200519 15

<h4>Background and purpose</h4>Various GPCRs have been described as being modulated in a voltage-dependent manner. Opioid analgesics act via activation of μ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on μ receptor signalling.<h4>Experimental approach</h4>We investigated potential voltage sensitivity of μ receptors in heterologous expression systems (HEK293T cells) using electrophysiolog ...[more]

PMID: 32297669

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Project description:G-protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR's activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular, we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp246(6.48)). Our findings agree with a previously proposed view that, during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G-protein binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity, and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in crystal structures. These results should aid in the discovery of more effective and selective GPCR ligands.

Dataset Information

Voltage modulates the effect of ?-receptor activation in a ligand-dependent manner.

Publications

Voltage modulates the effect of μ-receptor activation in a ligand-dependent manner.

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