The coupling of the M2 muscarinic receptor to its G protein is voltage dependent.
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ABSTRACT:
SUBMITTER: Ben-Chaim Y
PROVIDER: S-EPMC6822938 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage.
| S-EPMC5052511 | biostudies-literature
Project description:Muscarinic acetylcholine receptors are G protein-coupled receptors that respond to acetylcholine and play important signaling roles in the nervous system. There are five muscarinic receptor subtypes (M1R to M5R), which, despite sharing a high degree of sequence identity in the transmembrane region, couple to different heterotrimeric GTP-binding proteins (G proteins) to transmit signals. M1R, M3R, and M5R couple to the Gq/ 11 family, whereas M2R and M4R couple to the Gi/ o family. Here, we present and compare the cryo-electron microscopy structures of M1R in complex with G11 and M2R in complex with GoA The M1R-G11 complex exhibits distinct features, including an extended transmembrane helix 5 and carboxyl-terminal receptor tail that interacts with G protein. Detailed analysis of these structures provides a framework for understanding the molecular determinants of G-protein coupling selectivity.
| S-EPMC7034192 | biostudies-literature
Project description:Membrane potential controls the response of the M2 muscarinic receptor to its ligands. Membrane hyperpolarization increases response to the full agonist acetylcholine (ACh) while decreasing response to the partial agonist pilocarpine. We previously have demonstrated that the regulator of G-protein signaling (RGS) 4 protein discriminates between the voltage-dependent responses of ACh and pilocarpine; however, the underlying mechanism remains unclear. Here we show that RGS4 is involved in the voltage-dependent behavior of the M2 muscarinic receptor-mediated signaling in response to pilocarpine. Additionally we revealed structural determinants on the M2 muscarinic receptor underlying the voltage-dependent response. By electrophysiological recording in Xenopus oocytes expressing M2 muscarinic receptor and G-protein-gated inwardly rectifying K+ channels, we quantified voltage-dependent desensitization of pilocarpine-induced current in the presence or absence of RGS4. Hyperpolarization-induced desensitization of the current required for RGS4, also depended on pilocarpine concentration. Mutations of charged residues in the aspartic acid-arginine-tyrosine motif of the M2 muscarinic receptor, but not intracellular loop 3, significantly impaired the voltage-dependence of RGS4 function. Thus, our results demonstrated that voltage-dependence of RGS4 modulation is derived from the M2 muscarinic receptor. These results provide novel insights into how membrane potential impacts G-protein signaling by modulating GPCR communication with downstream effectors.
| S-EPMC5522400 | biostudies-literature
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| S-EPMC3980162 | biostudies-literature
Project description:G-protein-coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. Although significant advances have been made in structural studies of GPCRs, details of their activation mechanism remain unclear. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined recently in an inactive antagonist-bound state. Here, activation of the M2 receptor is directly observed via accelerated molecular dynamics simulation, in contrast to previous microsecond-timescale conventional molecular dynamics simulations in which the receptor remained inactive. Receptor activation is characterized by formation of a Tyr206(5.58)-Tyr440(7.53) hydrogen bond and ?6-Å outward tilting of the cytoplasmic end of transmembrane ?-helix 6, preceded by relocation of Trp400(6.48) toward Phe195(5.47) and Val199(5.51) and flipping of Tyr430(7.43) away from the ligand-binding cavity. Network analysis reveals that communication in the intracellular domains is greatly weakened during activation of the receptor. Together with the finding that residue motions in the ligand-binding and G-protein-coupling sites of the apo receptor are correlated, this result highlights a dynamic network for allosteric regulation of the M2 receptor activation.
| S-EPMC3703993 | biostudies-literature
Project description:G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M3-R interactions but rather the affinities of Gq and Go proteins to M3-Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.
| S-EPMC5948956 | biostudies-literature
Project description:The proto-oncogene and inhibitor of protein phosphatase 2A (PP2A), SET, interacts with the third intracellular loop of the M3 muscarinic receptor (M3-MR), and SET knockdown with small interfering RNA (siRNA) in Chinese hamster ovary (CHO) cells augments M3-MR signaling. However, the mechanism of this action of SET on receptor signaling has not been defined, and we initiated studies to address this question. Knockdown of SET by siRNA in CHO cells stably expressing the M3-MR did not alter agonist-induced receptor phosphorylation or receptor internalization. Instead, it increased the extent of receptor dephosphorylation after agonist removal by ∼60%. In competition binding assays, SET knockdown increased high-affinity binding of agonist in intact cells and membrane preparations. Glutathione transferase pull-down assays and site-directed mutagenesis revealed a SET binding site adjacent to and perhaps overlapping the G protein-binding site within the third intracellular loop of the receptor. Mutation of this region in the M3-MR altered receptor coupling to G protein. These data indicate that SET decreases M3-MR dephosphorylation and regulates receptor engagement with G protein, both of which may contribute to the inhibitory action of SET on M3-MR signaling.
| S-EPMC3382831 | biostudies-literature
Project description:The cardiac muscarinic receptor (M2R) regulates heart rate, in part, by modulating the acetylcholine (ACh) activated K+ current IK,ACh through dissociation of G-proteins, that in turn activate KACh channels. Recently, M2Rs were noted to exhibit intrinsic voltage sensitivity, i.e. their affinity for ligands varies in a voltage dependent manner. The voltage sensitivity of M2R implies that the affinity for ACh (and thus the ACh effect) varies throughout the time course of a cardiac electrical cycle. The aim of this study was to investigate the contribution of M2R voltage sensitivity to the rate and shape of the human sinus node action potentials in physiological and pathophysiological conditions. We developed a Markovian model of the IK,ACh modulation by voltage and integrated it into a computational model of human sinus node. We performed simulations with the integrated model varying ACh concentration and voltage sensitivity. Low ACh exerted a larger effect on IK,ACh at hyperpolarized versus depolarized membrane voltages. This led to a slowing of the pacemaker rate due to an attenuated slope of phase 4 depolarization with only marginal effect on action potential duration and amplitude. We also simulated the theoretical effects of genetic variants that alter the voltage sensitivity of M2R. Modest negative shifts in voltage sensitivity, predicted to increase the affinity of the receptor for ACh, slowed the rate of phase 4 depolarization and slowed heart rate, while modest positive shifts increased heart rate. These simulations support our hypothesis that altered M2R voltage sensitivity contributes to disease and provide a novel mechanistic foundation to study clinical disorders such as atrial fibrillation and inappropriate sinus tachycardia.
| S-EPMC6197694 | biostudies-literature
Project description:Background and purposeArtificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such 'superagonism' has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a 'superagonist'.Experimental approachSignalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies.Key resultsIn CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi /Gs signalling competence. In the orthosteric loss-of-function mutant M2 -Y104(3.33) A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. 'Superagonism' is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure-signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that 'superagonism' of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points.Conclusion and implicationsSupraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR 'superagonism' is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators.
| S-EPMC3651662 | biostudies-literature
Project description:Muscarinic M1/M4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (SD) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M1, M2, or M4 receptors (M1-/-, M2-/-, M4-/-), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M1, M2, or M4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M1/M4 receptors partially substituted for cocaine and increased the SD effect of cocaine, while M2-preferring antagonists did not substitute, and reduced the SD effect of cocaine. The cocaine-like effects of scopolamine were absent in M1-/- mice. The cocaine SD attenuating effects of methoctramine were absent in M2-/- mice and almost absent in M1-/- mice. The findings indicate that the cocaine-like SD effects of muscarinic antagonists are primarily mediated through M1 receptors, with a minor contribution of M4 receptors. The data also support our previous findings that stimulation of M1 receptors and M4 receptors can each attenuate the SD effect of cocaine, and show that this can also be achieved by blocking M2 autoreceptors, likely via increased acetylcholine release.
| S-EPMC5495997 | biostudies-literature