M1 and M2 Muscarinic Acetylcholine Receptor Subtypes Mediate Ca2+ Channel Current Inhibition in Rat Sympathetic Stellate Ganglion Neurons

2006 ◽  
Vol 96 (5) ◽  
pp. 2479-2487 ◽  
Author(s):  
Qing Yang ◽  
Andrew D. Sumner ◽  
Henry L. Puhl ◽  
Victor Ruiz-Velasco
Keyword(s):  
Acetylcholine Receptors ◽  
Stellate Ganglion ◽  
Muscarinic Acetylcholine Receptor ◽  
Muscarinic Acetylcholine Receptors ◽  
Receptor Subtypes ◽  
Muscarinic Acetylcholine ◽  
Peripheral Neurons ◽  
Voltage Dependent ◽  
Ganglion Neurons ◽  
Channel Currents

Muscarinic acetylcholine receptors (mAChRs) are known to mediate the acetylcholine inhibition of Ca2+ channels in central and peripheral neurons. Stellate ganglion (SG) neurons provide the main sympathetic input to the heart and contribute to the regulation of heart rate and myocardial contractility. Little information is available regarding mAChR regulation of Ca2+ channels in SG neurons. The purpose of this study was to identify the mAChR subtypes that modulate Ca2+ channel currents in rat SG neurons innervating heart muscle. Accordingly, the modulation of Ca2+ channel currents by the muscarinic cholinergic agonist, oxotremorine-methiodide (Oxo-M), and mAChR blockers was examined. Oxo-M–mediated mAChR stimulation led to inhibition of Ca2+ currents through voltage-dependent (VD) and voltage-independent (VI) pathways. Pre-exposure of SG neurons to the M1 receptor blocker, M1-toxin, resulted in VD inhibition of Ca2+ currents after Oxo-M application. On the other hand, VI modulation of Ca2+ currents was observed after pretreatment of cells with methoctramine (M2 mAChR blocker). The Oxo-M–mediated inhibition was nearly eliminated in the presence of both M1 and M2 mAChR blockers but was unaltered when SG neurons were exposed to the M4 mAChR toxin, M4-toxin. Finally, the results from single-cell RT-PCR and immunofluorescence assays indicated that M1 and M2 receptors are expressed and located on the surface of SG neurons. Overall, the results indicate that SG neurons that innervate cardiac muscle express M1 and M2 mAChR, and activation of these receptors leads to inhibition of Ca2+ channel currents through VI and VD pathways, respectively.

scholarly journals Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes

Science ◽  
2019 ◽  
Vol 364 (6440) ◽  
pp. 552-557 ◽  
Author(s):  
Shoji Maeda ◽  
Qianhui Qu ◽  
Michael J. Robertson ◽  
Georgios Skiniotis ◽  
Brian K. Kobilka
Keyword(s):  
G Protein ◽  
Acetylcholine Receptors ◽  
Protein Complexes ◽  
Transmembrane Helix ◽  
Muscarinic Acetylcholine Receptor ◽  
Muscarinic Acetylcholine Receptors ◽  
Receptor Subtypes ◽  
Muscarinic Acetylcholine ◽  
High Degree ◽  
Distinct Features

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.

scholarly journals Role of M1, M3, and M5 muscarinic acetylcholine receptors in cholinergic dilation of small arteries studied with gene-targeted mice

2011 ◽  
Vol 300 (5) ◽  
pp. H1602-H1608 ◽  
Author(s):  
Adrian Gericke ◽  
Jan J. Sniatecki ◽  
Veronique G. A. Mayer ◽  
Evgeny Goloborodko ◽  
Andreas Patzak ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscarinic Receptor ◽  
Acetylcholine Receptors ◽  
Muscarinic Acetylcholine Receptors ◽  
Receptor Subtypes ◽  
Wild Type ◽  
Luminal Diameter ◽  
Small Arteries ◽  
Muscarinic Acetylcholine

Acetylcholine regulates perfusion of numerous organs via changes in local blood flow involving muscarinic receptor-induced release of vasorelaxing agents from the endothelium. The purpose of the present study was to determine the role of M1, M3, and M5 muscarinic acetylcholine receptors in vasodilation of small arteries using gene-targeted mice deficient in either of the three receptor subtypes (M1R−/−, M3R−/−, or M5R−/− mice, respectively). Muscarinic receptor gene expression was determined in murine cutaneous, skeletal muscle, and renal interlobar arteries using real-time PCR. Moreover, respective arteries from M1R−/−, M3R−/−, M5R−/−, and wild-type mice were isolated, cannulated with micropipettes, and pressurized. Luminal diameter was measured using video microscopy. mRNA for all five muscarinic receptor subtypes was detected in all three vascular preparations from wild-type mice. However, M3 receptor mRNA was found to be most abundant. Acetylcholine produced dose-dependent dilation in all three vascular preparations from M1R−/−, M5R−/−, and wild-type mice. In contrast, cholinergic dilation was virtually abolished in arteries from M3R−/− mice. Deletion of either M1, M3, or M5 receptor genes did not affect responses to nonmuscarinic vasodilators, such as substance P and nitroprusside. These findings provide the first direct evidence that M3 receptors mediate cholinergic vasodilation in cutaneous, skeletal muscle, and renal interlobar arteries. In contrast, neither M1 nor M5 receptors appear to be involved in cholinergic responses of the three vascular preparations tested.

scholarly journals Ligand-binding propeties of the muscarinic acetylcholine receptor in mouse neuroblastoma cells

1978 ◽  
Vol 172 (3) ◽  
pp. 495-501 ◽  
Author(s):  
P G Strange ◽  
N J M Birdsall ◽  
A S V Burgen
Keyword(s):  
Acetylcholine Receptors ◽  
Neuroblastoma Cells ◽  
Muscarinic Acetylcholine Receptor ◽  
Muscarinic Acetylcholine Receptors ◽  
Mass Action ◽  
Similar Data ◽  
Muscarinic Acetylcholine ◽  
Mouse Neuroblastoma ◽  
Good Agreement ◽  
Quinuclidinyl Benzilate

1. Muscarinic acetylcholine receptors in a plasma-membrane fraction derived from mouse neuroblastoma clone NIE-115 bind [3-3H]quinuclidinyl benzilate according to the Law of Mass Action (Kdissociation 40 pM, h0.96). 2. Antagonist and agonist binding to the receptor was studied by displacement of [3-3H]quinuclidinyl benzilate with non-radioactive ligands. The data show good agreement with similar data obtained on rat brain and ideal smooth muscle [Birdsall & Hulme (1976) J. Neurochem. 27, 7-16] indicating that the receptor is very similar in these three tissues.

Intracellular transduction mechanisms for the slow synaptic events

1992 ◽  
Vol 70 (S1) ◽  
pp. S44-S50 ◽  
Author(s):  
Haruo Kobayashi ◽  
Sumiko Mochida ◽  
Susumu Y. Takahashi
Keyword(s):  
Superior Cervical Ganglion ◽  
Action Potentials ◽  
Acetylcholine Receptors ◽  
Second Messengers ◽  
Muscarinic Acetylcholine Receptors ◽  
Characteristic Change ◽  
Receptor Subtypes ◽  
Cervical Ganglion ◽  
Cervical Ganglia ◽  
Ganglion Neurons

Electrical activities of the postganglionic neurons in the superior cervical ganglia of rabbits are modulated in various ways following activation of the subtypes of muscarinic acetylcholine receptors, (i) M1 receptors mediate a slow depolarization consisting of at least three types of ionic conductance changes, and one of these is possibly mediated by cyclic GMP. (ii) M2 receptors mediate a slow hyperpolarization that seems to be generated by inositol triphosphate derived from phosphatidylinositol breakdown. (iii) M2 receptors also cause, through an activation of C kinase, a suppression of Ca entry during action potentials that results in a characteristic change in the action potentials and thereby modulates excitability of superior cervical ganglion neurons. Each subtype of muscarinic receptors thus regulates different pathways of intracellular transduction and modulates the electrical signaling of sympathetic neurons.Key words: superior cervical ganglion, electrical signals, muscarinic responses, muscarinic receptor subtypes, second messengers.

scholarly journals Application of Parallel Multiparametric Cell-Based FLIPR Detection Assays for the Identification of Modulators of the Muscarinic Acetylcholine Receptor 4 (M4)

2015 ◽  
Vol 20 (7) ◽  
pp. 858-868 ◽  
Author(s):  
Emery Smith ◽  
Peter Chase ◽  
Colleen M. Niswender ◽  
Thomas J. Utley ◽  
Douglas J. Sheffler ◽  
...  
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Muscarinic Acetylcholine Receptor ◽  
Muscarinic Acetylcholine Receptors ◽  
Response Analysis ◽  
Allosteric Modulators ◽  
Muscarinic Acetylcholine ◽  
Single Well ◽  
Well Assay ◽  
Receptor Modulators

Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer’s disease and other disorders involving impaired cognitive function. In an attempt to identify orthosteric and allosteric modulators of the muscarinic acetylcholine receptor M4 (M4), we developed a homogenous, multiparametric, 1536-well assay to measure M4 receptor agonism, positive allosteric modulation (PAM), and antagonism in a single well. This assay yielded a Z′ of 0.85 ± 0.05 in the agonist, 0.72 ± 0.07 in PAM, and 0.80 ± 0.06 in the antagonist mode. Parallel screening of the M1 and M5 subtypes using the same multiparametric assay format revealed chemotypes that demonstrate selectivity and/or promiscuity between assays and modalities. This identified 503 M4 selective primary agonists, 1450 PAMs, and 2389 antagonist hits. Concentration-response analysis identified 25 selective agonists, 4 PAMs, and 41 antagonists. This demonstrates the advantages of this approach to rapidly identify selective receptor modulators while efficiently removing assay artifacts and undesirable compounds.

scholarly journals Discovery of the first selective M4 muscarinic acetylcholine receptor antagonists with in vivo anti-parkinsonian and anti-dystonic efficacy

2020 ◽  
Author(s):  
Mark S. Moehle ◽  
Aaron M. Bender ◽  
Jonathan W. Dickerson ◽  
Daniel J. Foster ◽  
Yuping Donsante ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Acetylcholine Receptor ◽  
Movement Disorders ◽  
Intracellular Signaling ◽  
Acetylcholine Receptors ◽  
Muscarinic Acetylcholine Receptor ◽  
Muscarinic Acetylcholine Receptors ◽  
Muscarinic Acetylcholine ◽  
Selective Antagonists

AbstractNon-selective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson disease and dystonia. Despite their efficacy in these and other central nervous system disorders, anti-muscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the anti-parkinsonian and anti-dystonic efficacy observed with the use of non-selective anti-muscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of non-selective anti-muscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here we utilize genetic mAChR knockout animals in combination with non-selective mAChR antagonists to confirm that the M4 receptor underlies the locomotor-stimulating and anti-parkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have anti-parkinsonian and anti-dystonic efficacy in pharmacological and genetic models of movement disorders.

scholarly journals Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor

2018 ◽  
Vol 115 (10) ◽  
pp. E2419-E2428 ◽  
Author(s):  
Magdalena Korczynska ◽  
Mary J. Clark ◽  
Celine Valant ◽  
Jun Xu ◽  
Ee Von Moo ◽  
...  
Keyword(s):  
Acetylcholine Receptors ◽  
Muscarinic Acetylcholine Receptor ◽  
Dissociation Rate ◽  
Muscarinic Acetylcholine Receptors ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Muscarinic Acetylcholine ◽  
Adult Rat ◽  
Subtype Selectivity ◽  
Subtype Selective

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound ’628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, ’628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of ’628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between ’628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like ’628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

scholarly journals Single Channel Studies of Inward Rectifier Potassium Channel Regulation by Muscarinic Acetylcholine Receptors

2000 ◽  
Vol 116 (5) ◽  
pp. 645-652 ◽  
Author(s):  
Joel Bard ◽  
Maya T. Kunkel ◽  
Ernest G. Peralta
Keyword(s):  
G Protein ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Muscarinic Acetylcholine Receptor ◽  
Inward Rectifier ◽  
Muscarinic Acetylcholine Receptors ◽  
Muscarinic Acetylcholine ◽  
Basal Activity ◽  
Inward Rectifier Potassium Channel ◽  
Direct Binding

Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (IKACh) by G protein–coupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of Gβγ subunits to the NH2- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate IKACh. Because of the very low basal activity of native IKACh, it has been difficult to determine the single channel effect of Gβγ subunit binding on IKACh activity. Through analysis of a novel G protein–activated chimeric inward rectifier channel that displays increased basal activity relative to IKACh, we find that single channel activation can be explained by a G protein–dependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state.

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