Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.

• MeSH
• agonisté muskarinových receptorů * farmakologie   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• karbachol farmakologie   MeSH
• lidé MeSH
• proteiny vázající GTP - alfa-podjednotky metabolismus   genetika   MeSH
• proteiny vázající GTP metabolismus   MeSH
• pyridiny farmakologie   MeSH
• receptory muskarinové * metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• agonisté muskarinových receptorů * MeSH
• karbachol MeSH
• proteiny vázající GTP - alfa-podjednotky MeSH
• proteiny vázající GTP MeSH
• pyridiny MeSH
• receptory muskarinové * MeSH

A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G16 protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα16 limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα16 mediated pathway more specifically. Our data demonstrated agonist-specific activation of G16 pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα16 pathway at the M2 receptor and at the same time impaired Gα16 signaling of iperoxo at M5 receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.

• Klíčová slova
• fusion proteins, muscarinic receptors, non-canonical signaling, signaling bias,
• MeSH
• AMP cyklický metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• inhibiční koncentrace 50 MeSH
• isoxazoly chemie   MeSH
• křečci praví MeSH
• kvartérní amoniové sloučeniny chemie   MeSH
• lidé MeSH
• molekulární konformace MeSH
• oxotremorin chemie   MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus   MeSH
• receptory muskarinové metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   MeSH
• signální transdukce * MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AMP cyklický MeSH
• G protein alpha 16 MeSH Prohlížeč
• iperoxo MeSH Prohlížeč
• isoxazoly MeSH
• kvartérní amoniové sloučeniny MeSH
• oxotremorin MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• receptory muskarinové MeSH
• rekombinantní fúzní proteiny MeSH

BACKGROUND AND PURPOSE: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. EXPERIMENTAL APPROACH: We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M2 receptors was modelled in silico. KEY RESULTS: Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M2 muscarinic receptors and displaying bias towards the Gi signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor. CONCLUSIONS AND IMPLICATIONS: The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M2 and M4 muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

• MeSH
• agonisté muskarinových receptorů * farmakologie   MeSH
• antagonisté muskarinových receptorů farmakologie   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• křečci praví MeSH
• krysa rodu Rattus MeSH
• receptor muskarinový M2 MeSH
• receptory muskarinové * MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• agonisté muskarinových receptorů * MeSH
• antagonisté muskarinových receptorů MeSH
• receptor muskarinový M2 MeSH
• receptory muskarinové * MeSH

Allosteric modulators are ligands that bind to a site on the receptor that is spatially separated from the orthosteric binding site for the endogenous neurotransmitter. Allosteric modulators modulate the binding affinity, potency, and efficacy of orthosteric ligands. Muscarinic acetylcholine receptors are prototypical allosterically-modulated G-protein-coupled receptors. They are a potential therapeutic target for the treatment of psychiatric, neurologic, and internal diseases like schizophrenia, Alzheimer's disease, Huntington disease, type 2 diabetes, or chronic pulmonary obstruction. Here, we reviewed the progress made during the last decade in our understanding of their mechanisms of binding, allosteric modulation, and in vivo actions in order to understand the translational impact of studying this important class of pharmacological agents. We overviewed newly developed allosteric modulators of muscarinic receptors as well as new spin-off ideas like bitopic ligands combining allosteric and orthosteric moieties and photo-switchable ligands based on bitopic agents.

• Klíčová slova
• acetylcholine, allosteric modulation, muscarinic receptors,
• MeSH
• agonisté muskarinových receptorů metabolismus   MeSH
• alosterická regulace fyziologie   MeSH
• antagonisté muskarinových receptorů metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• receptory muskarinové metabolismus   fyziologie   MeSH
• receptory spřažené s G-proteiny MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• agonisté muskarinových receptorů MeSH
• antagonisté muskarinových receptorů MeSH
• ligandy MeSH
• receptory muskarinové MeSH
• receptory spřažené s G-proteiny MeSH

We have found earlier that changes in membrane cholesterol content have distinct impact on signaling via the M1, M2, or M3 receptors expressed in CHO cells (CHO-M1 through CHO-M3). Now we investigated whether gradual changes in membrane cholesterol exerts differential effects on coupling of the M1 and M3 muscarinic receptors to preferential signaling pathways through Gq/11 and non-preferential Gs G-proteins signaling. Changes in membrane cholesterol resulted in only marginal alterations of antagonist and agonist affinity of the M1 and M3 receptors, and did not influence precoupling of either subtype. Changes in membrane cholesterol did not influence parameters of carbachol-stimulated GTP-γ(35)S binding in CHO-M1 membranes while reduction as well as augmentation of membrane cholesterol lowered the efficacy but increased the potency of carbachol in CHO-M3 membranes. Gradual increase or decrease in membrane cholesterol concentration dependently attenuated agonist-induced inositolphosphates release while only cholesterol depletion increased basal values in both cell lines. Similarly, membrane cholesterol manipulation modified basal and agonist-stimulated cAMP synthesis via Gs in the same way in both cell lines. These results demonstrate that changes in membrane cholesterol concentration differentially impact preferential and non-preferential M1 and M3 receptor signaling. They point to the activated G-protein/effector protein interaction as the main site of action in alterations of M1 receptor-mediated stimulation of second messenger pathways. On the other hand, modifications in agonist-stimulated GTP-γ(35)S binding in CHO-M3 membranes indicate that in this case changes in ligand-activated receptor/G-protein interaction may also play a role.

• Klíčová slova
• Agonist binding, Cholesterol, G-Proteins, Muscarinic receptors, Signal transduction, cAMP synthesis,
• MeSH
• CHO buňky MeSH
• cholesterol metabolismus   MeSH
• Cricetulus MeSH
• karbachol farmakologie   MeSH
• lidé MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptor muskarinový M1 účinky léků   metabolismus   MeSH
• receptor muskarinový M3 účinky léků   metabolismus   MeSH
• signální transdukce MeSH
• systémy druhého messengeru fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• karbachol MeSH
• proteiny vázající GTP MeSH
• receptor muskarinový M1 MeSH
• receptor muskarinový M3 MeSH

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

• Klíčová slova
• G protein-coupled receptor (GPCR), computer modeling, nuclear magnetic resonance (NMR), protein dynamic, recombinant protein expression, site-directed mutagenesis, snake neurotoxin,
• MeSH
• Elapidae MeSH
• inzerční mutageneze MeSH
• jedy hadů čeledi Elapidae chemie   MeSH
• konformace proteinů MeSH
• molekulární sekvence - údaje MeSH
• neurotoxiny chemie   genetika   metabolismus   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• receptory muskarinové metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jedy hadů čeledi Elapidae MeSH
• neurotoxiny MeSH
• receptory muskarinové MeSH

Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

• MeSH
• AMP cyklický metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• časové faktory MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• extracelulární prostor účinky léků   metabolismus   MeSH
• intracelulární prostor účinky léků   metabolismus   MeSH
• kinetika MeSH
• křečci praví MeSH
• lidé MeSH
• N-methylskopolamin farmakologie   MeSH
• pyridiny farmakologie   MeSH
• receptor muskarinový M1 agonisté   antagonisté a inhibitory   metabolismus   MeSH
• receptor muskarinový M4 agonisté   antagonisté a inhibitory   metabolismus   MeSH
• thiadiazoly farmakologie   MeSH
• vápník metabolismus   MeSH
• vazebná místa účinky léků   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AMP cyklický MeSH
• N-methylskopolamin MeSH
• pyridiny MeSH
• receptor muskarinový M1 MeSH
• receptor muskarinový M4 MeSH
• thiadiazoly MeSH
• vápník MeSH
• xanomeline MeSH Prohlížeč

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex "collides" with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G(q/11), while even-numbered receptors prefer coupling to G(i/o). We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [(3)H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M(1) and M(3) receptors also pre-couple with non-preferential G(i/o) G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.

• MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• karbachol metabolismus   MeSH
• kinetika MeSH
• kompetitivní vazba MeSH
• křečci praví MeSH
• lidé MeSH
• mutace MeSH
• N-methylskopolamin metabolismus   MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptory muskarinové genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• guanosin 5'-O-(3-thiotrifosfát) MeSH
• karbachol MeSH
• N-methylskopolamin MeSH
• proteiny vázající GTP MeSH
• receptory muskarinové MeSH

BACKGROUND AND PURPOSE: Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH: Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. KEY RESULTS: Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. CONCLUSIONS AND IMPLICATIONS: These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding.

• MeSH
• agonisté muskarinových receptorů metabolismus   MeSH
• alosterická regulace MeSH
• antagonisté muskarinových receptorů metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• guanosindifosfát metabolismus   MeSH
• kinetika MeSH
• křečci praví MeSH
• lidé MeSH
• N-methylskopolamin metabolismus   MeSH
• proteiny vázající GTP klasifikace   metabolismus   MeSH
• radioligandová zkouška MeSH
• receptor muskarinový M2 genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• transfekce MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• agonisté muskarinových receptorů MeSH
• antagonisté muskarinových receptorů MeSH
• guanosin 5'-O-(3-thiotrifosfát) MeSH
• guanosindifosfát MeSH
• N-methylskopolamin MeSH
• proteiny vázající GTP MeSH
• receptor muskarinový M2 MeSH
• rekombinantní proteiny MeSH

BACKGROUND: Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity. RESULTS: At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 microM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [3H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [35S]GTPgammaS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [35S]GTPgammaS binding at M2 and M4 receptors. In contrast, 0.1 microM rapacuronium significantly increased ACh potency at M1, M3, and M5 receptors. Kinetic measurements at M3 receptors showed acceleration of the rate of ACh-induced [35S]GTPgammaS binding by rapacuronium. CONCLUSIONS: Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 microM) but not at high concentrations (10 microM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M3 receptor-mediated bronchoconstriction.

• MeSH
• acetylcholin metabolismus   MeSH
• agonisté muskarinových receptorů farmakologie   MeSH
• alosterická regulace účinky léků   MeSH
• alosterické místo účinky léků   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• kompetitivní vazba účinky léků   MeSH
• křečci praví MeSH
• N-methylskopolamin metabolismus   MeSH
• nedepolarizující myorelaxancia farmakologie   MeSH
• radioligandová zkouška metody   MeSH
• receptory muskarinové účinky léků   fyziologie   MeSH
• vekuronium analogy a deriváty   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylcholin MeSH
• agonisté muskarinových receptorů MeSH
• guanosin 5'-O-(3-thiotrifosfát) MeSH
• N-methylskopolamin MeSH
• nedepolarizující myorelaxancia MeSH
• rapacuronium MeSH Prohlížeč
• receptory muskarinové MeSH
• vekuronium MeSH