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

Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation.

• MeSH
• agonisté muskarinových receptorů farmakologie   MeSH
• alkuronium farmakologie   MeSH
• alosterická regulace MeSH
• AMP cyklický metabolismus   MeSH
• antagonisté muskarinových receptorů farmakologie   MeSH
• chinuklidinylbenzilát farmakologie   MeSH
• CHO buňky MeSH
• inositolfosfáty fyziologie   MeSH
• křečci praví MeSH
• periodicita MeSH
• proteiny vázající GTP fyziologie   MeSH
• receptory muskarinové fyziologie   MeSH
• rekombinantní proteiny MeSH
• signální transdukce MeSH
• strychnin farmakologie   MeSH
• transfekce MeSH
• triethojodid gallaminia farmakologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• agonisté muskarinových receptorů MeSH
• alkuronium MeSH
• AMP cyklický MeSH
• antagonisté muskarinových receptorů MeSH
• chinuklidinylbenzilát MeSH
• inositolfosfáty MeSH
• proteiny vázající GTP MeSH
• receptory muskarinové MeSH
• rekombinantní proteiny MeSH
• strychnin MeSH
• triethojodid gallaminia MeSH