Abstract
Muscarinic acetylcholine receptors contain two distinct ligand binding sites, i.e. the orthosteric site for acetylcholine and other conventional ligands, and an allosteric site located at the entrance of the ligand binding pocket. We used a set of allosteric agents to probe whether muscarinic M2 receptors whose orthosteric site is occupied by an agonist still reveal the common allosteric site that has been identified in M2 receptors being occupied by an orthosteric antagonist (N-methylscopolamine, NMS). Equilibrium and dissociation binding experiments were carried out in porcine heart homogenates using either the agonist [3H]oxotremorine M ([3H]OxoM) or the antagonist [3H]NMS. The affinities of the allosteric agents were determined for the radioligand-occupied receptor states and, additionally, for the radioligand-free (ground state) M2 receptor. The archetypal agent W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide] and its bispyridinio middle chain analogue WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl]propane dibromide) had a clearly lower affinity for [3H]OxoM-liganded receptors compared with [3H]NMS-liganded and ground state receptors. In contrast, a derivative resembling only one half of W84 had equal affinities for both radioligand-occupied receptor states. Also, the agents gallamine and obidoxime did not discriminate between [3H]OxoM- and [3H]NMS-occupied receptors. The allosteric antagonistic tool obidoxime inhibited WDuo3 action in [3H]OxoM-liganded receptors with the same potency as in [3H]NMS-liganded receptors. We conclude that the common allosteric site is still present in OxoM-liganded M2 receptors, but its spatial conformation is considerably altered compared with NMS-liganded receptors.
Similar content being viewed by others
References
Arunlakshana O, Schild (1950) The air-perfused bronchial tree. J Physiol. 16,3(3–4):48p
Botero Cid HM, Holzgrabe U, Kostenis E, Mohr K, Tränkle C (1994) Search for the pharmacophore of bispyridinium-type allosteric modulators of muscarinic receptors. J Med Chem 37:1439–1445
Burgmer U, Schulz U, Tränkle C, Mohr K (1998) Interaction of Mg2+ with the allosteric site of muscarinic M2 receptors. Naunyn Schmiedebergs Arch Pharmacol 357:363–370
Buller S, Zlotos DP, Mohr K, Ellis J (2002) Allosteric site on muscarinic acetylcholine receptors: a single amino acid in TM7 is critical to the subtype selectivities of caracurine V derivatives and alkane-bisammonium ligands. Mol Pharmacol 61:160–168
Birdsall NJ, Lazareno S (2005) Allosterism at muscarinic receptors: ligands and mechanisms. Mini Rev Med Chem 5:523–543
Christopoulos A, Kenakin T (2002) G protein-coupled receptor allosterism and complexing. Pharmacol Rev 54:323–374
Ehlert FJ (1988) Estimation of the affinities of allosteric ligands using radioligand binding and pharmacological null methods. Mol Pharmacol 35:187–194
Ellis J (1997) Allosteric binding sites on muscarinic receptors. Drug Dev Res 40:193–204
Ellis J, Seidenberg M (1992) Two allosteric modulators interact at a common site on cardiac muscarinic receptors. Mol Pharmacol 42:638–641
Ellis J, Seidenberg M (2000) Interactions of alcuronium, TMB-8, and other allosteric ligands with muscarinic acetylcholine receptors: studies with chimeric receptors. Mol Pharmacol 58:1451–1460
Gilsbach R, Großmüller M, Alptüzün V, Erciyas E, Tränkle C, Holzgrabe U, Mohr K (2003) Cooperative interactions at M2 muscarinic acetylcholine interactions: Structure/activity-relationships in stepwise shortened bispyridinium- and bis(ammonio)alkane-type allosteric modulators. Neurochem Res 28:667–673
Gnagey A, Ellis J (1996) Allosteric regulation of the binding of [3H]acetylcholine to m2 muscarinic receptors. Biochem Pharmacol 52:1767–1775
Gnagey AL, Seidenberg M, Ellis J (1999) Site-directed mutagenesis reveals two epitopes involved in the subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors. Mol Pharmacol 56:1245–1253
Heitz F, Holzwarth JA, Gies JP, Pruss RM, Trumpp-Kallmeyer S, Hibert MF, Guenet C (1999) Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site. Eur J Pharmacol 380:183–195
Holzgrabe U, Mohr K (1998) Allosteric modulators of ligand binding to muscarinic acetylcholine receptors. Drug Discovery Today 3:214–222
Huang XP, Prilla S, Mohr K, Ellis J (2005) Critical amino acid residues of the common allosteric site on the M2 muscarinic acetylcholine receptor: more similarities than differences between the structurally divergent agents gallamine and bis(ammonio)alkane-type W84. Mol Pharmacol 68:769–778
Hulme EC, Lu ZL, Saldanha JW, Bee MS (2003) Structure and activation of muscarinic acetylcholine receptors. Biochem Soc Trans. 31(Pt 1):29–34
Jakubík J, Bačáková L, El-Fakahany EE, Tuček S (1997) Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors. Mol Pharmacol 52:172–179
Jöhren K, Höltje H-D (2002) A model of the human M2 muscarinic acetylcholine receptor. J Comput Aided Mol Des 16:795–801
Kostenis E, Holzgrabe U, Mohr K (1994) Allosteric effect on muscarinic M2-receptors of derivatives of the alkane-bis-ammonium compound W84. Comparison with bispyridinium-type allosteric modulators. Eur J Med Chem 29:947–953
Kostenis E, Mohr K (1996) Composite action of allosteric modulators on ligand binding. Trends Pharmacol Sci 17:443–444
Krejči A, Tuček S (2001) Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors. Mol Pharmacol 60:761–767
Krejčí A, Michal P, Jakubík J, Říčný J, Doležal V (2004) Regulation of signal transduction at M2 muscarinic receptor. Physiol Res 53 Suppl 1:131–140
Lazareno S, Birdsall NJM (1995) Detection, quantification and verification of allosteric interactions of agents with labeled and unlabeled ligands at G-protein-coupled receptors: Interaction of strychnine and acetylcholine at muscarinic receptors. Mol Pharmacol 48:362–378
Leppik RA, Miller RC, Eck M, Paquet JL (1994) Role of acidic amino acids in the allosteric modulation by gallamine of antagonist binding at the m2 muscarinic acetylcholine receptor. Mol Pharmacol 45:983–990
Lu ZL, Saldanha JW, Hulme EC (2002) Seven-transmembrane receptors: crystals clarify. Trends Pharmacol Sci 23:140–146
Maaß A, Mohr K (1996) Opposite effects of alcuronium on agonist and on antagonist binding to muscarinic receptors. Eur J Pharmacol 305:231–234
Melchiorre C, Minarini A, Angeli P, Giardina D, Gulini U, Quaglia W (1989) Polymethylene tetraamines as muscarinic receptor probes. Trends Pharmacol Sci (Suppl) 55–59
Mohr K, Tränkle C, Holzgrabe U (2003) Structure/activity relationships of M2 muscarinic allosteric modulators. Receptors Channels 9:229–240
Mohr M, Heller E, Ataie A, Mohr K, Holzgrabe U (2004) Development of a new type of allosteric modulator of muscarinic receptors: hybrids of the antagonist AF-DX 384 and the hexamethonio derivative W84 J Med Chem 47:3324–3327
Muth M, Bender W, Scharfenstein O, Holzgrabe U, Balatková E, Tränkle C, Mohr K (2003) Systematic development of high affinity bis(ammonio)alkane-type allosteric enhancers of muscarinic ligand binding. J Med Chem 46:1031–1040
Muth M, Sennwitz M, Mohr K, Holzgrabe U (2005) Muscarinic allosteric enhancers of ligand binding: pivotal pharmacophoric elements in hexamethonio-type agents. J Med Chem 48:2212–2217
Potter LT, Ballesteros LA, Bichajian LH, Ferrendelli CA, Fisher A, Hanchett HE, Zhang R (1991) Evidence of paired M2 muscarinic receptors. Mol Pharmacol 39:211–221
Schröter A, Tränkle C, Mohr K (2000) Modes of allosteric interactions with free and [3H]N-methylscopolamine-occupied muscarinic M2 receptors as deduced from buffer-dependent potency shifts. Naunyn Schmiedebergs Arch Pharmacol 362:512–519
Soudijn W, van Wijngaarden I, IJzerman AP (2004) Allosteric modulation of G protein-coupled receptors: perspectives and recent developments. Drug Discov Today 9:752–758
Stockton JM, Birdsall NJM, Burgen ASV, Hulme EC (1983) Modification of the binding properties of muscarinic receptors by gallamine. Mol Pharmacol 23:551–557
Tränkle C, Mohr K (1997) Divergent modes of action among cationic allosteric modulators of muscarinic M2 receptors. Mol Pharmacol 51(4):674–682
Tränkle C, Kostenis E, Burgmer U, Mohr K (1996) Search for lead structures to develop new allosteric modulators of muscarinic receptors. J Pharmacol Exp Ther 279:926–933
Tränkle C, Andresen I, Lambrecht G, Mohr K (1998) M2 receptor binding of the selective antagonist AF-DX 384: possible involvement of the common allosteric site. Mol Pharmacol 53:304–312
Tränkle C, Dittmann A, Schulz U, Weyand O, Buller S, Jöhren K, Heller E, Birdsall NJM, Holzgrabe U, Ellis J, Höltje HDH, Mohr K (2005) Atypical muscarinic allosteric modulation: cooperativity between modulators and their atypical binding topology in muscarinic M2 and M2/M5 chimeric receptors. Mol Pharmacol 68:1597–1610
Tuček S, Musílková J, Nedoma J, Proška J, Shelkovnikov S, Vorlíček J (1990) Positive cooperativity in the binding of alcuronium and N-methylscopolamine to muscarinic acetylcholine receptors. Mol Pharmacol 38:674–680
Voigtländer U, Jöhren K, Mohr M, Raasch A, Tränkle C, Buller S, Ellis J, Höltje HD, Mohr K (2003) Allosteric site on muscarinic acetylcholine receptors: Identification of two amino acids in the muscarinic M2 receptor that account entirely for the M2/M5 subtype selectivities of some structurally diverse allosteric ligands in N-methylscoplamine occupied receptors. Mol Pharmacol 64:21–31
Wess J (2005) Allosteric binding sites on muscarinic acetylcholine receptors (Relates to article by Tränkle et al.) Mol Pharmacol 68:1506–1509
Zahn K, Eckstein N, Tränkle C, Sadeé W, Mohr, K. (2002). Allosteric modulation of muscarinic receptor signaling: Alcuronium-induced conversion of pilocarpine from an agonist into an antagonist. J Pharmacol Exp Ther 301:720–728
Acknowledgement
This work was supported by the Deutsche Forschungsgemeinschaft (Mo821/1, Ho1368/7, GRK677).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Großmüller, M., Antony, J., Tränkle, C. et al. Allosteric site in M2 acetylcholine receptors: evidence for a major conformational change upon binding of an orthosteric agonist instead of an antagonist. Naunyn Schmied Arch Pharmacol 372, 267–276 (2006). https://doi.org/10.1007/s00210-005-0023-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00210-005-0023-4