Allosteric Interactions with Muscarinic Acetylcholine Receptors: Complex Role of the Conserved Tryptophan M₂⁴²²Trp in a Critical Cluster of Amino Acids for Baseline Affinity, Subtype Selectivity, and Cooperativity

Abstract

In general, the M 2 subtype of muscarinic acetylcholine receptors has the highest sensitivity for allosteric modulators and the M 5 subtype the lowest. The M 2 /M 5 selectivity of some structurally diverse allosteric agents is known to be completely explained by M 2 177 Tyr and M 2 423 Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS Trp by alanine revealed a pronounced contribution of these epitopes to subtype independent baseline affinity in NMSbound and NMS-free receptors for all agents except diallylcaracurine V. In a few instances, this tryptophan also influenced cooperativity and subtype selectivity. Docking simulations using a three-dimensional M 2 receptor model revealed that the aromatic rings of M 2 177 Tyr and M 2 422 Trp, in a concerted action, might fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M 2 422 Trp and the spatially adjacent M 2 177 Tyr, as well as M 2 423 Thr, form a cluster of amino acids within the allosteric binding cleft that is pivotal for both M 2 /M 5 subtype selectivity and baseline affinity of allosteric agents.All five subtypes of muscarinic acetylcholine receptors contain an allosteric site apart from the orthosteric site that is addressed by acetylcholine and conventional muscarinic agonists and antagonists. Binding of an allosteric modulator allows formation of ternary complexes consisting of the allosteric agent, the orthosteric ligand, and the receptor protein. Through ternary complex formation, allosteric agents may evoke particular actions that cannot be induced by orthosteric ligands alone and that may have therapeutic potential. For instance, allosteric modulators may increase the binding of orthosteric agonists or antagonists (positive cooperativity) or they may inhibit orthosteric ligand binding (negative cooperativity). In either case, the magnitude of the cooperativity will define an intrinsic limit on the magnitude of the positive or negative effect, in marked contrast to the unconstrained action of orthosteric agonists and antagonists. It is also possible for allosteric modulators to leave orthosteric ligand binding unchanged (neutral cooperativity) while nevertheless changing the kinetics of binding (Ellis, 1997;Christopoulos and Kenakin, 2002;Krejčí et al., 2004;Soudijn et al., 2004;Birdsall and Lazareno, 2005;Wess, 2005). Finally, in addition to modulating orthosteric ligand binding properties, allosteric agents also may modulate agonist induced intrinsic efficacy (Zahn et al., 2002). A better understanding of the molecular topology and mechanisms of allosteric

Exploration of the Orthosteric/Allosteric Interface in Human M1 Muscarinic Receptors by Bitopic Fluorescent Ligands

“…In contrast, the W400A/F mutations led to a more than 10-fold decrease in gallamine affinity at free-and [ 3 H]NMS-occupied receptors, with little impact on binding cooperativity. These data are in agreement with previous reports on gallamine properties at the W400A/F M1 mutants (Matsui et al, 1995) and at the alanine mutants of the homologous W400 and W405 residues in M2 and M5 receptors (Prilla et al, 2006). Alterations in brucine modulation were more difficult to ascertain given its neutral cooperativity with [ 3 H]NMS binding.…”

“…High [ 3 H]NMS affinity was preserved, as reported for the M1 W400 mutant (Matsui et al, 1995) and for homologous W400 ( M2 W422, M5 W477) and W405 ( M2 W427, M5 W482) mutations in M2 and M5 receptors (Prilla et al, 2006).…”

“…Accumulating evidence supports the bitopic character of the BoPz derivatives: they recognize epitopes within both orthosteric (competitive character when tested against orthosteric ligands in functional and equilibrium binding assays) and allosteric (retardation of [ To address this question, we considered an ensemble of observations pointing to the M1 W400 and W405 residues as interesting mutagenesis probes: 1) the Bodipy fluorophore may bind to an allosteric site [Bo(5) data; Fig. 5B]; 2) the aromatic Bodipy moiety is well suited to elicit a p-p stacking with the indole ring of a Trp residue; 3) the EGFP (donor)-Bodipy (acceptor) distances for the Bo(10,12)Pz and Bo(15,22)Pz subgroups, as determined from FRET efficacy (Tahtaoui et al, 2004), only differ by 3-5 Å; 4) the conserved W400 (7.35) and W405 (7.40) residues [numbering according to Ballesteros and Weinstein (1995) convention indicated under parentheses] are spatially close, one a-helical turn apart on top of TM7; and, most importantly, 5) the conserved Trp at position 7.35 is regarded as a key component of the prototypical gallamine allosteric site of mAChRs (Matsui et al, 1995;Prilla et al, 2006).…”

Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

“…LY2033298 binds to the M 4 mAChR at a Region that Overlaps with the 'Prototypical' Allosteric-Binding Site Extensive mutagenesis studies have determined a function for residues in the second and third extracellular loops and at the top of transmembrane domain VII to be important for the binding of prototypical allosteric modulators such as C 7 /3-phth and gallamine at mAChRs (Ellis et al, 1993;Huang et al, 2005;Krejci and Tucek, 2001;Leppik et al, 1994;May et al, 2007a;Prilla et al, 2006;Voigtlander et al, 2003). Preliminary mutagenesis experiments with LY2033298 also implicated extracellular loop regions in the actions of the modulator (Chan et al, 2008).…”

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

“…The incubation time was calculated as described previously (41). Atropine (10 M) was used to determine unspecific binding.…”