Allosteric interactions with muscarinic acetylcholine receptors: Complex role of the conserved tryptophan M2Trp in a critical cluster of amino acids for baseline affinity, subtype selectivity, and cooperativity

In general, the M2 subtype of muscarinic acetylcholine receptors has the highest sensitivity for allosteric modulators and the M5 subtype the lowest. The M2/M5 selectivity of some structurally diverse allosteric agents is known to be completely explained by M2 Tyr and M2 Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS). This study explored the role of the conserved M2 Trp and the adjacent M2 Thr in the binding of alkanebisammonio type modulators, gallamine, and diallylcaracurine V. Experiments were performed with human M2 or M5 receptors or mutants thereof. It was found that M2 Trp and M2 Thr independently influenced allosteric agent binding. The presence of M2 Thr may enhance the affinity of binding, depending on the allosteric agent, either directly or indirectly (by avoiding sterical hindrance through its M5 counterpart His). Replacement of M2 Trp and of the corresponding M5 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 M2 receptor model revealed that the aromatic rings of M2 Tyr and M2 Trp, in a concerted action, might fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M2 Trp and the spatially adjacent M2 Tyr, as well as M2 Thr, form a cluster of amino acids within the allosteric binding cleft that is pivotal for both M2/M5 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; Krejčı́ 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 This work was supported by the Deutsche Forschungsgemeinschaft (Mo821/ 1–4), and by United States Public Health Service grant R01-AG05214 (to J.E.). □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.023481. ABBREVIATIONS: M2, M2 subtype of the muscarinic acetylcholine receptor; M5, M5 subtype of the muscarinic acetylcholine receptor; NMS, N-methylscopolamine; o2, second outer (extracellular) loop of the receptor; o3, third outer (extracellular) loop of the receptor; PB, sodiumpotassium phosphate buffer (5 mM, pH 7.4); W84, hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide. 0026-895X/06/7001-181–193$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 1 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 23481/3124530 Mol Pharmacol 70:181–193, 2006 Printed in U.S.A. 181 http://molpharm.aspetjournals.org/content/suppl/2006/04/27/mol.106.023481.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from agent binding and action will help to design new allosteric agents with improved properties and will lead to a better insight into the principles of muscarinic receptor function. The M2 subtype of muscarinic receptors generally displays highest affinity for allosteric modulators, whereas the M5 subtype has lowest sensitivity. A rather good insight into the allosteric binding area has now been achieved by combining three strategies [i.e., development of allosteric agents with high affinity and selectivity for M2 receptors that presumably fit tightly in a fixed position at the receptor protein (Mohr et al., 2003), receptor mutagenesis starting from M2/M5 chimeric receptor constructs to identify essential epitopes for allosteric agent binding (Ellis et al., 1993; Gnagey et al., 1999; Buller et al., 2002; Huang et al., 2005), and generation of a three-dimensional M2 receptor model based on the crystal structure of the inactive bovine rhodopsin (Jöhren and Höltje, 2002; Voigtländer et al., 2003)]. This approach has allowed the visualization of different binding topologies for typical and atypical allosteric agents (Tränkle et al., 2005; Wess, 2005). However, the mode by which certain epitopes affect binding affinity of allosteric agents is still in question. For instance, an amino acid may directly serve as a docking point or alternatively constitute a sterical hindrance, or it may indirectly contribute to ligand binding by governing the conformation of amino acid strands that contain a relevant point of attachment. We have found that two amino acids are sufficient to account completely for the 100-fold M2/M5 selectivity of structurally different allosteric agents (Voigtländer et al., 2003). These amino acids are M2 Tyr and M2 Thr, corresponding to the M5 amino acids Gln and His. The receptor model suggested M2 Thr to be a direct docking point for caracurine V-type agents. For alkane-bisammonio–type compounds such as W84 (Fig. 1), however, the model suggested an indirect influence, in that M2 Thr induces a favorable spatial adjustment of the adjacent M2 Trp for its interaction with one of the phthalimide moieties of W84. The involvement of M2 Trp is supported by a broad mutagenesis study by Matsui et al. (1995), who found the corresponding tryptophan of M1 to be relevant to the binding of the allosteric agent gallamine. Therefore, we set out to clarify the role of this conserved tryptophan and its neighboring M2 Thr or M5 His. As a major outcome of this study, we found that this tryptophan is of crucial importance for M2/M5 subtype independent baseline affinity of alkane-bisammonio type allosteric modulators and of gallamine. Thus, an epitope of high relevance to M2/M5 subtype independent baseline affinity of allosteric agents has been discovered. Furthermore, we found that this tryptophan in certain instances may provide subtype selectivity for allosteric agents or may modulate their cooperativity with an orthosteric antagonist. Taken together, the findings reveal that relevant epitopes for subtype dependent and subtype independent allosteric agent binding are clustered in close spatial proximity at the junction between the allosteric and the orthosteric binding areas of muscarinic acetylcholine receptors. Fig. 1. Structures of the applied allosteric agents. 182 Prilla et al. at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from Materials and Methods