Two epitopes have been identified recently to be responsible for the high-affinity binding of alkane-bisammonium and caracurine V type allosteric ligands to N-methylscopolamine (NMS)-occupied M 2 muscarinic acetylcholine receptors, relative to M 5 receptors: the amino acid M 2 -Thr 423 at the top of transmembrane region (TM) 7 and an epitope comprising the second extracellular loop (o2) of the M 2 receptor including the flanking regions of TM4 and TM5. We aimed to find out whether a single amino acid could account for the contribution of this epitope to binding affinity. Allosteric interactions were investigated in wildtype and mutant receptors in which the orthosteric binding site was occupied by [ 3 H]NMS (5 mM Na,K,P i buffer, pH 7.4, 23°C were replaced by the corresponding amino acids of M 5 revealed that these two amino acids account entirely for the (approximately 100-fold) M 2 /M 5 selectivity of the alkane-bisammonium and the caracurine V type allosteric ligands. At NMSfree M 2 receptors, the caracurine V derivative also displayed approximately 100-fold M 2 /M 5 selectivity, but the double point mutation reduced the M 2 affinity by only ϳ10-fold; thus, additional epitopes may influence selectivity for the free receptors.A three-dimensional model of the M 2 receptor was used to simulate allosteric agent docking to NMS-occupied receptors. M 2 -Tyr 177 and M 2 -Thr 423 seem to be located near the junction of the allosteric and the orthosteric areas of the M 2 receptor ligand binding cavity.Muscarinic acetylcholine receptors are members of the rhodopsin-like family of G protein-coupled receptors, which share general structural motifs, including seven hydrophobic transmembrane helices connected by intracellular and extracellular loops, an extracellular amino terminus, and a cytoplasmic carboxyl terminus. Molecular cloning studies revealed the existence of five (M 1 -M 5 ) muscarinic acetylcholine receptors (Bonner et al., 1987), all of which are susceptible to allosteric modulation (Ellis et al., 1991). The orthosteric acetylcholine binding site seems to be lined by the transmembrane helices (Wess, 1993) and seems to be highly conserved among the five subtypes (Hulme et al., 1990). The allosteric binding site is located at the entrance of the ligand binding pocket (Ellis et al., 1993;Leppik et al., 1994) and is likely to be less well conserved than the orthosteric ligand binding site, thus potentially allowing the design of ligands with greater subtype selectivity (Tuček and Proška, 1995). The family of the muscarinic acetylcholine receptors has been widely studied as a model system for the interaction of allosteric modulators with G protein-coupled receptors (Christopoulos and Kenakin, 2002).It is striking that muscarinic allosteric ligands exhibit generally the highest affinity to the M 2 receptor subtype (Lee and El-Fakahany, 1991;Ellis et al., 1991;Ellis and Seidenberg, 2000). A number of studies aimed to identify receptor This article is dedicated to the late Dr. Stanislav Tuček, Academy of Scien...

The binding and function of muscarinic acetylcholine receptors can be modulated allosterically. Some allosteric muscarinic ligands are "atypical", having steep concentration-effect curves and not interacting competitively with "typical" allosteric modulators. For atypical agents, a second allosteric site has been proposed. Different approaches have been used to gain further insight into the interaction with M 2 receptors of two atypical agents, tacrine and the bispyridinium compound 4,4Ј-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1Ј-propane-1,3-diyl-bispyridinium dibromide (Duo3). Interaction studies, using radioligand binding assays and the allosteric ligands obidoxime, Mg 2ϩ , and the new tool hexamethonium to antagonize the allosteric actions of the atypical ligands, showed different modes of interaction for tacrine and Duo3 at M 2 receptors. A negatively cooperative interaction was observed between hexamethonium and tacrine (but not Duo3). A tacrine dimer that exhibited increased allosteric potency relative to tacrine but behaved like a typical allosteric modulator was competitively inhibited by hexamethonium. M 2 /M 5 -receptor mutants revealed a dependence of tacrine and Duo3 affinity on different receptor epitopes. This was confirmed by docking simulations using a three-dimensional model of the M 2 receptor. These showed that the allosteric site could accommodate two molecules of tacrine simultaneously but only one molecule of Duo3, which binds in different mode from typical allosteric agents. Therefore, the atypical actions of tacrine and Duo3 involve different modes of receptor interaction, but their sites of attachment seem to be the "common" allosteric binding domain at the entrance to the orthosteric ligand binding pocket of the M 2 -receptor. Additional complex behavior may be rationalized by allosteric interactions transmitted within a receptor dimer.A rapidly increasing number of G protein-coupled receptors have been discovered to be sensitive to allosteric modulation (Christopoulos and Kenakin, 2002). Potentially favorable features for a clinical application of such modulation include the enhancement of the binding of endogenous ligands (but also exogenous agonists and antagonists), absolute subtype selectivity of action, and self-limiting effects on receptor function (see Christopoulos and Kenakin, 2002 for review).Over the past decade, allosteric interactions at muscarinic acetylcholine receptors have been intensively studied (e.g., Ellis, 1997;Mohr et al., 2003, Birdsall andLazareno, 2005). All five muscarinic receptor subtypes are sensitive to alloste-

The M2 muscarinic acetylcholine receptor belongs to the family of rhodopsin like G-Protein Coupled Receptors. This subtype of muscarinic receptors is of special interest because it bears, aside from an orthosteric binding site, also an allosteric binding site. Based on the X-ray structure of bovine rhodopsin a complete homology model of the human M2 receptor was developed. For the orthosteric binding site point mutations and binding studies with different agonists and antagonists are available. This knowledge was utilized for an initial verification of the M2 model. Allosteric modulation of activity is mediated by structurally different ligands such as gallamine, caracurine V salts or W84 (a hexamethonium-derivative). Caracurine V derivatives with different affinities to M2 were docked using GRID-fields. Subsequent molecular dynamics simulations yielded different binding energies based on diverse electrostatic and lipophilic interactions. The calculated affinities are in good agreement to experimentally determined affinities.

A model of the human M(2) muscarinic receptor was taken as an example for a class A G-protein coupled receptor to explore the influence of different environments in a molecular dynamics simulation (MDS) on the protein structure. The most commonly used environment is the vacuum, although it is very unnatural for a transmembrane protein. As an alternative a membrane-like system, consisting of a lipophilic central layer and two aqueous flanking layers, was tested. The most realistic system that can be applied is a phospholipid bilayer with a surrounding physiological sodium chloride solution. From all systems good protein structures were received, nevertheless clear differences between the systems were detected in the structural comparison of the models. Subsequently it was analyzed whether the observed structural differences influence ligand binding. For this purpose the antagonist (S)-scopolamine was docked into the binding cavity, which is well known by many reported single and multiple point mutations. As expected from the observed structural variations triggered by the type of environment employed in MDS, also differences in the binding mode of (S)-scopolamine were detected, all contacts, however, which are known to be important were found.