Asparagine, Valine, and Threonine in the Third Extracellular Loop of Muscarinic Receptor Have Essential Roles in the Positive Cooperativity of Strychnine-Like Allosteric Modulators

Abstract

We have investigated allosteric interactions of four closely related strychnine-like substances: Wieland-Gumlich aldehyde (WGA), propargyl Wieland-Gumlich aldehyde, strychnine, and brucine with N-methylscopolamine (NMS) on M 3 subtype of muscarinic receptor genetically modified in the second or the third extracellular loop to corresponding loops of M 2 subtype (M 3 o2 and M 3 o3 chimera). The M 3 o2 chimeric receptor exhibited no change in either affinity of strychnine, brucine, and WGA or in cooperativity of brucine or WGA, whereas both parameters for propargyl-WGA changed. In contrast, there was a change in affinity of all tested modulators (except for brucine) and in their cooperativity in the M 3 o3 chimera. Directions of affinity changes in both chimeras were always toward values of the donor M 2 subtype, but changes in cooperativity were variable. Compared with the native M 3 receptor, strychnine displayed a slight increase in positive cooperativity and propargyl-WGA a robust decrease in negative cooperativity at M 3 o2 chimera. Similar changes were found in the M 3 o3 chimera. Interestingly, cooperativity of brucine and WGA at the M 3 o3 chimera changed from negative to positive. This is the first evidence of constitution of positive cooperativity of WGA by switching sequences of two parental receptors, both exhibiting negative cooperativity. Gradual replacement of individual amino acids revealed that only three residues (NVT of the o3 loop of the M 2 receptor) are involved in this effect. Data suggest that these amino acids are essential for propagation of a conformation change resulting in positive cooperativity induced by these modulators.Muscarinic receptors belong to the superfamily of seven transmembrane spanning segment, G protein-coupled receptors (Fredriksson et al., 2003). Five subtypes of muscarinic receptors that share a high degree of homology in transmembrane segments while connecting extracellular loops displaying more diversity have been identified (Bonner et al., 1987(Bonner et al., , 1988Peralta et al., 1987; Bonner, 1989a,b). Selectivity of individual subtypes for classical ligands and particularly for agonists is rather low because the orthosteric binding site is formed by highly homologous transmembrane segments (Lu et al., 2002;Hulme et al., 2003). Besides the classical binding site, muscarinic receptors possess one or more allosteric binding sites (Ellis and Seidenberg, 1989;Ellis et al., 1993;Lazareno et al., 2000;Christopoulos and Kenakin, 2002) whose binding determinants are located on less conserved extracellular loops. Binding of allosteric ligands can induce conformational changes of the receptor that result in changes in receptor affinity for orthosteric agonists and antagonists (Tuček et al., 1990Proška and Tuček, 1994;Dong et al., 1995;Jakubík et al., 1995Jakubík et al., , 1997Lazareno and Birdsall, 1995). These effects exhibit remarkable receptor subtype selectivity that is also reflected in the functional outcome of receptor stimulation by orthosteric agonists Dol...

Allosteric modulation of the muscarinic M 4 receptor as an approach to treating schizophrenia

“…To date, limited mutagenesis studies of muscarinic receptors have focused on a common allosteric binding site on M 1 , M 2 , and M 5 receptor subtypes (33,35) and M 3 (36,37) and M 4 receptors (38) used by prototypical negative allosteric modulators, such as gallamine, alcuronium, and C 7 /3-phth (21). Functional screening of a wide range of receptor mutants systematically pointed to the acidic residue D 432 of the hM 4 receptor as being critical to the potentiation of ACh by LY2033298 (Fig.…”

Critical Amino Acid Residues of the Common Allosteric Site on the M2Muscarinic Acetylcholine Receptor: More Similarities than Differences between the Structurally Divergent Agents Gallamine and Bis(ammonio)alkane-Type Hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide

“…The importance of these two positions in the receptor may be related to the fact that, if the region containing these residues is helical, their side groups will be adjacent on the same face of the helix. In addition, both 419 Asn and 423 Thr seem to be involved in mechanisms of cooperativity between NMS and several allosteric ligands, especially when comparisons are drawn between the M 2 and M 3 receptor subtypes (Krejci and Tucek, 2001;Jakubik et al, 2005).…”

Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors

“…Several lines of evidence suggest that muscarinic allosteric ligands (e.g., gallamine and alcuronium) bind to the extracellular domain of muscarinic receptors (Jakubík and Tucek, 1994;Leppik et al, 1994;Proska and Tucek, 1994), and that amino acid residues in the second and third extracellular loops are important for this mode of binding (Leppik et al, 1994;Matsui et al, 1995;Gnagey et al, 1999;Krejcí and Tucek, 2001;Jakubík et al, 2005). In contrast, virtually nothing is known on the nature of the selectivity of orthosteric muscarinic ligands.…”