The Y82A mutant of the M1 receptor has increased affinity and cooperativity with acetylcholine for win 62,577 and win 51,708

Asma Baig, Ray Leppik & Nigel J.M. Birdsall, National Institute for Medical Research, Mill Hill, London, NW7 1AA.

Muscarinic acetylcholine receptors have two allosteric sites (Lazareno et al., 2002). One site binds ligands such as gallamine and brucine (the ‘gallamine’ site) and the other binds WIN 62,577, WIN 51,708 and staurosporine (the ‘WIN’ site). During studies on mutants of the M1 receptor it was discovered that the Y82A mutant in the second transmembrane domain exhibited enhanced affinity for WIN 51,708 and WIN 62,577 and strong positive cooperativity with acetylcholine in binding and functional studies.

Membranes from COS-7 cells transiently expressing the wild-type (WT) and Y82A mutant receptors (0.5-3 pmoles/mg protein) were harvested as described previously (Matsui et al., 1995). Radioligand binding assays were carried out at 30o in a 100mM NaCl/20mM Hepes buffer pH 7.4 containing 10mM MgCl2 and 0.2mM GTP. The equilibrium and kinetic assays to determine the affinities and cooperativities of allosteric ligands with acetylcholine (ACh) and the radiolabelled antagonist [3H]N-methylscopolamine (3H]NMS) have been described in detail (Lazareno 2004). Assays of muscarinic receptor function measured 3H]inositol phosphates generated by ACh stimulation under Li+ block of whole cells prelabelled with 3H]inositol (Lu et al., 2001). Data are expressed as means ± sem (n).

The Y82A mutation had ca 3-fold lower affinity for NMS and ACh [0.43 ± 0.07 (10) and 0.55 ± 0.08 (4) log units respectively]. The affinity of gallamine increased 7 fold [0.89 ± 0.05 log units (3)] at the unoccupied receptors but less at the NMS occupied receptors [0.32 ± 0.21 log units (5)]. Gallamine thus exhibited increased negative cooperativity with NMS at Y82A. In contrast, both WIN 62,577 and WIN 51,708 switched from being negatively cooperative with NMS on WT [cooperativity = 0.12 ± 0.03 (3) and 0.11 ± 0.02 3 respectively] to having neutral cooperativity with NMS [cooperativity = 1.0 (6)] at Y82A. These two compounds also exhibited high potency in slowing down the dissociation rate of 3H]NMS from Y82A [log affinities 6.79 ± 0.14 (5) and 6.34 ± 0.05 (5) respectively]. Relative to the WT receptor, the affinities of WIN 62,577 and WIN 51,708 were calculated to have increased 15- and 9-fold respectively at the unoccupied receptors and 100- and 40- fold at the NMS-occupied receptors. The Y82A mutation also resulted in the cooperativities of ACh with WIN 62,577 or WIN 51,708 switching from negative to positive cooperativity. In the presence of 10-6M WIN 62,577 or WIN 51,708, ACh potency in the binding and functional assays increased 5-7 fold [0.74 ± 0.09 (7) and 0.90 ± 0.13 (3) log units respectively). The affinity of these analogues for the ACh-occupied Y82A receptor are in the 30-50 nanomolar range.

The high affinities of these allosteric agents and their large positive cooperativities with ACh at the Y82A receptor allow a more detailed exploration of cooperative interactions at muscarinic receptors.

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