Comparative studies of the binding of ligands to mutant M1 muscarinic receptors

J.A.Goodwin and E.C. Hulme, Division of Physical Biochemistry, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K.

Ala-substitution mutagenesis has located the transmembrane (TM) amino acids that form the ACh binding site of the M1 mAChR. Tyr106 (TM 3) and Tyr381 (TM6) are particularly important constituents. Homology modeling has proposed additional targets in the second extracellular loop, including Cys178 and Ile180. In addition, we have mutated Cys98 (TM3) that forms a disulphide-bond anchor to Cys178 and Asp99, possibly part of the ligand access channel. We report the effects of these mutations on the binding of ACh, and selected structurally distinct muscarinic ligands.

Rat M 1 mAChRs controlled by an SV40 promoter were expressed in COS-7 cells. Mutations were made by the QuickChange method. Washed cell homogenates were prepared after 3 days. Measurements of radioligand binding to membrane suspensions (15-60 μg/ml in 20 mM NaHepes, 100 mM NaCl, 1 mM MgCl2 pH 7.5) were performed at 30 ºC using (-)- 3H-N-methylscopolamine (NMS; 80 Ci/mmol) or (-)- 3H-3-quinuclidinyl benzilate (QNB; 42 Ci/mmol) and terminated by rapid filtration. The binding of unlabelled ligands was measured by radioligand competition. Binding curves were analysed by non-linear least-squares fitting using SigmaPlot. The results are summarised in Table 1.

* 2-19 independent measurements. a p<0.05, bp<0.01 wrt corresponding WT (1-way anova/ 2-tailed t-test).

The Y381A mutation decreased the affinities of the antagonists NMS and scopolamine by more than 200-fold. Interestingly, Y381A did not reduce QNB affinity. Mutation of the other residues, including the disulphide-bonded cysteines, caused smaller (up to 50-fold) reductions in antagonist affinity. Of the agonists, ACh showed the greatest sensitivity to mutation; the differential effect of C178A (60-fold) vs C98A (13-fold) may implicate the disulfide bonded C178 and its neighbouring residue I180 in the ACh binding site, as well as Y106 and Y381. The agonists oxo-M and oxotremorine, and the partial agonist pilocarpine followed an attenuated ACh-like pattern. Interestingly, the affinities of the functionally M 1- selective agonists McN-A-343, and AC-42 (Spalding et al, 2002) were little affected, or increased by these mutations. These ligands may bind outside the ACh binding pocket.

Spalding, T.A. et al (2002) Mol. Pharmacol 61, 1297-1302.

Supported by a MRC studentship to A. Goodwin. We are grateful to ACADIA Pharmaceuticals Inc. for the kind gift of AC-42.