Modulation of the activity of wild-type and mutant M3 muscarinic receptors in CHO cells by allosteric ligands – a cautionary tale

Laura Iarriccio, Nigel J. M. Birdsall
MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom

During an investigation of the effects of mutations in the 1st and 3rd extracellular loops of M3 receptors on the binding and function of allosteric ligands, relatively high levels of apparent constitutive activity were found. This activity could be modulated in a ligand- and mutant- dependent manner.

Wild-type M3 receptor (WT) and its N132G and K523E mutants were stably expressed using the Flp-In expression vector and CHO cell lines (Invitrogen). Cells were prelabelled with 3H-inositol (2μCi/ml) for 18h. The 3H-inositol-phosphates (3H-IP) generated in 60-80 minutes under Li block was measured as described by Jones et al. (1995). The results are expressed as means ± sem, n ≥ 3. Statistically significant differences of fold-changes relative to controls are shown with asterisks (P < 0.05, paired t-test).

Acetylcholine (ACh) potencies at the WT and N132G cell lines were similar (pEC50 7.11 ± 0.06(11)), the pEC50 at the K523E cell line being lower (6.53 ± 0.08(9)). ‘Basal’ activity was relatively high and could be inhibited 3.5 ± 0.6*, 2.8 ± 0.7* and 1.9 ± 0.2* fold respectively by atropine (10-7M). Initial studies indicated that this apparent constitutive activity could be enhanced or inhibited by different allosteric muscarinic ligands, sometimes in a mutant sensitive manner. N-chloromethylbrucine (300μM) enhanced 3H-IP production at WT, N132G and K523E 1.5 ± 0.2*, 1.5 ± 0.2* and 1.8 ± 0.3* fold respectively whereas brucine (300μM) was neutral at WT and N132G (fold increase 1.0 ± 0.1, 0.9 ± 0.1) and enhanced 3H-IP production 2.1 ± 0.4* fold at K523E. A similar pattern was shown by 30μM WIN62,577 (1.0 ± 0.2, 1.0 ± 0.1 and 1.6 ± 0.1* fold respectively). In contrast, strychnine (100μM) and N-desmethylclozapine (a possible selective orthosteric M1 agonist, 10μM) inhibited 3H-IP production at WT (0.61 ± 0.03* and 0.83 ± 0.05*, respectively) but had no effect at the two mutated receptors.

As this behaviour of the four allosteric ligands mimicked their ability to act as allosteric enhancers or inhibitors of ACh at WT, N132G and K523E, cells were incubated with acetylcholinesterase (AChase, 0.05-5U/ml) and this reduced 3H-IP production to the levels found in the presence of atropine in the three cell lines. In the presence of AChase (2.5U/0.5ml), N-chloromethylbrucine (300μM), brucine (300μM) and strychnine (100μM) had no effect on basal activity. However WIN62,577 (30μM) did stimulate WT, N132G and K523E receptors (2.3 ± 0.2*, 1.6 ± 0.2* and 1.9 ± 0.6* fold respectively) and N-desmethylclozapine (10μM) also revealed low levels of stimulation (1.9 ± 0.3*, 1.8 ± 0.3* and 1.8 ± 0.2* fold respectively) at the 3 receptor species. Both ligands behaved as low efficacy partial agonists.

This study reveals that surprisingly, under certain conditions, low concentrations of ACh (estimated levels 2-10 nM) can be present in assays of muscarinic receptor function and this may give misleading interpretations that allosteric enhancers can act as allosteric agonists. In the absence of ACh, an allosteric agonist action of WIN62,577 is revealed at M3 muscarinic receptors and at the two mutants.

Jones P.G. et al., (1995) Eur J Pharmacol 288: 251-257