Binding parameters of wild-type M2 and M3 and mutated M3 receptors
Values of equilibrium dissociation constants (Kd) were obtained by nonlinear regression analysis of saturation binding experiments. Values of negative logarithms of methoctramine inhibition constants (pKi) were obtained by nonlinear regression of data shown in Figs. 2 and 5. Equation 2 was fitted to the data to obtain IC50. Ki was computed according eq. 3. Data are means ± S.E.M. from four to six independent experiments performed in quadruplicates.
| Receptora | [3H]NMS KD | Methoctramine pKi | SMP pKi | LMP pKi |
|---|---|---|---|---|
| nM | ||||
| M3 wt | 0.32 ± 0.01 | 5.69 ± 0.12 | 4.41 ± 0.15 | 5.43 ± 0.13 |
| M2 wt | 0.78 ± 0.04 | 7.29 ± 0.04 | 5.25 ± 0.12 | 6.78 ± 0.08 |
| Mutations in o3 of M3 | ||||
| M3 S | 0.32 ± 0.02b | 5.95 ± 0.15b,c | 4.58 ± 0.14b | 5.64 ± 0.12 |
| M3 K | 0.34 ± 0.02b | 6.49 ± 0.11b,d | 4.96 ± 0.15b,d | 6.15 ± 0.11b,d |
| M3 KFN | 0.51 ± 0.03b,d | 6.51 ± 0.09b,d | n.d. | n.d. |
| M3 DKFN | 0.49 ± 0.03b,d | 6.51 ± 0.16b,d | n.d. | n.d. |
| M3 SK | 0.34 ± 0.02b | 7.02 ± 0.19c,d | 5.33 ± 0.09d | 6.65 ± 0.09d |
| M3 SKFN | 0.68 ± 0.05d | 7.04 ± 0.15c,d | n.d. | n.d. |
| M3 DSKFN | 0.76 ± 0.05d | 7.17 ± 0.04c,d | 5.20 ± 0.11d | 6.68 ± 0.08d |
| Mutations in o3 of M2 | ||||
| M2 P | 0.62 ± 0.05b | 6.31 ± 0.12b | n.d. | n.d. |
n.d., not determined.
↵a For the mutants nomenclature, see Materials and Methods and Supplemental Fig. 3.
↵b Significantly different from M2 wt (P < 0.01, multiparametric one-way analysis of variance with Tukey-Kramer post-test).
↵c Significantly different from M3 K (P < 0.01, multiparametric one-way analysis of variance with Tukey-Kramer post-test).
↵d Significantly different from M3 wt (P < 0.01, multiparametric one-way analysis of variance with Tukey-Kramer post-test).