Table 1. Pharmacological parameters from radioligand binding and functional experiments.
| [3H]-NMS saturation binding on stable M4 mAChR CHO cells | |||||||
|---|---|---|---|---|---|---|---|
| Constructs | Sites per cell* | pKD† | |||||
| Human WT M4 mAChR | 598,111 ± 43,067 (7) | 9.76 ± 0.05 (7) | |||||
| Mouse WT M4 mAChR | 21,027 ± 2188 (3) | 9.76 ± 0.05 (3) | |||||
| Human D432E M4 mAChR | 126,377 ± 10,066 (3) | 9.60 ± 0.07 (3) | |||||
| Human T433R M4 mAChR | 157,442 ± 36,658 (6) | 9.64 ± 0.09 (6) | |||||
| Human V91L, D432E, T433R M4 mAChR | 205,771 ± 20,975 (4) | 9.58 ± 0.08 (4) | |||||
| [3H]-NMS interaction binding assays between ACh or Ipx and LY298 or VU154 on stable M4 mAChR constructs in Flp-In CHO cells | |||||||
| Constructs | PAM | pKi ACh ‡ | pKi Ipx ‡ | pKB PAM ‡ | log αACh § | log αIpx § | |
| Human WT M4 mAChR | LY298 | 4.50 ± 0.06 (4) | 8.30 ± 0.06 (4) | 5.65 ± 0.07 (8) ¶ | 2.59 ± 0.10 (4) | 1.86 ± 0.10 (4) | |
| VU154 | 4.40 ± 0.09 (4) | 8.19 ± 0.06 (8) | 5.83 ± 0.11 (12) ¶ | 1.61 ± 0.13 (4) | 1.03 ± 0.10 (8) | ||
| Mouse WT M4 mAChR | LY298 | 4.52 ± 0.07 (4) | 8.55 ± 0.06 (4) | 5.74 ± 0.07 (8) ¶ | 1.78 ± 0.10 (4) | 1.30 ± 0.11 (4)* | |
| VU154 | 4.59 ± 0.06 (4) | 8.57 ± 0.06 (3) | 6.07 ± 0.09 (7) ¶ | 2.43 ± 0.10 (4) | 1.75 ± 0.12 (3)* | ||
| Human D432E M4 mAChR | LY298 | N.T. | 8.28 ± 0.04 (5) | 5.86 ± 0.07 (5) | N.T. | 1.59 ± 0.06 (5) | |
| VU154 | N.T. | 8.27 ± 0.06 (6) | 6.21 ± 0.12 (6) | N.T. | 1.04 ± 0.09 (6) | ||
| Human T433R M4 mAChR | LY298 | N.T. | 8.05 ± 0.08 (5) | 5.04 ± 0.04 (5)* | N.T. | 1.91 ± 0.11 (5) | |
| VU154 | N.T. | 7.88 ± 0.04 (5) | 5.50 ± 0.08 (5) | N.T. | 1.67 ± 0.07 (5)* | ||
| Human V91L, D432E, T433R M4 mAChR | LY298 | N.T. | 7.95 ± 0.10 (4) | 5.29 ± 0.26 (4) | N.T. | 1.80 ± 0.22 (4) | |
| VU154 | N.T. | 7.89 ± 0.12 (4) | 6.34 ± 0.16 (4)* | N.T. | 1.35 ± 0.16 (4) | ||
| Gαi1 activation (TruPath) interaction assays between ACh or Ipx and LY298 or VU154 on transiently expressed M4 mAChR constructs in HEK293A cells | |||||||
| Constructs | PAM | log τ ACh** | log τ Ipx** | pKB PAM ‡ | log τ PAM** | log αβACh†† | log αβIpx†† |
| Human WT M4 mAChR | LY298 | 2.71 ± 0.14 (4) | 1.49 ± 0.12 (4) | = 5.65 | 1.02 ± 0.03 (8) ¶ | 2.01 ± 0.14 (4) | 1.96 ± 0.16 (4) |
| VU154 | = 5.83 | –0.55 ± 0.08 (8) ¶ | 1.22 ± 0.13 (4) | 0.20 ± 0.13 (4) | |||
| pERK1/2 interaction assays between ACh or Ipx and LY298 or VU154 on stable M4 mAChR constructs in Flp-In CHO cells | |||||||
| Constructs | PAM | log τ ACh** | log τ Ipx** | pKB PAM ‡ | log τC PAM ‡ ‡ | log αβACh†† | log αβIpx†† |
| Human WT M4 mAChR | LY298 | 3.27 ± 0.06 (8) ¶ | 1.74 ± 0.03 (16) ¶ | = 5.65 | 1.19 ± 0.05 (12)** | 2.29 ± 0.22 (4) | 1.08 ± 0.28 (8) |
| VU154 | = 5.83 | 0.11 ± 0.05 (12)** | 0.88 ± 0.23 (4) | 0.66 ± 0.15 (8) | |||
| Mouse WT M4 mAChR | LY298 | N.T. | N.D. | = 5.74 | 1.32 ± 0.07 (5) | N.T. | 1.24 ± 0.12 (4) |
| VU154 | N.T. | N.D. | = 6.07 | 1.47 ± 0.08 (5) § § | N.T. | 2.08 ± 0.15 (5) § § | |
| Human D432E M4 mAChR | LY298 | N.T. | N.D. | = 5.86 | 1.34 ± 0.08 (5) | N.T. | 1.37 ± 0.28 (5) |
| VU154 | N.T. | N.D. | = 6.21 | 0.78 ± 0.08 (5) § § | N.T. | 1.02 ± 0.15 (5) | |
| Human T433R M4 mAChR | LY298 | N.T. | N.D. | = 5.04 | 1.73 ± 0.13 (5) § § | N.T. | 1.85 ± 0.28 (5) |
| VU154 | N.T. | N.D. | = 5.50 | 0.95 ± 0.12 (5) § § | N.T. | 1.18 ± 0.14 (5) | |
| Human V91L, D432E, T433R M4 mAChR | LY298 | N.T. | N.D. | = 5.29 | 1.62 ± 0.09 (5) § § | N.T. | 1.64 ± 0.30 (5) |
| VU154 | N.T. | N.D. | = 6.34 | 0.68 ± 0.06 (5) § § | N.T. | 1.34 ± 0.11 (5) § § | |
Values represent the mean ± SEM with the number of independent experiments shown in parenthesis.
N.T.: not tested; N.D.: not determined; Ach, acetylcholine; Ipx: iperoxo; PAM: positive allosteric modulator.
Number of [3H]-NMS binding sites per cell.
Negative logarithm of the radioligand equilibrium dissociation constant.
Negative logarithm of the orthosteric (pKi) or allosteric (pKB) equilibrium dissociation constant.
Logarithm of the binding cooperativity factor between the agonist (ACh or Ipx) and the PAM (LY298 or VU154).
Parameter was determined in a shared global analysis between agonists.
Logarithm of the operational efficacy parameter determined using the Operational Model of Agonism.
Logarithm of the functional cooperativity factor between the agonist (ACh or Ipx) and the PAM (LY298 or VU154).
logτC = logarithm of the operational efficacy parameter corrected for receptor expression (methods in Appendix 1).
Values from pKB PAM, log αIpx, log τC PAM, and log αβIpx that are significantly different from human WT M4 mAChR (p<0.05) calculated by a one-way ANOVA with a Dunnett’s post-hoc test.