Table 1.
Binding parameters for the interaction between ACh and M1 PAMs, BQCA, PF-06767832, VU6004256 or MIPS1780, at the WT and mutant M1 mAChRs. Values represent the mean ± S.E.M. of four experiments performed in duplicate, and were estimated from binding interaction studies using an allosteric ternary complex model (Eq. (1)). The pKA of [3H]NMS was constrained to the values listed in Table 3. ND, not determined.
| PAMs | pKBa | Logα (α)NMSb | Logα (α)AChc | |
|---|---|---|---|---|
| BQCA | WT | 5.78 ± 0.05 | −1.07 ± 0.10 (0.06) |
1.32 ± 0.11 (13.5) |
| F772.56I | 5.82 ± 0.06 | −0.90 ± 0.07 (0.08) |
1.31 ± 0.12 (13.5) |
|
| Y822.61A | 5.76 ± 0.06 | −0.84 ± 0.06 (0.11) |
1.58 ± 0.11 (30.2) |
|
| Y179ECL2A | ND | ND | ND | |
| W4007.35A | ND | ND | ND | |
| PF-06767832 | WT | 6.51 ± 0.03 | −1.05 ± 0.06 (0.06) |
1.40 ± 0.08 (16.2) |
| F772.56I | 6.38 ± 0.07 | −1.02 ± 0.12 (0.06) |
1.83 ± 0.14* (44.7) |
|
| Y822.61A | 5.61 ± 0.12* | −0.16 ± 0.02 (0.62) |
1.53 ± 0.11 (30.9) |
|
| Y179ECL2A | ND | ND | ND | |
| W4007.35A | ND | ND | ND | |
| VU6004256 | WT | 6.32 ± 0.03 | = −2 | 1.40 ± 0.09 (15.5) |
| F772.56I | 6.38 ± 0.03 | = −2 | 1.45 ± 0.10 (14.1) |
|
| Y822.61A | 5.65 ± 0.19* | −0.09 ± 0.03 (0.76) |
1.08 ± 0.13 (11.2) |
|
| Y179ECL2A | ND | ND | ND | |
| W4007.35A | ND | ND | ND | |
| MIPS1780 | WT | 6.18 ± 0.03 | = −2 | 1.60 ± 0.08 (25.7) |
| F772.56I | 6.18 ± 0.03 | = −2 | 1.60 ± 0.09 (25.7) |
|
| Y822.61A | 5.00 ± 0.24* | −0.16 ± 0.06 (0.69) |
1.66 ± 0.19 (45.7) |
|
| Y179ECL2A | ND | ND | ND | |
| W4007.35A | ND | ND | ND | |
Negative logarithm of the allosteric modulator equilibrium dissociation constant.
Logarithm of binding cooperativity between [3H]NMS and each modulator. Where determined as the preferred model by F-test, logαNMS was constrained to −2, consistent with high negative cooperativity between the two ligands.
Logarithm of binding cooperativity between ACh and each modulator.
Significantly different compared to WT, p < 0.05, one-way ANOVA with Dunnett’s post-hoc test.