Table 4. Pharmacological parameters of LY298 and VU154 at key M4 muscarinic acetylcholine receptor (mAChR) mutants.
| [3H]-NMS saturation binding on stable M4 mAChR Flp-In CHO cells | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Constructs | Sites per cell* | pKD† | |||||||
| Human WT M4 mAChR (from Table 1) | 598,111 ± 43,067 (7) | 9.76 ± 0.05 (7) | |||||||
| Y89A2.61 | 32,674 ± 4174 (4) | 9.88 ± 0.06 (4) | |||||||
| Q184A45.49 | 88,728 ± 3056 (3) | 9.99 ± 0.06 (3) | |||||||
| F186A45.51 | 36,907 ± 4170 (4) | 9.75 ± 0.16 (4) | |||||||
| W435A7.35 | 34,861 ± 3510 (3) | 9.81 ± 0.22 (3) | |||||||
| Y439A7.39 | 42,690 ± 4547 (3) | 8.31 ± 0.14 (3) | |||||||
| [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§ | log αNMS¶ | ||
| Human WT M4 | LY298 | 5.09 ± 0.07 (7) | 8.54 ± 0.04 (11) | = 5.65 | 1.57 ± 0.11 | 1.71 ± 0.09 | = 0 | ||
| VU154 | 5.06 ± 0.05 (7) | 8.54 ± 0.03 (11) | = 5.83 | 1.44 ± 0.07 | 1.11 ± 0.06 | = 0 | |||
| Y89A2.61 | LY298 | 5.25 ± 0.05 (6) | 8.48 ± 0.05 (6) | N.D. | N.D. | N.D. | N.D. | ||
| VU154 | 5.27 ± 0.05 (6) | 8.47 ± 0.05 (6) | N.D. | N.D. | N.D. | N.D. | |||
| Q184A45.49 | LY298 | 5.24 ± 0.06 (6) | 8.74 ± 0.04 (10) | 6.23 ± 0.06 | 1.28 ± 0.13 | 1.27 ± 0.11 | –1.10 ± 0.07 | ||
| VU154 | 5.28 ± 0.05 (6) | 8.69 ± 0.04 (10) | 5.87 ± 0.17 | 1.07 ± 0.09 | 0.81 ± 0.07 | = 0 | |||
| F186A45.51 | LY298 | 4.91 ± 0.05 (6) | 8.12 ± 0.05 (8) | N.D. | N.D. | N.D. | N.D. | ||
| VU154 | 4.91 ± 0.05 (6) | 8.12 ± 0.05 (8) | N.D. | N.D. | N.D. | N.D. | |||
| W4357.35 | LY298 | 3.79 ± 0.07 (7) | 6.88 ± 0.07 (7) | N.D. | N.D. | N.D. | N.D. | ||
| VU154 | 3.79 ± 0.07 (7) | 6.88 ± 0.07 (7) | N.D. | N.D. | N.D. | N.D. | |||
| Y439A7.39 | LY298 | 3.23 ± 0.22 (8) | 5.36 ± 0.25 (8) | N.D. | N.D. | N.D. | N.D. | ||
| VU154 | 3.23 ± 0.22 (8) | 5.36 ± 0.25 (8) | N.D. | N.D. | N.D. | N.D. | |||
Values represent the mean ± SEM with the number of independent experiments shown in parenthesis.
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. pKi values for ACh and Ipx are shared at each M4 mAChR construct. pKB values for the PAMs at Q184A are shared across the agonist data sets.
§
Logarithm of the binding cooperativity factor between the agonist (ACh or Ipx) and the PAM (LY298 or VU154).
¶
Logarithm of the binding cooperativity factor between the [3H]-NMS and the PAM (LY298 or VU154).