Antagonist binding parameters of wild-type, fragmented, and mutant muscarinic receptors coexpressed in COS-7 cells
| Transfected Receptors | High-Affinity Binding | IC50H | IC50L |
|---|---|---|---|
| % | nM | ||
| Pirenzepine | |||
| M2(1.6 μg) υ M3-tail (2.4 μg) | 34 ± 4 | 114 ± 8 | 1572 ± 56 |
| M2 (1.6 μg) − M3-tail (2.4 μg) | N.D. | 1462 ± 61 | |
| Tripitramine | |||
| M3 (0.5 μg) υ M2-trunc (3.5 μg) | 22 ± 6 | 2.22 ± 0.29 | 402 ± 33 |
| M3(0.5 μg) − M2-trunc (3.5 μg) | N.D. | 215 ± 21 | |
| M3-short (0.5 μg) υ M2-trunc (3.5 μg) | N.D. | 307 ± 23 | |
| M3-short (4 μg) | N.D. | 255 ± 29 | |
| M2(Asn404 → Ser) (3.3 μg) υ M3 (0.7 μg) | 17 ± 5 | 1.18 ± 0.22 | 408 ± 25 |
| M2 (Asn404 → Ser) (0.9 μg) υ M3 (0.2 μg)2-a | 21 ± 3 | 1.56 ± 0.15 | 333 ± 29 |
| M2 (Asn404 → Ser) (3.3 μg) υ M3 (0.7 μg) | N.D. | 225 ± 31 | |
| IC50 | Hill Coefficient | |
| nM | ||
| Methoctramine | ||
| M2 (1.6 μg) υ M3-tail (2.4 μg) | 57.5 ± 3.91 | 0.89 ± 0.07 |
| M3 (0.5 μg) υ M2-trunc (3.5 μg) | 392 ± 26 | 0.86 ± 0.09 |
| p-F-HHSiD | ||
| M2 (1.6 μg) υ M3-tail (2.4 μg) | 318 ± 22.3 | 0.99 ± 0.05 |
| M3 (0.5 μg) υ M2-trunc (3.5 μg) | 88.4 ± 12.2 | 1.03 ± 0.25 |
COS-7 cells were cotransfected (υ) or separately transfected (−) with plasmid DNAs coding for M2 and M3 wild-type receptors and for different muscarinic receptor mutants. A total amount of 4 μg of DNA was transfected (the relative amount of each plasmid is given in parentheses). IC50 values were obtained in displacement curves against 200 pM [3H]NM. Competition curves were fitted to one- or two-site binding models; to determine whether the data were best fitted by a one- or two-site model, the residual sums of squares were compared by Scheffé’s F test. Data are presented as mean ± S.E. of at least four experiments, each performed in duplicate.
IC50H and IC50L, high and low IC50 values. N.D., not detected.
↵2-a Supplemented with 2.9 μg of vector DNA.