TABLE 1.
Ligand binding and functional properties of mutant M3Rs containing single Cys substitutions within the C-terminal segment of the i2 loop
COS-7 cells were transiently transfected with the indicated M3′(3C)-Xa-derived Cys-substituted mutant M3R constructs. Radioligand binding and phosphoinositide assays were performed and analyzed as described under ”Experimental Procedures.“ In all cases, carbachol inhibition binding curves were characterized by Hill coefficients ranging from 0.7 to 0.8. Basal inositol monophosphate (IP) levels for the M3′(3C)-Xa construct were 1905 ± 279 dpm (=100%). Data are shown as mean ± S.E. of 2–4 independent experiments, each performed in duplicate.
| Receptor | [3H]NMS binding |
Carbachol binding, |
Carbachol-induced IP production |
|||
|---|---|---|---|---|---|---|
| KD | Bmax | Ki | EC50 | Emax | Basal activity | |
| pm | pmol/mg of protein | μm | nm | Fold above basal | % | |
| M3′(3C)-Xa | 403 ± 128 | 16.2 ± 3.9 | 24.6 ± 7.6 | 11.3 ± 1.1 | 5.5 ± 0.7 | 100 |
| R176C | 484 ± 29 | 11.4 ± 0.4 | 22.0 ± 5.9 | 264 ± 11 | 7.9 ± 1.3 | 76 ± 3 |
| A177C | 521 ± 18 | 12.8 ± 1.9 | 41.3 ± 16.2 | 21.4 ± 5.7 | 5.2 ± 2.7 | 84 ± 9 |
| K178C | 499 ± 46 | 12.1 ± 1.1 | 29.5 ± 8.8 | 40.1 ± 9.6 | 4. 9 ± 2.3 | 109 ± 12 |
| R179C | 493 ± 33 | 11.5 ± 1.1 | 8.2 ± 1.9 | 2.6 ± 0.8 | 2.9 ± 1.4 | 106 ± 1 |
| T180C | 751 ± 27 | 13.0 ± 2.0 | 20.4 ± 3.7 | 22.9 ± 2.3 | 7.7 ± 0.4 | 90 ± 3 |