TABLE 2.
Ligand binding and functional properties of mutant M3Rs containing single Cys substitutions within the H8 helix
Radioligand binding and phosphoinositide assays were performed with COS-7 cells transiently transfected with the indicated M3′(3C)-Xa-derived Cys-substituted mutant M3Rs. Data were 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 2683 ± 118 dpm ( = 100%). Data are given as mean ± S.E. of 2–5 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 | 555 ± 15 | 14.9 ± 3.0 | 23.8 ± 1.2 | 28.0 ± 2.7 | 8.4 ± 0.3 | 100 |
| K548C | 555 ± 92 | 12.4 ± 2.9 | 17.0 ± 1.6 | 23.6 ± 3.27 | 6.9 ± 0.3 | 111 ± 10 |
| T549C | 564 ± 75 | 13.7 ± 0.9 | 12.6 ± 2.2 | 108 ± 5 | 8.5 ± 0.3 | 107 ± 15 |
| F550C | 556 ± 38 | 9.3 ± 1.2 | 6.7 ± 0.1 | 101 ± 22 | 11.9 ± 1.2 | 81 ± 7 |
| R551C | 524 ± 42 | 12.8 ± 0.9 | 19.5 ± 4.7 | 116 ± 24 | 10.9 ± 1.7 | 81 ± 7 |
| T552C | 438 ± 77 | 11.0 ± 0.6 | 14.6 ± 2.3 | 31.3 ± 2.7 | 9.8 ± 2.5 | 73 ± 6 |
| T553C | 548 ± 18 | 11.2 ± 0.7 | 13.7 ± 2.1 | 21.5 ± 2.4 | 5.8 ± 0.4 | 133 ± 17 |
| F554C | 583 ± 53 | 12.0 ± 0.7 | 8.2 ± 2.5 | 46.8 ± 11.2 | 7.7 ± 2.4 | 149 ± 10 |
| K555C | 553 ± 17 | 10.8 ± 1.1 | 18.5 ± 0.8 | 7.2 ± 0.3 | 11.8 ± 2.3 | 76 ± 1 |
| T556C | 462 ± 30 | 13.4 ± 3.0 | 18.2 ± 3.8 | 23.6 ± 2.5 | 9.4 ± 0.2 | 97 ± 3 |