Comparison of BoPz ligand binding properties at the EGFP(Δ17)hM1 receptor and its four mutants as assessed from equilibrium [3H]NMS and FRET binding measurements
Affinity constants (mean ± S.E. values for 3–5 independent determinations) for the fluorescent ligands are from competition experiments (Ki values) performed in the presence of a fixed [3H]NMS concentration or from saturation experiments (Kd values) taking each BoPz ligand as a FRET tracer over a 1 nM to 10 µM concentration range (Materials and Methods).
| EGFP-wt hM1 | EGFP-W405A | EGFP-W405F | EGFP-W400A | EGFP-W400F | |
|---|---|---|---|---|---|
| Bo(10)Pz (pK; −log M) | |||||
| [3H]NMS | 8.02 ± 0.04 | 7.77 ± 0.05 | 7.42 ± 0.05 | 8.25 ± 0.02 | 8.00 ± 0.01 |
| FRET | 7.91 ± 0.04 | 7.60 ± 0.10 | 7.42 ± 0.06 | 8.12 ± 0.08 | 8.01 ± 0.01 |
| Bo(12)Pz (pK; −log M) | |||||
| [3H]NMS | 7.51 ± 0.01 | 7.20 ± 0.01 | 6.99 ± 0.09 | 7.49 ± 0.06 | 7.19 ± 0.05 |
| FRET | 7.67 ± 0.01 | 7.35 ± 0.06 | 7.08 ± 0.04 | 7.66 ± 0.09 | 7.51 ± 0.03 |
| Bo(15)Pz (pK; −log M) | |||||
| [3H]NMS | 7.77 ± 0.05 | 7.06 ± 0.04 | 6.80 ± 0.06 | 6.60 ± 0.05 | 6.94 ± 0.04 |
| FRET | 7.97 ± 0.01 | 7.19 ± 0.07 | 6.94 ± 0.08 | 6.88 ± 0.02 | 7.41 ± 0.01 |
| Bo(22)Pz (pK; −log M) | |||||
| [3H]NMS | 7.74 ± 0.07 | 7.60 ± 0.06 | 7.18 ± 0.04 | 7.17 ± 0.07 | 7.14 ± 0.09 |
| FRET | 7.92 ± 0.09 | 7.77 ± 0.07 | 7.15 ± 0.12 | 7.19 ± 0.01 | 7.27 ± 0.04 |