Summary of kinetics
| Step | Probes | Resting FRETr % | ΔFRETr % | Delayon s | τon s | Delayoff s | τoff s | EC50 (oxo-M) nM |
| M1R activation | M1R-YFP-CFP | 0.88 | +6 | <0.1 | 0.18 | |||
| M1R/Gβ interaction | M1R-CFP | 0.42 ± 0.07 | +33 ± 6 | 0.20 ± 0.03 | 3.7 ± 0.2 | 330 ± 150 | ||
| Gβ1-YFP | ||||||||
| Gαq/Gβ1 separation | Gαq-CFP | 0.15 ± 0.01 | −10 | 2.0 | 5.8 | 35 | ||
| Gβ1-YFP (with GRK2) | 0.26 ± 0.03 | −17 ± 2 | 0.14 ± 0.05 | 2.8 ± 0.3 | 9.9 ± 2.9 | 28 ± 2 | 160 ± 100 | |
| Gαq/PLCβ1 interaction | Gαq-CFP | 0.14 ± 0.03 | +20 ± 2 | 0.38 ± 0.25 | 1.3 ± 0.3 | 0.34 ± 0.14 | 3.6 ± 0.5 | 260 ± 190 |
| PLCβ1-YFP | ||||||||
| PIP2 hydrolysis | PH(PLCδ1)-CFP | 0.14 ± 0.03 | −44 ± 3 | 1.3 ± 0.2 | 5.4 ± 1.6 | 29 ± 2 | 59 ± 7 | 28 ± 14 |
| PH(PLCδ1)-YFP | ||||||||
| Kv7.2/7.3 closure | M current | 1.4 ± 0.3 | 5.0 ± 0.6 | 34 ± 6 | 123 ± 20 | 120 ± 100 | ||
| Kv7.2/7.3 closure with PLCβ | M current | 0.78 ± 0.07 | 1.2 ± 0.1 | 11 ± 7 | 62 ± 22 | |||
| PLCβ1-YFP | ||||||||
| Kv7.2/7.3 closure with PH probes | M current | 2.1 ± 0.1 | 5.7 ± 0.7 | 11 ± 5 | 63 ± 9 | |||
| PH(PLCδ1)-CFP | ||||||||
| PH(PLCδ1)-YFP |
| Step | Probes | Resting FRETr % | ΔFRETr % | Delayon s | τon s | Delayoff s | τoff s | EC50 (oxo-M) nM |
| M1R activation | M1R-YFP-CFP | 0.88 | +6 | <0.1 | 0.18 | |||
| M1R/Gβ interaction | M1R-CFP | 0.42 ± 0.07 | +33 ± 6 | 0.20 ± 0.03 | 3.7 ± 0.2 | 330 ± 150 | ||
| Gβ1-YFP | ||||||||
| Gαq/Gβ1 separation | Gαq-CFP | 0.15 ± 0.01 | −10 | 2.0 | 5.8 | 35 | ||
| Gβ1-YFP (with GRK2) | 0.26 ± 0.03 | −17 ± 2 | 0.14 ± 0.05 | 2.8 ± 0.3 | 9.9 ± 2.9 | 28 ± 2 | 160 ± 100 | |
| Gαq/PLCβ1 interaction | Gαq-CFP | 0.14 ± 0.03 | +20 ± 2 | 0.38 ± 0.25 | 1.3 ± 0.3 | 0.34 ± 0.14 | 3.6 ± 0.5 | 260 ± 190 |
| PLCβ1-YFP | ||||||||
| PIP2 hydrolysis | PH(PLCδ1)-CFP | 0.14 ± 0.03 | −44 ± 3 | 1.3 ± 0.2 | 5.4 ± 1.6 | 29 ± 2 | 59 ± 7 | 28 ± 14 |
| PH(PLCδ1)-YFP | ||||||||
| Kv7.2/7.3 closure | M current | 1.4 ± 0.3 | 5.0 ± 0.6 | 34 ± 6 | 123 ± 20 | 120 ± 100 | ||
| Kv7.2/7.3 closure with PLCβ | M current | 0.78 ± 0.07 | 1.2 ± 0.1 | 11 ± 7 | 62 ± 22 | |||
| PLCβ1-YFP | ||||||||
| Kv7.2/7.3 closure with PH probes | M current | 2.1 ± 0.1 | 5.7 ± 0.7 | 11 ± 5 | 63 ± 9 | |||
| PH(PLCδ1)-CFP | ||||||||
| PH(PLCδ1)-YFP |
For each kinetic step, the probes used, average resting FRETr ratio, percent change, delays, and single-exponential time constants from onset and washout of 10 µM oxo-M are given. The reported EC50 is based on a Hill fit to steady-state responses.