Table 5.
Mutations to aligned residues in each M4 α-helix have independent effects on function
| Dose responsea |
Fold change | |||||
|---|---|---|---|---|---|---|
| Mutation(s) | EC50 (μM) | pEC50 (M) | Hill slope | n | ||
| WT | 7.61 | 5.12 ± 0.07b | 1.70 ± 0.47 | 50 | Observed | Predictedc |
| αF426A | 2.02 | 5.71 ± 0.14b | 1.80 ± 0.76 | 11 | 3.76 | — |
| βF464A | 8.55 | 5.08 ± 0.11 | 1.38 ± 0.19 | 10 | 0.89 | — |
| δF468A | 5.83 | 5.25 ± 0.10 | 1.60 ± 0.27 | 7 | 1.31 | — |
| εF454A | 3.90 | 5.42 ± 0.10b | 1.80 ± 0.25 | 8 | 1.95 | — |
| αF426A + βF464A + δF468A + εF454A | 0.67 | 6.17 ± 0.17b | 2.04 ± 0.15 | 8 | 11.4 | 8.54 |
| αV425A | 4.30 | 5.37 ± 0.05b | 1.89 ± 0.28 | 10 | 1.77 | — |
| βI463A | 3.67 | 5.44 ± 0.16b | 1.78 ± 0.11 | 8 | 2.07 | — |
| δI467A | 4.63 | 5.34 ± 0.07b | 1.91 ± 0.79 | 7 | 1.65 | — |
| εI453A | 4.50 | 5.35 ± 0.06b | 1.83 ± 0.27 | 8 | 1.69 | — |
| αV425A + βI463A + δI467A + εI453A | 0.63 | 6.20 ± 0.17b | 2.11 ± 0.28 | 7 | 12.1 | 10.2 |
| αC418W | 0.47 | 6.33 ± 0.13b | 1.54 ± 0.23 | 50 | 16.2 | — |
| βT456W | 4.50 | 5.36 ± 0.10b | 1.73 ± 0.17 | 10 | 1.69 | — |
| δM460W | 12.2 | 4.93 ± 0.12b | 1.41 ± 0.16 | 9 | 0.63 | — |
| εF446W | 4.26 | 5.39 ± 0.14b | 1.75 ± 0.13 | 9 | 1.79 | — |
| αC418W + βT456W + δM460W + εF446W | 0.25 | 6.60 ± 0.08b | 1.90 ± 0.58 | 9 | 30.0 | 30.7 |
a
Measurements performed 1 to 4 days after cRNA injection (Vhold ranging from −20 to −80 mV). Error values are represented as standard deviation.
b
p < 0.001 relative to WT via one-way ANOVA followed by Dunnet’s post hoc test.
c
Predicted fold change if individual mutations affect function independently.