Double-mutant cycle analysis of A652(NR1) and A651(NR2B) mutations
| Constitutive open index (%) | ΔG(kcal/mol) | ΔΔG(kcal/mol) | ΔΔGint(kcal/mol) | |
|---|---|---|---|---|
| Wild type | 2.0 ± 0.4 | −2.35 ± 0.02 | ||
| A652E(NR1) | 25 ± 1.2 | −0.67 ± 0.01 | 1.68 ± 0.03 | |
| A651R(NR2B) | 96 ± 2.2 | 1.91 ± 0.08 | 4.26 ± 0.07 | |
| A652E(NR1)+A651R(NR2B)a | 65 ± 1.1 | 0.37 ± 0.01 | 2.72 ± 0.03 | −3.22 ± 0.06 |
| A652R(NR1) | 71 ± 1.7 | 0.54 ± 0.01 | 2.89 ± 0.02 | |
| A651E(NR2B) | 15 ± 0.3 | −1.03 ± 0.02 | 1.32 ± 0.03 | |
| A652R(NR1)+A651E(NR2B)a | 30 ± 0.7 | −0.50 ± 0.01 | 1.85 ± 0.02 | −2.36 ± 0.04 |
| A652D(NR1) | 69 ± 0.9 | 0.48 ± 0.01 | 2.83 ± 0.03 | |
| A651K(NR2B) | 90 ± 1.6 | 1.32 ± 0.02 | 3.67 ± 0.02 | |
| A652D(NR1)+A651K(NR2B) | 85 ± 1.2 | 1.04 ± 0.01 | 3.39 ± 0.03 | −3.11 ± 0.09 |
| A652K(NR1) | 79 ± 1.1 | 0.79 ± 0.01 | 3.14 ± 0.03 | |
| A651D(NR2B) | 93 ± 1.7 | 1.55 ± 0.02 | 3.90 ± 0.005 | |
| A652K(NR1)+A651D(NR2B) | 90 ± 1.8 | 1.32 ± 0.02 | 3.67 ± 0.03 | −3.37 ± 0.06 |
| A652C(NR1) | 2.4 ± 0.4 | −2.21 ± 0.01 | 0.14 ± 0.01 | |
| A651C(NR2B) | 1.8 ± 0.2 | −2.41 ± 0.01 | −0.06 ± 0.01 | |
| A652C(NR1)+A651C(NR2B) | 0.8 ± 0.1 | −2.90 ± 0.03 | −0.55 ± 0.02 | −0.63 ± 0.02 |
| A652T(NR1) | 1.8 ± 0.2 | −2.41 ± 0.05 | −0.06 ± 0.02 | |
| A651T(NR2B) | 2.2 ± 0.3 | −2.26 ± 0.03 | 0.09 ± 0.02 | |
| A652T(NR1)+A651T(NR2B) | 0.9 ± 0.1 | −2.83 ± 0.06 | −0.48 ± 0.04 | −0.45 ± 0.01 |
| A652F(NR1) | 0.9 ± 0.2 | −2.83 ± 0.07 | −0.48 ± 0.06 | |
| A651F(NR2B) | 1.1 ± 0.3 | −2.71 ± 0.05 | −0.36 ± 0.04 | |
| A652F(NR1)+A651F(NR2B)b | 46 ± 1.4 | −0.10 ± 0.01 | 2.25 ± 0.03 | 3.09 ± 0.06 |
| A652L(NR1) | 0.8 ± 0.2 | −2.90 ± 0.07 | −0.55 ± 0.02 | |
| A651L(NR2B) | 1.2 ± 0.2 | −2.65 ± 0.06 | −0.30 ± 0.02 | |
| A652L(NR1)+A651L(NR2B)b | 56 ± 1.9 | 0.14 ± 0.02 | 2.49 ± 0.01 | 3.34 ± 0.07 |
| A652W(NR1) | 10 ± 0.5 | −1.33 ± 0.04 | 1.02 ± 0.02 | |
| A651W(NR2B) | 30 ± 1.1 | −0.50 ± 0.01 | 1.85 ± 0.02 | |
| A652W(NR1)+A651W(NR2B)b | 97 ± 2.1 | 2.09 ± 0.07 | 4.44 ± 0.08 | 1.57 ± 0.04 |
Δ G, The difference in free energy between closed and activated channels; Δ Δ G, the change in Δ G caused by each mutation; Δ Δ Gint, the coupling energy (for more details, see Results).
↵aDouble mutations showing a significantly (p < 0.05) smaller constitutive open index than one of the two component single mutations.
↵bDouble mutations showing a significantly (p < 0.05) larger constitutive open index than the addition of the two component single mutations (data are expressed as mean ± SEM; n = 8–12 for each mutation).