Kinetic Analysis of Wild-Type and α P272 Mutant Receptors
| Mutant . | k+1 . | k-1 . | K1 . | k+2 . | k-2 . | K2 . | β1 . | α1 . | Θ1 . | β2 . | α2 . | Θ2 . | k+b . | k-b . | KB . | ΔG° . |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| μM | μM | mM | kcal/mol | |||||||||||||
| Wild type | 271 ± 16 | 7190 ± 545 | 26 | 192 ± 6 | 19300 ± 309 | 101 | 67 ± 8 | 2190 ± 160 | 0.03 ± 0.0043 | 43700 ± 999 | 1650 ± 32 | 26.5 ± 0.79 | 26 ± 1 | 113000 ± 1690 | 4.3 | – |
| αP272G | 31 ± 19 | 895 ± 653 | 29 | 115 ± 26 | 13400 ± 2440 | 117 | NA | NA | – | 1260 ± 29 | 7410 ± 72 | 0.17 ± 0.004 | 17 ± 2 | 95800 ± 4920 | 5.6 | 2.96 ± 0.04 |
| αP272A | 48 ± 3 | 139 ± 14 | 2.9 | 84 ± 2 | 7100 ± 171 | 85 | 119 ± 9 | 475 ± 28 | 0.25 ± 0.024 | 40100 ± 1140 | 461 ± 12 | 87 ± 3.35 | 24 ± 1 | 130000 ± 1780 | 5.4 | −0.70 ± 0.05 |
| αP272V | 168 ± 29 | 1620 ± 298 | 9.6 | 109 ± 4 | 24600 ± 615 | 226 | NA | NA | – | 32400 ± 1240 | 1010 ± 24 | 32 ± 1.45 | 22 ± 1 | 107000 ± 2200 | 4.9 | −0.11 ± 0.06 |
| αP272L | 12 ± 8 | 228 ± 162 | 19 | NA | NA | – | NA | NA | – | 60.1 ± 1.9 | 1420 ± 26 | 0.042 ± 0.002 | 19 ± 2 | 110000 ± 4490 | 5.8 | 3.77 ± 0.05 |
| αP272I | 40 ± 12 | 478 ± 168 | 12 | 25 ± 1 | 8200 ± 363 | 328 | 18 ± 2 | 3850 ± 391 | 0.005 ± 0.0007 | 3410 ± 152 | 1030 ± 14 | 3.3 ± 0.15 | 31 ± 1 | 99400 ± 1710 | 3.2 | 1.22 ± 0.06 |
| αP272S | 153 ± 27 | 1120 ± 224 | 7.3 | 72 ± 2 | 18300 ± 500 | 254 | 69 ± 6 | 2450 ± 125 | 0.028 ± 0.0028 | 13200 ± 391 | 917 ± 12 | 14 ± 0.47 | 22 ± 1 | 120000 ± 1870 | 5.5 | 0.36 ± 0.04 |
| Mutant . | k+1 . | k-1 . | K1 . | k+2 . | k-2 . | K2 . | β1 . | α1 . | Θ1 . | β2 . | α2 . | Θ2 . | k+b . | k-b . | KB . | ΔG° . |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| μM | μM | mM | kcal/mol | |||||||||||||
| Wild type | 271 ± 16 | 7190 ± 545 | 26 | 192 ± 6 | 19300 ± 309 | 101 | 67 ± 8 | 2190 ± 160 | 0.03 ± 0.0043 | 43700 ± 999 | 1650 ± 32 | 26.5 ± 0.79 | 26 ± 1 | 113000 ± 1690 | 4.3 | – |
| αP272G | 31 ± 19 | 895 ± 653 | 29 | 115 ± 26 | 13400 ± 2440 | 117 | NA | NA | – | 1260 ± 29 | 7410 ± 72 | 0.17 ± 0.004 | 17 ± 2 | 95800 ± 4920 | 5.6 | 2.96 ± 0.04 |
| αP272A | 48 ± 3 | 139 ± 14 | 2.9 | 84 ± 2 | 7100 ± 171 | 85 | 119 ± 9 | 475 ± 28 | 0.25 ± 0.024 | 40100 ± 1140 | 461 ± 12 | 87 ± 3.35 | 24 ± 1 | 130000 ± 1780 | 5.4 | −0.70 ± 0.05 |
| αP272V | 168 ± 29 | 1620 ± 298 | 9.6 | 109 ± 4 | 24600 ± 615 | 226 | NA | NA | – | 32400 ± 1240 | 1010 ± 24 | 32 ± 1.45 | 22 ± 1 | 107000 ± 2200 | 4.9 | −0.11 ± 0.06 |
| αP272L | 12 ± 8 | 228 ± 162 | 19 | NA | NA | – | NA | NA | – | 60.1 ± 1.9 | 1420 ± 26 | 0.042 ± 0.002 | 19 ± 2 | 110000 ± 4490 | 5.8 | 3.77 ± 0.05 |
| αP272I | 40 ± 12 | 478 ± 168 | 12 | 25 ± 1 | 8200 ± 363 | 328 | 18 ± 2 | 3850 ± 391 | 0.005 ± 0.0007 | 3410 ± 152 | 1030 ± 14 | 3.3 ± 0.15 | 31 ± 1 | 99400 ± 1710 | 3.2 | 1.22 ± 0.06 |
| αP272S | 153 ± 27 | 1120 ± 224 | 7.3 | 72 ± 2 | 18300 ± 500 | 254 | 69 ± 6 | 2450 ± 125 | 0.028 ± 0.0028 | 13200 ± 391 | 917 ± 12 | 14 ± 0.47 | 22 ± 1 | 120000 ± 1870 | 5.5 | 0.36 ± 0.04 |
Kinetic parameters and error estimates are derived from global fitting of kinetic scheme to data obtained over a wide range of ACh concentrations (Materials and methods). Units are μM−1s−1 for the association rate constants, s−1 for all others. Gating equilibrium constants (Θ) are ratios of channel opening (β) to closing rate (α) constants. NA, state is not included in the kinetic scheme. Free energy change (ΔG°) = RTln(Θmutant/Θwild-type), where R is gas constant (1.987 cal/K·mol) and T is absolute temperature (295°K).