Kinetic Analysis of Wild-Type, α V46, and α V132 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 | - |
| αE45A | 296 ± 37 | 5300 ± 770 | 18 | 173 ± 6 | 19500 ± 447 | 113 | NA | NA | - | 28900 ± 504 | 13700 ± 153 | 2.1 ± 0.04 | 21 ± 1 | 110000a | 5.2 | 1.48 ± 0.05 |
| αV46G | NA | NA | - | NA | NA | - | NA | NA | - | 115 ± 12 | 7870 ± 168 | 0.015 ± 0.002 | 40 ± 8 | 128000 ± 8670 | 3.2 | 4.40 ± 0.12 |
| αV46A | 111 ± 55 | 3100 ± 1560 | 28 | 55 ± 31 | 9490 ± 5130 | 173 | 51 ± 7 | 6560 ± 320 | 0.008 ± 0.001 | 155 ± 5 | 2740 ± 51 | 0.057 ± 0.002 | 26 ± 1 | 95500 ± 1990 | 3.7 | 3.60 ± 0.05 |
| αV46L | 328 ± 27 | 3140 ± 319 | 9.6 | 267 ± 11 | 7130 ± 144 | 27 | NA | NA | - | 40200 ± 636 | 4880 ± 81 | 8.2 ± 0.19 | 83 ± 8 | 120000 ± 4240 | 1.4 | 0.69 ± 0.05 |
| αV46I | 434 ± 39 | 4740 ± 486 | 11 | 217 ± 5 | 14300 ± 204 | 66 | NA | NA | - | 36500 ± 412 | 5360 ± 54 | 6.8 ± 0.10 | 17 ± 1 | 92900 ± 1960 | 5.5 | 0.80 ± 0.05 |
| αV132G | 718 ± 384 | 5180 ± 3000 | 7.2 | 222 ± 9 | 7860 ± 296 | 35 | NA | NA | - | 12900 ± 123 | 7830 ± 51 | 1.7 ± 0.02 | 19 ± 1 | 82600 ± 2110 | 4.4 | 1.63 ± 0.04 |
| αV132A | 324 ± 31 | 3040 ± 401 | 9.4 | 267 ± 11 | 10200 ± 255 | 38 | 77 ± 34 | 6840 ± 1030 | 0.011 ± 0.005 | 26600 ± 313 | 5430 ± 61 | 4.9 ± 0.08 | 18 ± 1 | 98700 ± 3130 | 5.5 | 0.99 ± 0.03 |
| αV132L | 97 ± 65 | 2030 ± 1430 | 21 | 107 ± 16 | 32300 ± 4510 | 302 | NA | NA | - | 2540 ± 91 | 2640 ± 30 | 0.96 ± 0.04 | 22 ± 1 | 99300 ± 2420 | 4.5 | 1.94 ± 0.06 |
| αV132I | 86 ± 44 | 3220 ± 1810 | 37 | 194 ± 69 | 40000 ± 13900 | 206 | NA | NA | - | 2280 ± 73 | 1580 ± 20 | 1.4 ± 0.05 | 24 ± 1 | 95700 ± 1910 | 4.0 | 1.71 ± 0.05 |
| 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 | - |
| αE45A | 296 ± 37 | 5300 ± 770 | 18 | 173 ± 6 | 19500 ± 447 | 113 | NA | NA | - | 28900 ± 504 | 13700 ± 153 | 2.1 ± 0.04 | 21 ± 1 | 110000a | 5.2 | 1.48 ± 0.05 |
| αV46G | NA | NA | - | NA | NA | - | NA | NA | - | 115 ± 12 | 7870 ± 168 | 0.015 ± 0.002 | 40 ± 8 | 128000 ± 8670 | 3.2 | 4.40 ± 0.12 |
| αV46A | 111 ± 55 | 3100 ± 1560 | 28 | 55 ± 31 | 9490 ± 5130 | 173 | 51 ± 7 | 6560 ± 320 | 0.008 ± 0.001 | 155 ± 5 | 2740 ± 51 | 0.057 ± 0.002 | 26 ± 1 | 95500 ± 1990 | 3.7 | 3.60 ± 0.05 |
| αV46L | 328 ± 27 | 3140 ± 319 | 9.6 | 267 ± 11 | 7130 ± 144 | 27 | NA | NA | - | 40200 ± 636 | 4880 ± 81 | 8.2 ± 0.19 | 83 ± 8 | 120000 ± 4240 | 1.4 | 0.69 ± 0.05 |
| αV46I | 434 ± 39 | 4740 ± 486 | 11 | 217 ± 5 | 14300 ± 204 | 66 | NA | NA | - | 36500 ± 412 | 5360 ± 54 | 6.8 ± 0.10 | 17 ± 1 | 92900 ± 1960 | 5.5 | 0.80 ± 0.05 |
| αV132G | 718 ± 384 | 5180 ± 3000 | 7.2 | 222 ± 9 | 7860 ± 296 | 35 | NA | NA | - | 12900 ± 123 | 7830 ± 51 | 1.7 ± 0.02 | 19 ± 1 | 82600 ± 2110 | 4.4 | 1.63 ± 0.04 |
| αV132A | 324 ± 31 | 3040 ± 401 | 9.4 | 267 ± 11 | 10200 ± 255 | 38 | 77 ± 34 | 6840 ± 1030 | 0.011 ± 0.005 | 26600 ± 313 | 5430 ± 61 | 4.9 ± 0.08 | 18 ± 1 | 98700 ± 3130 | 5.5 | 0.99 ± 0.03 |
| αV132L | 97 ± 65 | 2030 ± 1430 | 21 | 107 ± 16 | 32300 ± 4510 | 302 | NA | NA | - | 2540 ± 91 | 2640 ± 30 | 0.96 ± 0.04 | 22 ± 1 | 99300 ± 2420 | 4.5 | 1.94 ± 0.06 |
| αV132I | 86 ± 44 | 3220 ± 1810 | 37 | 194 ± 69 | 40000 ± 13900 | 206 | NA | NA | - | 2280 ± 73 | 1580 ± 20 | 1.4 ± 0.05 | 24 ± 1 | 95700 ± 1910 | 4.0 | 1.71 ± 0.05 |
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).
Value was constrained.