Inhibition and enhancement (+bicuculline) by para-substituted propofol analogs of α1β3γ2 GABAAR photolabeling by [3H]azietomidate and [3H]R-mTFD-MPAB
When drugs only inhibited photolabeling, data were fit to eq. 1 (Materials and Methods) to determine IC50, the total drug concentration producing 50% inhibition of GABAAR photolabeling, and Hill coefficients (nH) and, when indicated, to eq. 2, a two-site model. For inhibition in the presence of bicuculline, nH was set to 1. When enhancement was seen in the presence of bicuculline, data were fit to eqs. 3A or 3B, as noted in the figure legends, to determine EC50 and IC50 values for 50% maximal enhancement and inhibition. Parameters [best fit ± S.E. or 95% confidence interval (CI)] were determined from fits of data from N independent experiments from two or more GABAAR purifications. As bicuculline and GABA experiments were paired, their values of N are the same.
| Drug (NR-mTFD-MPAB, NAziET) | Ratio IC50(AziEt)/IC50(TFD-MPAB)a | [3H]R-mTFD-MPAB IC50 {95% CIs} (nH) | [3H]R-Azietomidate IC50 {95% CIs} (nH) | ||
|---|---|---|---|---|---|
| +GABA | +GABA | +Bicuculline EC50/IC50 | +GABA | +Bicuculline EC50/IC50 | |
| μM | μM | μM | μM | ||
| Etomidate (5, 3) | 0.01 ± 0.002 | 430 {330, 580} (1.5 ± 0.3) | 3.0 ± 0.7/440 ± 50 | 2.0 {1.8, 2.3} (1.00 ± 0.06) | 5.5 {3.4, 9} |
| R-mTFD-MPAB (5, 3) | 45 ± 12 | 0.7 {0.6, 0.8} (1.2 ± 0.1) | 3.1 {1.5, 5.0} | 38 {29, 50} (1.0 ± 0.1) | 1.1 ± 0.3/18 ± 3 |
| Propofol (6, 6)b | 0.2 ± 0.04 | 44 {36, 53} (1.1 ± 0.1) | 2.5 ± 0.8/57 ± 11 | 7.8 {6, 10} (1.1 ± 0.1) | 29 {12, 97} |
| 4-Cl-propofol (4, 4)b | 0.25 ± 0.08 | 20 {14, 28} (1.2 ± 0.2) | 1.5 ± 0.4/19 ± 3 | 4.9 {3.4, 7.0} (0.9 ± 0.2) | 20 {7, 90} |
| 4-acetyl-propofol (4, 3)c | 1.5 ± 0.4 | 32 {25, 41} (0.9 ± 0.1) | 250 {100, 790} | 48 {38, 61} (1.1 ± 0.3) | 17 ± 2/41 ± 4 |
| 4-(tert-butyl)-propofol (4, 3)c | 8 ± 3 | 17 {13, 23} (1.0 ± 0.1) | 120 {68, 220} | 134 {100, 226} (1.2 ± 0.2) | 5.1 ± 0.7/72 ± 8 |
| 4-benzyl-propofol (2, 3) | 5 ± 1 | 5.7 {4.7, 6.9} (1.3 ± 0.1) | ND | 30 {21, 53} (0.8 ± 0.1) | ND |
| 4-benzoyl-propofol (6, 5)d | 200 ± 140e | 21 {11, 38} (0.27 ± 0.04) eq. 2 IC50H = 0.5 ± 0.3 (45% ± 4%)/IC50L = 330 ± 120 | eq. 3A: 1.0 ± 0.9/156 ± 54 Bmax= 61% ± 5% eq. 3B: 1.0 ± 0.8/158 ± 54 Bmax= 87% ± 5% | 99 {82, 120} (2.0 ± 0.3) | 0.6 ± 0.2/89 ± 8 Bmax = 81% ± 3%f |
| 4-[hydroxyl(phenyl)methyl]-propofol (3,3) R-isomer (2) S-isomer (2)d | 16 ± 5 | 10 {8.5, 12} (1.1 ± 0.1) 12 ± 1; 7 ± 1 | 30 {15, 63} ND; ND | 157 ± 33 (1.1 ± 0.3) ND; ND | 8 ± 2/41 ± 8 ND; ND |
ND, not determined;
↵a For the ratio of IC50 values (+GABA), uncertainties were determined by propagation of error from the individual parameter uncertainties.
↵b Data from Fig. 3.
↵c Data from Fig. 4.
↵d Data from Fig. 5, with parameters for 4-benzoyl-propofol inhibition of [3H]R-mTFD-MPAB (+GABA) also fit to a two-site model, eq. 2.
↵e The ratio calculated for the high-affinity component (IC50H) of [3H]R-mTFD-MPAB inhibition.
↵f Parameters based upon fit of the data to eq. 3A with nI = 2, the Hill coefficient determined for inhibition in the presence of GABA.