Article Figures & Data
Figures
- Fig. 1.
Low concentrations of propofol potentiate GABA-induced currents but do not directly activate the receptors. Effects of propofol application on the currents recorded by two-electrode voltage clamp from oocytes expressing the α1V297C (A) and α1L301C (B) mutants. Bars above the current traces indicate the reagent applied and the duration of application. Time between current traces is 3–5 min. Holding potential, −80 mV. Note that propofol potentiates the GABA-induced currents but does not elicit a current when applied by itself at these concentrations. Also, the propofol effects wash out so that the final GABA test current is similar to the initial current.
- Fig. 2.
The effect of coapplication of 0.5 mmpCMBS− with a potentiating concentration of propofol on the subsequent GABA-induced currents. Currents recorded by two-electrode voltage clamp from oocytes expressing the α1V297C (A), α1I302C (B), α1E303C (C), and α1F304C (D) mutants. Bars above the current traces indicate the reagent applied and the duration of application. Time between current traces is 3–5 min. Holding potential, −80 mV. The propofol concentration was 0.1 μm inA and 0.5 μm in B–D. Note that in A and D the subsequent GABA-induced currents were potentiated; in B the subsequent currents were inhibited, and in B–D the subsequent currents were unchanged.
- Fig. 3.
The irreversible effect of a 1 min application of 0.5 mm pCMBS− applied in the presence of a potentiating concentration of propofol on subsequent GABA-induced currents of wild-type and mutant GABAA receptors. For each mutant an EC50 GABA concentration was used for the test pulses. Black bars indicate effects that are statistically significantly different from the effect on wild type by a one-way ANOVA. A negative effect indicates inhibition, and a positive effect indicates potentiation of subsequent GABA currents. The mean and SEM values are shown. For each mutant, the effects on three to six oocytes are averaged.
- Fig. 4.
The lack of effect of a 1 min application of 0.5 mm pCMBS− applied in the presence of 2,6 di-tert-butyl-phenol (2,6 dtbp), a nonanesthetic analog of propofol, on GABA-induced currents from oocytes expressing α1V297C (A) and α1A305C (B). The concentration of 2,6 di-tert-butyl-phenol was similar to a potentiating propofol concentration, 0.5 μm in A, and similar to an activating propofol concentration, 50 μm inB. Time between current traces is 3–5 min. Holding potential, −80 mV. Note that the initial and final GABA currents are similar.
- Fig. 5.
The effect of coapplication of 0.5 mmpCMBS− with 50 μm propofol, a directly activating concentration, on the subsequent GABA-induced currents. Currents recorded by two-electrode voltage clamp from oocytes expressing the α1I302C (A), α1E303C (B), and α1A305C (C) mutants. Bars above the current traces indicate the reagent applied and the duration of application. Time between current traces is 3–5 min. Holding potential, −80 mV. Note that the subsequent GABA-induced currents evoked by both saturating and EC50 GABA concentrations are inhibited. The data in A and C suggest that pCMBS− modification reduced the GABA EC50.
- Fig. 6.
The irreversible effect of a 1 min application of 0.5 mm pCMBS− applied in the presence of 50 μm propofol, a directly activating concentration, on subsequent GABA-induced currents of wild-type and mutant GABAA receptors. Saturating GABA concentrations were used for the test pulses. Black bars indicate effects that are statistically significantly different from the effect on wild type by a one-way ANOVA. A negative effect indicates inhibition, and a positive effect indicates potentiation of subsequent GABA currents. The mean and SEM values are shown. For each mutant, the effects on three to seven oocytes are averaged.
Tables
- Table 1.
Extent of potentiation and direct activation of the α1 M3 Cys substitution mutants by propofol
Mutant Percentage of potentiation1-a Percentage of direct activation1-b V292C 13 ± 4 (4) 26 ± 5 (3) C293, WT 46 ± 12 (7) 41 ± 5 (5) Y294C 102 ± 46 (3) 65 ± 8 (3) A295C 28 ± 14 (3) 24 ± 7 (6) F296C 71 ± 20 (3) 33 ± 11 (4) V297C 57 ± 15 (5) 50 ± 7 (6) F298C 22 ± 5 (4) 49 ± 7 (4) S299C 13 ± 5 (3) 88 ± 3 (4) A300C 34 ± 4 (4) 146 ± 20 (10) L301C 51 ± 5 (3) 57 ± 6 (3) I302C 27 ± 16 (4) 25 ± 11 (4) E303C 40 ± 6 (6) 63 ± 5 (6) F304C 43 ± 11 (3) 65 ± 4 (3) A305C 42 ± 12 (5) 51 ± 7 (4) T306C 39 ± 20 (4) 85 ± 6 (5) V307C 33 ± 9 (6) 41 ± 2 (4) ↵F1-a The percentage of potentiation was {(IGABA + Propofol/IGABA) − 1}*100. Propofol (0.5 μm) was used for all mutants except V297C, where 0.1 μm propofol was used. EC50 GABA concentration was used. Mean ± SEM is given. Number of oocytes is indicated in parentheses.
↵F1-b The percentage of direct activation was calculated as {(IPropofol/IGABA) − 1}*100. The propofol concentration was 50 μm in all cases. The GABA concentration was 100 μm.
- Table 2.
Summary of α1 M3 segment Cys substitution mutant accessibility to pCMBS− applied in the presence of the indicated reagents2-a
Mutant Resting state2-b GABA2-b Diazepam2-c Potentiating propofol Activating propofol A291C ↓ ↓ ↓ nt nt V292C C293, WT Y294C ↓ ↓ ↓ ↓ ↓ A295C F296C ↓ ↑ V297C ↑ F298C ↑ ↑ S299C ↓ A300C ↑ L301C ↑ ↑ I302C ↓ ↓ E303C ↓ ↓ F304C ↑ A305C ↓ T306C V307C ↵F2-a Symbols indicate the effect of pCMBS− modification on the subsequent GABA-induced currents. ↓ inhibition; ↑ potentiation; nt, not tested; absence of a symbol indicates that there was no effect of pCMBS−application.
↵F2-b Data from Williams and Akabas (1999).
↵F2-c Data from Williams and Akabas (2000,2001).
