Article Figures & Data
Figures
- Fig. 1.
Constitutive activity modifies receptor potentiation by propofol. (A) Sample current responses from α1β3 wild-type and α1β3(Y143W) mutant receptors. To measure potentiation, the drug concentrations were 0.3 μM GABA and 1 μM propofol for the wild type and 0.02 μM GABA and 0.1 μM propofol for α1β3(Y143W). For each receptor, sample traces showing responses to saturating GABA [dashed lines; 30 μM for the wild type, 10 μM for α1β3(Y143W)] and 300 μM picrotoxin are also shown. Recovery from picrotoxin-elicited block of constitutive activity in the mutant receptor was complete after a 10-minute washout. Only the first 100 seconds of the trace are shown. (B) Summary of potentiation data from α1β3 and α1β3(Y143W) receptors. The graph shows data from each cell tested (open circles) and the mean ± S.D. (filled circles and error bars), normalized to responses to saturating GABA. The number of cells tested was six for the wild type and eight for the mutant. (C) Potentiation of GABA-activated α1β3 receptors by propofol was simulated at GABA and propofol concentrations eliciting responses equal to 5% of the response to saturating GABA. The potentiation response ratio was calculated as the ratio of the response to the combination of GABA plus propofol to that to GABA alone. The simulated potentiation response ratio decreases as the level of constitutive activity increases. The dashed line shows the response to GABA alone (1). PRO, propofol; PTX, picrotoxin.
- Fig. 2.
The simulated relationship between the properties of a potentiator and the extent of potentiation. (A) Simulated concentration-response curves for potentiation of EC5 GABA-activated wild-type β2α1γ2L+β2α1 receptors by propofol. The KPRO was 21 μM and cPRO was 0.22 (Shin et al., 2018). To mimic potentiation by different modulators, the number of propofol binding sites was varied from two to six (bottom to top). (B) Simulated potentiation response ratios for receptors activated by EC5 GABA and potentiated by propofol at a concentration that, when applied alone, elicits a response with the same peak current (EC5%), at different Po,const. The simulations show that the potentiation response ratio is identical for all imposed values of NPRO, suggesting that different allosteric potentiators potentiate the receptor by the same degree as long as the direct activating response to the potentiator is constant. The dashed line shows the response to GABA alone (1). NPRO, number of propofol binding sites.
- Fig. 3.
The relationship between constitutive activity, direct activation, and the extent of potentiation. (A) Sample current traces from the wild-type β2α1γ2L+β2α1 receptor. The receptors were exposed to GABA (EC9–10), propofol (EC6), or pentobarbital (EC6) and the combination of GABA plus propofol or GABA plus pentobarbital. (B) Sample current traces from the β2α1γ2L+β2α1(L263S) receptor. The receptors were exposed to GABA (EC12–13), propofol (EC6), or pentobarbital (EC8.5) and the combination of GABA plus propofol or GABA plus pentobarbital. (C) Sample current traces from the β2α1(L263S)γ2L+β2α1(L263S) receptor. The receptors were exposed to GABA (EC11–13), propofol (EC7), or pentobarbital (EC6) and the combination of GABA plus propofol or GABA plus pentobarbital. (A–C) Responses to 300 μM picrotoxin and saturating GABA (the dashed trace) from a same cell are shown to demonstrate differences in constitutive activity in the wild-type and mutant receptors. The mean Po,const is 0.0001 in the wild-type receptor (Akk et al., 2018), 0.014 in β2α1γ2L+β2α1(L263S) (Akk et al., 2018), and 0.1 in β2α1(L263S)γ2L+β2α1(L263S) (Shin et al., 2018). (D) Summary of potentiation data from β2α1γ2L+β2α1, β2α1γ2L+β2α1(L263S), and β2α1(L263S)γ2L+β2α1(L263S) receptors. The graph shows data from each cell tested (open circles) and the mean ± S.D. (filled circles and error bars), normalized to responses to saturating GABA. The number of cells tested was six for the wild type tested with propofol or pentobarbital. In β2α1γ2L+β2α1(L263S), the number of cells was six for propofol and pentobarbital. In β2α1(L263S)γ2L+β2α1(L263S), the number of cells was five for propofol and six for pentobarbital. The findings indicate that 1) apparent potentiation is greater in the receptor with less constitutive activity (wild-type) and 2) the extent of potentiation is similar for propofol and pentobarbital. PEB, pentobarbital; PRO, propofol; PTX, picrotoxin.
- Fig. 4.
Constitutive activity modifies the predicted isoboles of additivity. The figure shows simulated isobolograms for activation of the α1β3 receptor by GABA, propofol, and combinations of GABA plus propofol with the target open probability of 0.5 at different levels of constitutive activity. The straight line in each panel represents the linear isobole traditionally associated with additive effects of the two drugs. The data points show the simulated isoboles based on energetic additivity using the affinity and efficacy data published previously (Eaton et al., 2016). The KGABA was 1.6 μM, cGABA was 0.02, KPRO was 4.7 μM, and cPRO was 0.24. The numbers of binding sites were constrained to two and five for GABA and propofol, respectively. The data indicate that as Po,const increases, the predicted isoboles approach linearity.
- Fig. 5.
Constitutive activity is predicted to affect apparent synergy. Constitutive open probability affects the simulated ratio of [propofol]linear over [propofol]CTM, defined as the displacement between the linear isobole of additivity and the isobole predicted by the concerted transition model. A ratio greater than unity (shown with a dashed line) indicates that energetic additivity predicts a synergistic interaction between GABA and propofol. The simulations indicate that the apparent degree of synergy decreases as the difference between the target (Po,target) and basal open probabilities (Po,const) decreases.
- Fig. 6.
Constitutive activity affects the positions of the concentration-response relationships for direct activation and potentiation. (A) Simulated propofol EC50 values for direct activation and potentiation of receptors activated by EC5 GABA at different levels of constitutive activity. The simulations were done using the affinity and efficacy values determined previously for the wild-type α1β3 receptor (Eaton et al., 2016). The KGABA was 1.6 μM, cGABA was 0.02, KPRO was 4.7 μM, and cPRO was 0.24. The numbers of binding sites were constrained to two and five for GABA and propofol, respectively. (B) Simulated EC50 values for activation by GABA in the absence and presence of propofol at concentrations that elicited a response that was 5% of the response to saturating GABA at the given Po,const. (C) Concentration-response curves for GABA in the absence and presence of 4 μM (wild-type) or 0.5 μM propofol [α1β3(Y143W)]. The data points (black circles: wild type; blue squares: mutant) show the mean ± S.D. from 5 to 12 cells. The solid-line curves show simulations using the following averaged fitting parameters: wild-type, GABA plus propofol (black line): Ymin = 0.09, EC50 = 0.19 μM, nH = 1.16; and α1β3(Y143W), GABA plus propofol (blue line): Ymin = 0.08, EC50 = 0.15 μM, nH = 1.01. The dashed black (wild-type) and blue (mutant) lines show the concentration-response data for GABA alone with EC50s of 1.4 μM for the wild type and 0.18 μM for the mutant from a prior study (Eaton et al., 2016).
In this issue
Effect of Constitutive Activity on Potentiation
