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Steady-state activation and modulation of the synaptic-type α1β2γ2L GABAA receptor by combinations of physiological and clinical ligands.
Germann AL
,
Pierce SR
,
Senneff TC
,
Burbridge AB
,
Steinbach JH
,
Akk G
.
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The synaptic α1β2γ2 GABAA receptor is activated phasically by presynaptically released GABA. The receptor is considered to be inactive between synaptic events when exposed to ambient GABA because of its low resting affinity to the transmitter. We tested the hypothesis that a combination of physiological and/or clinical positive allosteric modulators of the GABAA receptor with ambient GABA generates measurable steady-state activity. Recombinant α1β2γ2L GABAA receptors were expressed in Xenopus oocytes and activated by combinations of low concentrations of orthosteric (GABA, taurine) and allosteric (the steroid allopregnanolone, the anesthetic propofol) agonists, in the absence and presence of the inhibitory steroid pregnenolone sulfate. Steady-state activity was analyzed using the three-state cyclic Resting-Active-Desensitized model. We estimate that the steady-state open probability of the synaptic α1β2γ2L GABAA receptor in the presence of ambient GABA (1 μmol/L), taurine (10 μmol/L), and physiological levels of allopregnanolone (0.01 μmol/L) and pregnenolone sulfate (0.1 μmol/L) is 0.008. Coapplication of a clinical concentration of propofol (1 μmol/L) increases the steady-state open probability to 0.03. Comparison of total charge transfer for phasic and tonic activity indicates that steady-state activity can contribute strongly (~20 to >99%) to integrated activity from the synaptic GABAA receptor.
Figure 1. Resting‐Active‐Desensitized (RAD) model. The kinetic scheme is shown with two binding sites for agonist X. The receptor can occupy a resting (R), active (A), or desensitized (D) state. The active and desensitized states have high affinity to the agonist while the resting state has low affinity to the agonist. The parameter L (=R/A) describes the equilibrium between the resting and active states, and the parameter Q (=A/D) describes the equilibrium between the active and desensitized states. The parameter KX is the equilibrium dissociation constant for X in the resting receptor. The parameter c
XKX is the equilibrium dissociation constant for X in the active receptor, and the parameter d
X
c
XKX is the equilibrium dissociation constant for X in the desensitized receptor.
Figure 2. Peak and steady‐state activation of the α1β2γ2L receptor by GABA. Panel A shows sample current traces for receptors activated by 1, 10, or 100 μmol/L GABA. The dashed lines show the steady‐state current levels as determined by exponential fitting of the decay phase. Panel B shows the concentration‐response relationships for peak and steady‐state currents. The data points show mean ± SD from 5 to 8 cells per concentration. The curves were fitted with equation 4 (peak data) and 1 (steady‐state data). The best‐fit parameters for peak currents are: KGABA = 16 ± 3 μmol/L and c
GABA = 0.0042 ± 0.0003. The curve for steady‐state currents was fitted using the KGABA and cGABA values constrained to those obtained in fitting the peak currents. The best‐fit value for Q was 0.29 ± 0.02. The value of L was constrained to 8000 (Shin et al. 2017), the number of binding sites for GABA was constrained to 2 (Amin and Weiss 1993), and the value of d
GABA was assumed to be 1 (see text for discussion).
Figure 3. Peak activation of the α1β2γ2L receptor by taurine. Panel A shows sample current traces for receptors activated by 0.1 or 10 mmol/L taurine. Both traces are from the same cell. Panel B shows responses to 30 mmol/L taurine and 1 mmol/L GABA + 50 μmol/L propofol (Popen ~1) from the same cell. Panel C shows the taurine concentration‐response relationship for peak currents. The curve was fitted with equation 4(3), yielding estimates for Ktaurine of 5.1 ± 1.2 mmol/L and for c
taurine of 0.0075 ± 0.0006. The value of L was constrained to 8000 and the number of binding sites for taurine was constrained to 2.
Figure 4. Receptor inhibition by the steroid pregnenolone sulfate. Panel A shows a sample current trace demonstrating the effect of 0.1 and 2 μmol/L pregnenolone sulfate (PS) on steady‐state current from receptors activated by 1 mmol/L GABA. Panel B shows the PS concentration‐response relationship for receptors activated by 1 mmol/L GABA. The data points show mean ± SD from 4 to 5 cells per concentration. The curve was fitted with equation (6), yielding a Q of 0.21 ± 0.02, KPS of 1.9 ± 1.5 μmol/L, and d
PS of 0.11 ± 0.06. The number of binding sites for PS was held at 1 and c
PS was assumed to be 1 (see text). The term LΓ
GABA was constrained to 0.08. The inset more clearly demonstrates the incomplete block at high concentrations of PS. Fitting the concentration‐response data to the Hill equation yielded an IC50 of 0.25 ± 0.05 μmol/L.
Figure 5. Modulation of steady‐state current by propofol. Panel A shows sample current traces for receptors activated by 1 mmol/L GABA and exposed to 0.2 or 2 μmol/L propofol (PRO). Coapplication of propofol increases the steady‐state current level. Panel B shows the propofol concentration‐response relationship for modulation of steady‐state open probability in the presence of 1 mmol/L GABA. The data points show mean ± SD from 5 to 7 cells. The curve was fitted with equation 7, yielding a KPRO of 1.2 ± 0.4 μmol/L for the active state and a d
PRO (ratio of the equilibrium dissociation constants of the active and desensitized states) of 1.17 ± 0.01, with the number of binding sites constrained to 4. The fitted value of Q was 0.24 ± 0.01. The term LΓ
GABA was constrained to 0.08.
Figure 6. Steady‐state activation of the α1β2γ2L receptor by combinations of agonists. The graph shows the observed and predicted POpen of steady‐state responses in the presence of GABA and various combinations of allopregnanolone (3α5αP), pregnenolone sulfate (PS), and propofol (PRO). The predicted values were determined using equation (10) modified to reflect the agonistic effects of GABA, 3α5αP, and propofol, and the effects of PS and propofol on the equilibrium between Active and Desensitized states. The small symbols show data from individual cells. The large symbols show mean ± SD for each agonist combination. The solid line gives the linear fit to all data points (R
2 = 0.85, P < 0.0001). The dashed line shows ideal agreement between predicted and experimental POpen.
Figure 7. Predicted steady‐state and peak currents in the presence of combinations of GABAergic drugs. Panels A and B show steady‐state currents predicted using equation 10. In all simulations, the α1β2γ2L receptor was exposed to 1 μmol/L GABA and 10 μmol/L taurine, whereas the concentration of 3α5αP varied between 0.01 and 0.1 μmol/L (Panel A), or 1 nmol/L and 10 μmol/L (Panel B) to illustrate the full concentration‐response relationship. The simulations were done in the absence of propofol (PRO; black line) or in the presence of 0.1 (red line), 0.3 (green line), or 1 μmol/L propofol (blue line). The effect of coapplication of 0.1 μmol/L PS on steady‐state current is shown as dashed lines, color‐coded for the presence of propofol. Panel C shows the predicted peak currents in the presence of 1 μmol/L GABA and 10 μmol/L taurine combined with 1 nmol/L–10 μmol/L 3α5αP and 0–1 μmol/L propofol.
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