Abstract
Activation of GABAA receptors consisting of α4, β2 (or β3), and δ subunits is a major contributor to tonic inhibition in several brain regions. The goal of this study was to analyze the function of the α4β2δ receptor in the presence of GABA and other endogenous and clinical activators and modulators under steady-state conditions. We show that the receptor has a high constitutive open probability (~0.1), but is only weakly activated by GABA that has a maximal peak open probability (POpen,peak) of 0.4, taurine (maximal POpen,peak = 0.4), or the endogenous steroid allopregnanolone (maximal POpen,peak = 0.2). The intravenous anesthetic propofol is a full agonist (maximal POpen,peak = 0.99). Analysis of currents using a cyclic three-state Resting-Active-Desensitized model indicates that the maximal steady-state open probability of the α4β2δ receptor is ~0.45. Steady-state open probability in the presence of combinations of GABA, taurine, propofol, allopregnanolone and/or the inhibitory steroid pregnenolone sulfate closely matched predicted open probability calculated assuming energetic additivity. The results suggest that the receptor is active in the presence of physiological concentrations of GABA and taurine, but, surprisingly, that receptor activity is only weakly potentiated by propofol.
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Introduction
Activation of the Cl− permeable GABAA receptor contributes to cellular inhibition. The two principal types of the GABAA receptor in the central nervous system are the synaptic receptor that is activated phasically by presynaptically released GABA, and the extrasynaptic receptor that is activated tonically by ambient GABA. Native GABAA receptors in the brain are additionally exposed to a number of endogenous GABAergic agents including taurine (2-aminoethanesulfonic acid) and potentiating and inhibitory neurosteroids, that can amplify or inhibit the response to the transmitter. Furthermore, both the synaptic and extrasynaptic GABAA receptors are activated and modulated by clinically used GABAergic sedatives and anesthetics such as propofol and etomidate1,2. The two types of receptors differ in their subunit composition; synaptic receptors comprise α1-3, β2-3, and γ2 subunits, whereas extrasynaptic receptors typically consist of α4, β2-3, and δ subunits.
With few exceptions3,4, previous functional studies of the α4βδ receptor have concentrated on recording peak current responses, i.e., maximal responses to short-duration applications of one or more agonists. It may be argued that this approach does not accurately reflect native conditions, which can be characterized as essentially infinite-duration exposure to a low concentration of GABA with slowly developing changes in the concentrations of other endogenous agonists and modulators and, if so administered, GABAergic clinical agents. This discrepancy between typical experimental and the presumed in vivo conditions makes prediction of normal behavior of the native extrasynaptic receptor and properties of tonic inhibition challenging.
We recently described derivation and properties of a three-state Resting-Active-Desensitized (“RAD”) model5. The model (Fig. 1), which was initially employed to quantitatively describe steady-state activity in the synaptic-type α1β2γ2L GABAA receptor activated by a single agonist, could also be used to accurately predict steady-state activity in the presence of multiple potentiating and inhibitory agents. Here, we have employed the RAD model to investigate the properties of the human α4β2δ expressed in Xenopus oocytes. A major goal of the study was to elucidate steady-state activity in the presence of multiple endogenous and clinical activating (GABA, taurine, propofol, allopregnanolone) and inhibitory (pregnenolone sulfate) agents to predict the behavior of the extrasynaptic GABAA receptor under conditions mimicking the native pharmacological environment.
We show that the receptor has a constitutive open probability of ~0.1 and a steady-state open probability (POpen,S.S.) near 0.3 in the presence of saturating GABA. The receptor is potently activated by the transmitter GABA, the orthosteric agonist taurine, and the allosteric agonists propofol and allopregnanolone (3α5αP). An agreement between the POpen,S.S. calculated using equations derived from the RAD model and the POpen,S.S. observed experimentally upon coapplication of combinations of GABA, taurine, propofol, 3α5αP, and the inhibitory steroid pregnenolone sulfate (PS) indicates that the drugs act energetically additively.
Results
Activation and desensitization by the orthosteric agonists GABA and taurine
The oocytes expressing α4β2δ GABAA receptors respond to application of GABA with inward current. Concentration-response measurements carried out in the presence of 0.3–1000 nM GABA yielded an EC50 of 20 ± 10 nM and a Hill coefficient of 0.80 ± 0.09 (mean ± S.D.; n = 6 cells). Sample current traces in the presence of GABA are shown in Fig. 2A.
To convert the raw current amplitudes to units of open probability (POpen), we compared the response to saturating GABA (0.3 μM) to the response to 300 μM picrotoxin (PTX) and the response to 10 μM GABA + 50 μM propofol. The details of this approach have been reported previously6,7. Blockade of activity from constitutively active receptors by PTX is expected to lead to zero GABAergic activity (POpen approaching 0), and receptor activation by the combination of saturating GABA and a high concentration of propofol is expected to generate a maximal possible peak response with a POpen indistinguishable from 1. Comparison of the holding current and peak responses to PTX, GABA, and GABA + propofol, yielded an estimate of 0.13 ± 0.09 (n = 24 cells) for constitutive open probability (POpen,const), and an estimate of 0.35 ± 0.09 (n = 22 cells) for open probability in the presence of 0.3 μM GABA. Sample current responses to PTX, GABA, and GABA + propofol are given in Fig. 2B.
The activation parameters for peak responses were determined by fitting the POpen data to Eq. (1)8,9:
where [X] is the concentration of agonist X (GABA in this experiment), KX is the equilibrium dissociation constant for agonist X of the resting receptor, cX is the ratio of the equilibrium dissociation constant for X of the open receptor to KX, and NX is the number of agonist binding sites. L expresses the level of background activity, and can be calculated from constitutive activity as: (1 − POpen,const)/POpen,const.
Curve-fitting of pooled data from 6 cells to Eq. (1) yielded a KGABA of 15.7 ± 2.3 nM (best-fit parameter ± S.E. of the fit) and a cGABA of 0.45 ± 0.01. The number of GABA binding sites was held at two10. The concentration-response relationship for peak currents is given in Fig. 2C.
The data indicate that GABA is a weak agonist of the α4β2δ receptor. The binding of two GABA molecules contributes only 0.94 kcal/mol (\({{\rm{N}}}_{{\rm{GABA}}}{\rm{RT}}\times \,\mathrm{ln}({c}_{{\rm{GABA}}})\)) towards stabilization of the open state. For comparison, in the synaptic-type α1β2γ2L receptor, the binding of two GABA molecules contributes 6.4–7.5 kcal/mol of stabilization energy11,12. Thus, despite the relatively high constitutive open probability (i.e., low intrinsic energy barrier towards channel opening), the theoretical peak maximal open probability of the α4β2δ receptor in the presence of GABA, calculated as \(1/(1+{\rm{L}}{{c}_{{\rm{GABA}}}}^{{{\rm{N}}}_{{\rm{GABA}}}})\), is only 0.44. This is in agreement with previous estimates in single-channel and macroscopic studies demonstrating that GABA is a partial agonist of the α4βδ receptor13,14,15,16,17.
To analyze the desensitization properties of the α4β2δ receptor, we fitted the concentration-response relationship for steady-state currents to Eq. (2)5:
The parameter Q (=A/D) reflects the equilibrium between the active and desensitized states (Fig. 1). The other terms are as described for Eq. (1). Curve fitting of steady-state responses, using KGABA and cGABA constrained to the values determined for peak currents in the same set of cells, yielded an estimate of 0.78 ± 0.08 for Q. Thus, under steady-state conditions, the ratio of open/active vs. desensitized receptors is ~4:5.
Taurine, an endogenous sulfonic acid and a structural analog of GABA, can activate the GABAA receptor18,19,20,21. Its effects are likely mediated through interactions with the transmitter binding sites, as suggested by molecular modeling22 and the finding that the β2(Y205S) mutation in the transmitter binding site that abolishes receptor activation by GABA10 also eliminates activation of the α1β2γ2L and α4β2δ receptors by taurine (<0.2% of the response to GABA + propofol; data not shown).
Taurine concentration-response measurements on oocytes expressing the α4β2δ GABAA receptor yielded an EC50 of 9.8 ± 4.8 μM and a Hill coefficient of 0.70 ± 0.08 (n = 6 cells) for peak currents. Fitting the concentration-response data to Eq. (1) gave the estimates of Ktaurine of 10.0 ± 2.1 μM and a ctaurine of 0.47 ± 0.02. Thus, taurine and GABA have similar gating efficacies (i.e., ctaurine ≈ cGABA) on the α4β2δ receptor and maximal peak open probabilities (0.44 and 0.42, respectively). In recordings in the presence of long (190–410 s) applications of 1 mM taurine, the steady-state open probability was 0.23 ± 0.04 (n = 5 cells), yielding a calculated value of 0.52 for Q.
Activation and desensitization by the allosteric agonists propofol and 3α5αP
The propofol concentration-response relationship was obtained by exposing oocytes expressing the α4β2δ receptor to 0.2–20 μM propofol. Curve-fitting the peak response data with the Hill equation yielded an EC50 of 7.3 ± 2.0 μM and a Hill coefficient of 2.17 ± 0.65 (n = 6 cells). Fitting the pooled data to Eq. (1) gave a Kpropofol of 55.1 ± 6.6 μM and a cpropofol of 0.16 ± 0.01. The number of binding sites for propofol was constrained to 4. Thus, the binding of propofol to the α4β2δ receptor contributes 4.3 kcal/mol towards stabilization of the open state. The predicted maximal peak POpen in the presence of propofol is ~0.99. Sample current responses and the concentration-response curves are given in Fig. 3.
Curve-fitting the peak response data recorded in the presence of 0.01–3 μM 3α5αP yielded an EC50 of 0.23 ± 0.10 μM and a Hill coefficient of 1.17 ± 0.30 (n = 6 cells). Analysis of the peak currents using Eq. (1) gave a K3α5αP of 0.21 ± 0.04 μM and a c3α5αP of 0.68 ± 0.01 with the number of binding sites for 3α5αP held at 2. Sample current responses and the concentration-response relationships are shown in Fig. 4.
To determine receptor desensitization properties in the presence of propofol or 3α5αP, we analyzed the steady-state currents using Eq. (2). With the K and c values constrained to the values estimated by analyzing peak responses, we obtained the estimates for Q of 1.29 ± 0.14 in the presence of propofol, and 0.89 ± 0.33 in the presence of 3α5αP. A higher value of Q is associated with reduced desensitization, i.e., a higher steady-state to peak ratio.
We recently showed that propofol enhances steady-state activity elicited by saturating GABA in the α1β2γ2L receptor23. The effect, which is observed as an increase in the apparent value of Q, was attributed to propofol having a higher affinity to the open vs. desensitized state. To determine whether an analogous mechanism underlies the higher value of Q in the α4β2δ receptor activated by propofol, we compared the potentiating effect of propofol on peak and steady-state currents elicited by saturating GABA. We reasoned that if propofol potentiates the responses by enhancing receptor open probability then the potentiating effect will be similar for peak and steady-state activity. On the other hand, if propofol additionally reduces receptor desensitization, then the potentiating effect of propofol on steady-state current should exceed that on the peak response. In five cells, coapplication of 1 μM propofol enhanced the peak response to 0.3 μM GABA to 151 ± 12% of control. Application of 1 μM propofol on steady-state response elicited by 0.3 μM GABA augmented the response to 145 ± 14% of control (n = 5 cells). We infer that within the limits of our experimental precision, propofol does not modify the equilibrium between active and desensitized states.
Modulation of steady-state current by PS
The endogenous steroid PS promotes desensitization of the synaptic-type αβγ GABAA receptor5,24,25. Here, we determined the effect of PS on the α4β2δ receptor.
The receptors were activated by a prolonged application of 0.3 μM GABA. Once steady-state response was reached, the flow was switched to GABA + PS. The concentration of PS ranged from 0.1 to 10 μM. Curve-fitting of pooled data from 5–7 cells per concentration gave an IC50 of 1.4 ± 0.3 μM and a high concentration asymptote of 50 ± 4% of control.
In the framework of the RAD model, PS inhibits receptor activity by binding with high affinity to the desensitized state and with low affinity to the resting and active states. For receptors activated by GABA, the open probability in the presence of PS is:
where KPS is the equilibrium dissociation constant of the resting and active receptors to PS, and dPS is the ratio of the equilibrium dissociation constant of the desensitized receptor to KPS. The number of sites for PS was assumed to be 1. Other terms are as described above for Eqs (1,2).
In this model, PS does not modify the intrinsic properties of the receptor (i.e., L or Q) or the parameters of receptor activation by GABA (i.e., KGABA or cGABA). Fitting the PS concentration-response data to Eq. (3) yielded a KPS of 2.6 ± 0.6 μM, and a dPS of 0.14 ± 0.02. The KPS and dPS estimates are similar to the values previously determined for the α1β2γ2L receptor (1.9 μM and 0.11, respectively)23. Sample current traces, the concentration-response data, and the fitted curve are shown in Fig. 5.
Steady-state activation in the presence of combinations of GABA, taurine, propofol, 3α5αP and/or PS
We previously showed for the α1β2γ2L GABAA receptor that steady-state activity in the presence of multiple active agents is determined by energetic additivity5,23. To verify that the same mechanism determines steady-state activity of the α4β2δ receptor, and to gain insight into receptor function in the presence of multiple endogenous and clinical activators and modulators, we measured steady-state responses in the presence of combinations of orthosteric (GABA, taurine) and allosteric activators (propofol, 3α5αP) and inhibitors (PS). The experimentally observed POpen,S.S. was compared with the predicted POpen,S.S.. The latter can be calculated using Eq. (4):
where Γpropofol is:
and Γ3α5αP is:
In practice, however, the predicted POpen,S.S. was calculated using Eq. (7):
where ΠSum is a measure of peak activation by the mixture of agonists and is related to the peak open probability as:
Equations (7) and (8) express steady-state open probability as a dependent product of peak open probability, related to it through Q and the effect of PS (KPS and dPS) on steady-state current. This approach enabled us to compensate for cell-to-cell variability in the actions of agonist mixtures.
In total, 8 combinations of drugs and drug concentrations were tested. The concentration of GABA ranged from 10 nM to 10 μM, taurine from 10 to 100 μM, propofol from 1 to 50 μM, 3α5αP from 10 to 30 nM, and PS from 0.1 to 1 μM. Not all combinations included all 5 compounds. The data from 53 cells are shown in Fig. 6. A linear fit to all data points yielded an R2 of 0.82 (P < 0.0001) with a regression slope of 0.99 ± 0.10.
Discussion
Receptors consisting of α4, β2 or β3, and δ subunits are a major extrasynaptic type of GABAA receptors in several brain regions such as the hippocampus and the thalamus26,27,28,29. Prior studies have indicated that the α4βδ receptor has a high affinity to GABA, and is only moderately desensitized during prolonged application of agonist30,31,32,33. Both properties support its presumed function to mediate tonic Cl− conductance in response to ambient GABA, and the concentration profile of ambient GABA in the brain. The α4βδ receptor is also activated by taurine, endogenous potentiating steroids, and various GABAergic sedative and anesthetic agents3,14,20,30. The overall goal of this study was to analyze the function of the α4β2δ receptor in the presence of one or more activators and modulators under steady-state conditions.
Previous work has provided evidence for two types or isoforms of receptors resulting from the expression of α4, β (β2 or β3), and δ subunits. In electrophysiological recordings, this manifests as widely different sensitivities to the agonist. The high-affinity type has a GABA EC50 at <100 nM whereas the low-affinity type has a GABA EC50 at >1 μM. In some cases, concentration-response relationships show two components in a single cell indicating that both types of the receptor can express concurrently31. The underlying reason for differing sensitivity to the agonist is not fully established. Several studies have suggested that it is due to the “promiscuous” nature of the δ subunit that allows for variability in the assembly order of subunits and stoichiometry of the surface receptor. For example, Hartiadi et al.34 showed that reduction in the ratio of α4 to β2 cRNAs tends to generate receptors with high affinity to GABA whereas changes in δ have no effect. We previously showed that linking individual subunits to concatemeric constructs enables selective generation of low- or high-affinity receptors32. In contrast, for receptors activated by the conformationally constrained analog of GABA, THIP, Meera et al.35 proposed that the two types of behavior are simply due to a mixture of low-affinity αβ and high-affinity αβδ receptors, i.e., incomplete incorporation of δ in all surface receptors. We note that our study was conducted on the “high-affinity” isoform of the α4β2δ receptor.
We have shown that the α4β2δ receptor exhibits relatively high constitutive open probability (POpen,const = 0.1). In the cyclic MWC model, high background activity is associated with enhanced sensitivity to agonist because of a lower energy barrier that needs to be crossed during transition from closed/resting to open/active9,36. Despite the high POpen,const, the receptor is only weakly activated by the endogenous agonists GABA and taurine. The maximal peak open probabilities were ~0.4 for either agonist. However, both GABA and taurine are relatively potent agonists, and the equilibrium dissociation constants for GABA (~15 nM) and taurine (10 μM) are near their reported extracellular concentrations of 5–30 nM and 10–25 μM, respectively37,38,39. The receptor is weakly directly activated by the endogenous steroid 3α5αP (maximal peak POpen ~0.2), but the intravenous anesthetic propofol is a full agonist (POpen,max ~0.99).
The estimate for Q (=A/D in Fig. 1) was 0.52 in the presence of taurine, 0.78 in the presence of GABA, 0.89 with 3α5αP, and 1.29 when the receptors were activated by propofol. Followup experiments showed that propofol similarly potentiates peak and steady-state currents from receptors activated by GABA. We infer that the observed difference in Q for GABA vs. propofol is a result of experimental imprecision rather than higher affinity of propofol to the open state as previously observed for the α1β2γ2L receptor23. In subsequent simulations, we used a value of Q of 0.87, averaged from the individual estimates in the presence of taurine, GABA, 3α5αP, or propofol.
We tested the independence of the actions of orthosteric and allosteric agents by coapplying various combinations of such agents, and comparing the observed POpen,S.S. with a predicted value calculated using Eq. (7), which assumes additive effects of each agonist and inhibitor. Overall there was a good agreement between predicted and observed data (Fig. 6). We infer that the actions GABA, taurine, propofol, 3α5αP, and PS on the α4β2δ receptor follow the basic rules of energetic additivity. We did not test energetic additivity of the drugs on peak responses.
The data indicate that taurine is a potent agonist of the α4β2δ receptor with an EC50 (10 μM) near its extracellular concentration in the resting state in brain37. This is in agreement with a previous study that showed increased tonic current and reduced action potential firing in the presence of 10–100 μM taurine in the thalamus20. The reported EC50 for taurine on recombinant α4β2δ receptors in HEK cells was, however, higher by several orders of magnitude20. We propose that this discrepancy arises from the HEK cells preferentially expressing the low-affinity isoform of the α4β2δ receptor32.
Taurine and GABA act additively rather than synergistically because both agonists interact with the same binding site. The calculated (Eq. (4)) steady-state POpen of the α4β2δ receptor is 0.24 in the presence of 30 nM GABA, 0.21 in the presence of 10 μM taurine, and 0.25 in the presence of GABA + taurine. The predicted POpen,S.S. in the simultaneous presence of physiological concentrations of major endogenous GABAergic agonists and modulators - 30 nM GABA39, 10 μM taurine37, 30 nM 3α5αP40, and 0.1 μM PS40 - is 0.24. The addition of 1 μM propofol41 increases the POpen,S.S. to 0.28. Such a small potentiating effect may be expected given the low affinity of the receptor for propofol (Kpropofol > 50 μM). The full extent of physiological significance of these predictions is unclear, but the results tend to argue against the α4β2δ receptor being a significant target for propofol.
The overall predicted theoretical dynamic range of steady-state activity in the α4β2δ receptor is relatively small, ranging from ~0.10 (constitutive activity) to ~0.45 (maximal allowable steady-state activity with Q = 0.87). We speculate that the α4β2δ receptor acts to stabilize the membrane potential near the Cl− reversal potential, and that surface receptor turnover plays a relatively large role in regulation of its function.
It is not fully established which affinity isoform is the best recombinant model of the native, neuronally-expressed extrasynaptic receptor. Several lines of evidence support the idea that the “high-affinity” isoform is a better analog of the native receptor. Submicromolar concentrations of THIP activate tonic current in cerebellar granule cells that is missing in the cells from δ knockout mice35. The α4β3δ receptors expressed in oocytes produced THIP concentration-response curves with a high-affinity component at <100 nM (assumed to be analogous to high affinity to GABA) and a low-affinity component at >10 μM35. A low concentration (10–100 μM) of taurine elicits tonic inhibitory currents in thalamic neurons20. This agrees with our study of the high-affinity isoform in oocytes where we saw strong activation in the 1–100 μM range (see above), but not with concentration-response studies of the α4β2δ receptor expressed in HEK cells20, which preferentially express the low-affinity isoform32. The physiological relevance of the high-affinity isoform is indirectly supported by the finding that the steroid alfaxalone elicits large currents in the presence of picrotoxin in hippocampal neurons transfected with α4β2δ(T269Y) subunits32. Finally, we note that the high-affinity isoform of the α4β2δ receptor with a GABA EC50 at 20 nM is expected to be responsive to extracellular (5–30 nM38,39) GABA, unlike the low-affinity isoform with an EC50 > 1 μM.
Methods
Receptors and expression
The human α4β2δ GABAA receptors were expressed in Xenopus laevis oocytes. Harvesting of oocytes was conducted under the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. The animal protocol was approved by the Animal Studies Committee of Washington University in St. Louis (Approval No. 20170071).
The cDNAs of individual subunits in the pcDNA3 vector were linearized with Xba I (NEB Labs, Ipswich, MA). The cRNAs were generated using mMessage mMachine (Ambion, Austin, TX). The oocytes were injected with a total of 11 ng cRNA per oocyte in a 5:1:5 (α4:β2:δ) ratio. Following injection, the oocytes were incubated in bath solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES; pH 7.4) plus supplements (2.5 mM Na pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml gentamycin) at 15 °C for 3–4 days prior to conducting electrophysiological recordings.
Prior studies have indicated that the α4βδ receptors in oocytes can assemble as isoforms characterized by high affinity to GABA (EC50 at tens to hundreds of nM) or low affinity (EC50 in the μM range) to GABA32,33,34,35. The high-affinity isoform has been shown to be directly activated by the δ-specific drug DS-2 whereas the low-affinity isoform is potentiated but not directly activated by DS234,42. The underlying mechanism for this discrepancy is not fully understood, but distinct stoichiometries or subunit order in the two isoforms have been proposed as the cause32,33,34. The isoform investigated in the present study had a high affinity to the transmitter and was directly activated by DS-2.
Electrophysiology and analysis of current responses
The recordings were conducted at room temperature using standard two-electrode voltage clamp. The pipets were filled with 3 M KCl. The oocytes were clamped at −60 mV. The chamber (RC-1Z, Warner Instruments, Hamden, CT) was perfused with bath solution (see above) at 5–8 ml/min. Solutions were gravity-applied from 30-ml glass syringes with glass luer slips via Teflon tubing, and switched manually.
The current responses were amplified with an Axoclamp 900A (Molecular Devices, Sunnyvale, CA) or OC-725C amplifier (Warner Instruments, Hamden, CT), digitized with a Digidata 1320 or 1200 series digitizer (Molecular Devices), and stored using pClamp (Molecular Devices).
A typical experiment entailed recording of baseline current for 10–20 s, followed by application of a test compound or a combination of compounds for 60–270 s (1–4.5 min), and by application of bath solution to demonstrate recovery. Due to long exposure times, not all cells yielded a full range of concentration-response data. Thus, the concentration-response relationships shown may reflect mean responses from cells exposed to an incomplete range of agonist concentrations. In such cases, the number of cell provided is given as a range of cell numbers for each concentration point. The effects of the inhibitory steroid PS were determined by coapplying the steroid with 0.3 μM GABA. Each cell was tested with 1–3 concentrations of PS. Each cell was also tested with 10 μM GABA + 50 μM propofol to determine the maximal attainable peak response, which was assigned a POpen of 1, and to which the responses to test drugs were compared. This approach assumes that peak responses are not affected by desensitization, i.e., that desensitization develops slowly compared to activation, and that the combination of GABA + propofol activates all resting receptors. The level of constitutive activity was determined by exposing the cells to 100–300 μM picrotoxin.
The current traces were analyzed using Clampfit (Molecular Devices) to determine the amplitudes of peak and steady-state responses. If steady-state (defined as ΔI < 2% during the last 20 s of agonist application) was not reached by the end of the agonist application, an estimate was made by exponential fitting of the current decay. Fitting was done using pClamp, to a single exponential or sums of up to three exponentials. The constant offset is reported as the steady-state response. The estimated value of the offset was relatively insensitive to the number of exponentials used in fitting (up to ~10% variability in the fitted offset).
Materials and chemicals
The salts and HEPES used to prepare the bath solution, GABA, and 3α5αP were purchased from Sigma-Aldrich (St. Louis, MO). Propofol was purchased from MP Biomedicals (Solon, OH). Pregnenolone sulfate (PS) was bought from Tocris (Bio-Techne, Minneapolis, MN).
The stock solution of GABA was made in the bath solution at 500 mM, stored in aliquots at −20 °C, and diluted on the day of experiment. Stock solution of propofol was made in DMSO at 200 mM and stored at room temperature. 3α5αP was dissolved in DMSO at 10–20 mM and stored at room temperature. PS was dissolved in DMSO at 50 mM and stored at 4 °C.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
Supported by the National Institutes of Health (grant GM108580), and funds from the Taylor Family Institute for Innovative Psychiatric Research. We thank Joe Henry Steinbach for many stimulating discussions.
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S.R.P., T.C.S. and A.L.G. performed the experiments; S.R.P., T.C.S., A.L.G. and G.A. analyzed the data; A.L.G. and G.A. contributed to the study design; A.L.G. and G.A. prepared the figures and wrote the manuscript; all authors reviewed the manuscript.
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Pierce, S.R., Senneff, T.C., Germann, A.L. et al. Steady-state activation of the high-affinity isoform of the α4β2δ GABAA receptor. Sci Rep 9, 15997 (2019). https://doi.org/10.1038/s41598-019-52573-z
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DOI: https://doi.org/10.1038/s41598-019-52573-z
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