Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(−) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action.

GABA receptors can be directly activated and potentiated by the intravenous anesthetic propofol. Previous photolabeling, modeling, and functional data have identified two binding domains through which propofol acts on the GABA receptor. These domains are defined by the (M286) residue at the"+"-"-" interface in the transmembrane region and the (Y143) residue near the"-" surface in the junction between the extracellular and transmembrane domains. In the ternary receptor, there are predicted to be two copies of each class of sites, for a total of four sites per receptor. We used 212L and 21 concatemeric constructs to determine the functional effects of the (Y143W) and(M286W) mutations to gain insight into the number of functional binding sites for propofol and the energetic contributions stemming from propofol binding to the individual sites. A mutation of each of the four sites affected the response to propofol, indicating that each of the four sites is functional in the wild-type receptor. The mutations mainly impaired stabilization of the open state by propofol, i.e., reduced gating efficacy. The effects were similar for mutations at either site and were largely additive and independent of the presence of other Y143W or M286W mutations in the receptor. The two classes of sites appeared to differ in affinity for propofol, with the site affected by M286W having about a 2-fold higher affinity. Our analysis indicates there may be one or two additional functionally equivalent binding sites for propofol, other than those modified by substitutions at (Y143) and(M286).

The concerted transition model for multimeric proteins is a simple formulation for analyzing the behavior of transmitter-gated ion channels. We used the model to examine the relationship between the EC for activation of the GABA type A (GABA) receptor by the transmitter GABA and basal activity employing concatemeric ternary GABA receptors expressed in oocytes. Basal activity, reflecting the receptor function in the absence of the transmitter, can be changed either by mutation to increase constitutive activity or by the addition of a second agonist (acting at a different site) to increase background activity. The model predicts that either mechanism for producing a change in basal activity will result in identical effects on the EC We examined receptor activation by GABA while changing the level of basal activity with the allosterically acting anesthetics propofol, pentobarbital, or alfaxalone. We found that the relationship between EC and basal activity was well described by the concerted transition model. Changes in the basal activity by gain-of-function mutations also resulted in predictable changes in the EC Finally, we altered the number of GABA-binding sites by a mutation and again found that the relationship could be well described by the model. Overall, the results support the idea that interactions between the transmitter GABA and the allosteric agonists propofol, pentobarbital, or alfaxalone can be understood as reflecting additive and independent free energy changes, without assuming any specific interactions.

Drug interactions are often analyzed in terms of isobolograms. In the isobologram, the line connecting the axial points corresponding to the concentrations of two different drugs that produce an effect of the same magnitude is termed an isobole of additivity. Although the isobole of additivity can be a straight line in some special cases, previous work has proposed that it is curvilinear when the two drugs differ in their maximal effects or Hill slopes. Modulators of transmitter-gated ion channels have a wide range of maximal effects as well as Hill slopes, suggesting that the isoboles for drug actions on ion channel function are not linear. In this study, we have conducted an analysis of direct activation and potentiation of the human 122L GABA receptor to demonstrate that: 1) curvilinear isoboles of additivity are predicted by a concerted transition model where the binding of each GABAergic drug additively and independently reduces the free energy of the open receptor compared with the closed receptor; and 2) experimental data for receptor activation using the agonist pair of GABA and propofol or potentiation of responses to a low concentration of GABA by the drug pair of alfaxalone and propofol agree very well with predictions. The approach assuming independent energetic contributions from GABAergic drugs enables, at least for the drug combinations tested, a straightforward method to accurately predict functional responses to any combination of concentrations.

Under both physiologic and clinical conditions GABA A receptors are exposed to multiple agonists, including the transmitter GABA, endogenous or exogenous neuroactive steroids, and various GABAergic anesthetic and sedative drugs. The functional output of the receptor reflects the interplay among all active agents. We have investigated the activation of the concatemeric a1b2g2L GABA A receptor by combinations of agonists. Simulations of receptor activity using the coagonist concerted transition model demonstrate that the response amplitude in the presence of agonist combinations is highly dependent on whether the paired agonists interact with the same or distinct sites. The experimental data for receptor activation by agonist combinations were in agreement with the established views of the overlap of binding sites for several pairs of orthosteric (GABA, b-alanine, and piperidine-4-sulfonic acid) and/or allosteric agents (propofol, pentobarbital, and several neuroactive steroids). Conversely, the degree of potentiation when two GABAergic agents are coapplied can be used to determine whether the compounds act by binding to the same or distinct sites. We show that common interaction sites mediate the actions of 5a-and 5b-reduced neuroactive steroids, and natural and enantiomeric steroids. Furthermore, the results indicate that the anesthetics propofol and pentobarbital interact with partially shared binding sites. We propose that the findings may be used to predict the efficacy of drug mixtures in combination therapy and thus have potential clinical relevance.

BackgroundPropofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the “+” of the β subunit, in the β-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the “-” side of the β subunit, in the α-β interface (or β-β interface, in the case of homomeric β receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the β(H267) residue lines a cavity that docks propofol with favorable interaction energy.MethodWe used two-electrode voltage clamp to determine the functional effects of mutations to the 
“+” and “-” sides of the β subunit on activation of the α1β3 GABAA receptor by propofol.ResultsWe found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-β interface leads to strong reduction in gating efficacy for propofol.ConclusionWe conclude that α1β3 GABAA receptors can be activated by propofol interactions with the β-β, α-β, and β-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.

This study examines how site-specific binding to three identified neurosteroid binding sites in the α1β3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β3(+)–α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

The two-state coagonist model has been successfully used to analyze and predict peak current responses of the g-aminobutyric acid type A (GABA A) receptor. The goal of the present study was to provide a model-based description of GABA A receptor activity under steady-state conditions after desensitization has occurred. We describe the derivation and properties of the cyclic three-state resting-active-desensitized (RAD) model. The relationship of the model to receptor behavior was tested using concatemeric a1b2g2 GABA A receptors expressed in Xenopus oocytes. The receptors were activated by the orthosteric agonists GABA or b-alanine, the allosteric agonist propofol, or combinations of GABA, propofol, pentobarbital, and the steroid allopregnanolone, and the observed steady-state responses were compared with those predicted by the model. A modified RAD model was employed to analyze and describe the actions on steady-state current of the inhibitory steroid pregnenolone sulfate. The findings indicate that the steady-state activity in the presence of multiple active agents that interact with distinct binding sites follows standard energetic additivity. The derived equations enable prediction of peak and steady-state activity in the presence of orthosteric and allosteric agonists, and the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT The study describes derivation and properties of a three-state resting-active-desensitized model. The model and associated equations can be used to analyze and predict peak and steady-state activity in the presence of one or more active agents.