Multiple functional neurosteroid binding sites on GABAA receptors.

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 α1ß3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and ß3 (labeled residue ß3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues ß3-L294 and G308) in the interface between the ß3(+) 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 ß3-α1 intersubunit sites are critical for neurosteroid action.

Analysis of GABAA Receptor Activation by Combinations of Agonists Acting at the Same or Distinct Binding Sites.

Under both physiologic and clinical conditions GABAA 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 α1ß2γ2L GABAA 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, ß-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 5α- and 5ß-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.

Enhanced GABAergic actions resulting from the coapplication of the steroid 3α-hydroxy-5α-pregnane-11,20-dione (alfaxalone) with propofol or diazepam.

Many GABAergic drugs are in clinical use as anesthetics, sedatives, or anxiolytics. We have investigated the actions of the combinations of the neuroactive steroid 3α-hydroxy-5α-pregnane-11,20-dione (alfaxalone) with the intravenous anesthetic propofol or the benzodiazepine diazepam. The goal of the study was to determine whether coapplication of alfaxalone reduces the effective doses and concentrations of propofol and diazepam. Behavioral effects of alfaxalone, propofol, diazepam, and the combinations of the drugs were evaluated during a 30-min activity test in mice. Functional effects of the individual drugs and drug combinations were tested by measuring the decay times of spontaneous inhibitory postsynaptic currents in rat hippocampal neurons, and peak current responses from heterologously expressed concatemeric α1ß2γ2L GABAA receptors. Co-administration of alfaxalone increased the sedative actions of propofol and diazepam in mice. The combination of alfaxalone with propofol or diazepam increased the decay times of sIPSCs and shifted the concentration-response relationships for GABA-activated receptors to lower transmitter concentrations. We infer that alfaxalone acts as a co-agonist to enhance the GABAergic effects of propofol and diazepam. We propose that co-administration of alfaxalone, and possibly other neuroactive steroids, can be employed to reduce dosage requirements for propofol and diazepam.

High Constitutive Activity Accounts for the Combination of Enhanced Direct Activation and Reduced Potentiation in Mutated GABAA Receptors.

GABAA receptors activated by the transmitter GABA are potentiated by several allosterically acting drugs, including the intravenous anesthetic propofol. Propofol can also directly activate the receptor, albeit at higher concentrations. Previous functional studies have identified amino acid residues whose substitution reduces potentiation of GABA-activated receptors by propofol while enhancing the ability of propofol to directly activate the receptor. One interpretation of such observations is that the mutation has specific effects on the sites or processes involved in potentiation or activation. We show here that divergent effects on potentiation and direct activation can be mediated by increased constitutive open probability in the mutant receptor without any specific effect on the interactions between the allosteric drug and the receptor. By simulating GABAA receptor activity using the concerted transition model, we demonstrate that the predicted degree of potentiation is reduced as the level of constitutive activity increases. The model further predicts that a potentiating effect of an allosteric modulator is a computable value that depends on the level of constitutive activity, the amplitude of the response to the agonist, and the amplitude of the direct activating response to the modulator. Specific predictions were confirmed by electrophysiological data from the binary α1ß3 and concatemeric ternary ß2α1γ2L+ß2α1 GABAA receptors. The corollaries of reduced potentiation due to increased constitutive activity are isobolograms that conform to simple additivity and a loss of separation between the concentration-response relationships for direct activation and potentiation.

Mapping two neurosteroid-modulatory sites in the prototypic pentameric ligand-gated ion channel GLIC.

Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand-gated ion channels (pLGICs) and can be effective anesthetics, analgesics, or anti-epileptic drugs. The complex effects of neurosteroids on pLGICs suggest the presence of multiple binding sites in these receptors. Here, using a series of novel neurosteroid-photolabeling reagents combined with top-down and middle-down mass spectrometry, we have determined the stoichiometry, sites, and orientation of binding for 3α,5α-pregnane neurosteroids in the Gloeobacter ligand-gated ion channel (GLIC), a prototypic pLGIC. The neurosteroid-based reagents photolabeled two sites per GLIC subunit, both within the transmembrane domain; one site was an intrasubunit site, and the other was located in the interface between subunits. By using reagents with photoreactive groups positioned throughout the neurosteroid backbone, we precisely map the orientation of neurosteroid binding within each site. Amino acid substitutions introduced at either site altered neurosteroid modulation of GLIC channel activity, demonstrating the functional role of both sites. These results provide a detailed molecular model of multisite neurosteroid modulation of GLIC, which may be applicable to other mammalian pLGICs.

Propofol Is an Allosteric Agonist with Multiple Binding Sites on Concatemeric Ternary GABAA Receptors.

GABAA 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 GABAA receptor. These domains are defined by the ß(M286) residue at the ß"+"-α"-" interface in the transmembrane region and the ß(Y143) residue near the ß"-" 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 ß2α1γ2L and ß2α1 concatemeric constructs to determine the functional effects of the ß(Y143W) and ß(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 ß(Y143) and ß(M286).

GABA Type A Receptor Activation in the Allosteric Coagonist Model Framework: Relationship between EC50 and Basal Activity.

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 EC50 for activation of the GABA type A (GABAA) receptor by the transmitter GABA and basal activity employing concatemeric ternary GABAA receptors expressed in Xenopus 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 EC50 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 EC50 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 EC50 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.

The Actions of Drug Combinations on the GABAA Receptor Manifest as Curvilinear Isoboles of Additivity.

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 α1ß2γ2L GABAA 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.

Determination of the Residues in the Extracellular Domain of the Nicotinic α Subunit Required for the Actions of Physostigmine on Neuronal Nicotinic Receptors.

Physostigmine can potentiate and inhibit neuronal nicotinic receptors, in addition to inhibiting the activity of acetylcholinesterase. We found that receptors containing three copies of the α2 subunit are inhibited by low concentrations of physostigmine in contrast to receptors containing three copies of the α4 subunit that are potentiated. We exploited this observation to determine the regions required for the actions of physostigmine. Chimeric constructs of the α2 and α4 subunits located two regions in the extracellular amino-terminal domain of the subunit: the E loop (a loop of the transmitter-binding domain) and a region closer to the amino-terminus that collectively could completely determine the different effects of physostigmine. Point mutations then identified a single residue, α2(I92) versus α4(R92), that, when combined with transfer of the E loop, could convert the inhibition seen with α2 subunits to potentiation and the potentiation seen with α4 subunits to inhibition. In addition, other point mutations could affect the extent of potentiation or inhibition, indicating that a more extensive set of interactions in the amino-terminal domain plays some role in the actions of physostigmine.

Activation and modulation of recombinant glycine and GABAA receptors by 4-halogenated analogues of propofol.

BACKGROUND AND PURPOSE: Glycine receptors are important players in pain perception and movement disorders and therefore important therapeutic targets. Glycine receptors can be modulated by the intravenous anaesthetic propofol (2,6-diisopropylphenol). However, the drug is more potent, by at least one order of magnitude, on GABAA receptors. It has been proposed that halogenation of the propofol molecule generates compounds with selective enhancement of glycinergic modulatory properties. EXPERIMENTAL APPROACH: We synthesized 4-bromopropofol, 4-chloropropofol and 4-fluoropropofol. The direct activating and modulatory effects of these drugs and propofol were compared on recombinant rat glycine and human GABAA receptors expressed in oocytes. Behavioural effects of the compounds were compared in the tadpole loss-of-righting assay. KEY RESULTS: Concentration-response curves for potentiation of homomeric α1, α2 and α3 glycine receptors were shifted to lower drug concentrations, by 2-10-fold, for the halogenated compounds. Direct activation by all compounds was minimal with all subtypes of the glycine receptor. The four compounds were essentially equally potent modulators of the α1ß3γ2L GABAA receptor with EC50 between 4 and 7 µM. The EC50 for loss-of-righting in Xenopus tadpoles, a proxy for loss of consciousness and considered to be mediated by actions on GABAA receptors, ranged from 0.35 to 0.87 µM. CONCLUSIONS AND IMPLICATIONS: We confirm that halogenation of propofol more strongly affects modulation of homomeric glycine receptors than α1ß3γ2L GABAA receptors. However, the effective concentrations of all tested halogenated compounds remained lower for GABAA receptors. We infer that 4-bromopropofol, 4-chloropropofol and 4-fluoropropofol are not selective homomeric glycine receptor modulators.

Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol.

BACKGROUND: Propofol 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 ß subunit, in the ß-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 ß subunit, in the α-ß interface (or ß-ß interface, in the case of homomeric ß receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the ß(H267) residue lines a cavity that docks propofol with favorable interaction energy. METHOD: We used two-electrode voltage clamp to determine the functional effects of mutations to the "+" and "-" sides of the ß subunit on activation of the α1ß3 GABAA receptor by propofol. RESULTS: We 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-ß interface leads to strong reduction in gating efficacy for propofol. CONCLUSION: We conclude that α1ß3 GABAA receptors can be activated by propofol interactions with the ß-ß, α-ß, and ß-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.