Structure and function of the GABAA receptor
The GABAA receptor is composed of five different subunits (α1-6, β1-3, γ1-3, δ, epsilon, φ, π and ρ1-3) which are encoded by at least 19 mammalian genes, with additional diversity arising in certain regions. In most GABAA receptors, the most common combination of subunits is α, β, and γ, with a ratio of 2:2:1 although the γ subunit may be replaced by δ or epsilon subunits, particularly in brain regions, as shown in Figure 1. These GABAA receptor subunits are densely packed in the cortex, and receptors with the γ2 subunit comprise more than 40% of all GABAA receptors in the brain.
Figure 1 Structure and function of γ- aminobutyric acid type A receptor.
A: γ- aminobutyric acid type A (GABAA) receptors commonly contain two α subunits, two β subunits and one γ subunit. Chloride influx through the pore could hyperpolarize the postsynaptic membrane; B: Left: Extra-membrane region of GABAA receptor. The binding sites for GABA are located between α and β subunits and the binding site for benzodiazepines is located between γ and α subunits; Right: Trans-membrane region of GABAA receptor. Four trans-membrane segments form the α subunit. It has been shown that the trans-membrane segment of β subunit is the binding site for propofol and etomidate. This binding site is close to a binding site for volatile anesthetics; C: Activation of the GABAA receptor could increase conductance of the postsynaptic membrane and alter the potential of the membrane because of influx of chloridion. Synaptic receptors could detect GABA at mmol concentration to produce fast inhibitory postsynaptic potentials (IPSPs), and extra-synaptic receptors that detect GABA at μmol concentrations to produce slower IPSPs.
The GABA system is the main inhibitory neurotransmitter pathway in the CNS of mammalian brain, and one-third of all synapses are GABAergic. The GABA system induces inhibition of the central nervous system by generating fast, transient inhibitory postsynaptic currents. Activation of GABAA receptors decreases excitability of the neurons by an influx of chloride, hyperpolarization of the membrane, and shunting of excitatory input. This synaptic inhibition of the GABA system maintains neuronal communication and induces precise timing of action potentials and synchronization of neuronal populations[4,5]. For many years, enhancement of fast inhibition at synapses was widely regarded as the dominate mechanism underlying the effects of many GABAergic drugs.
The α1 and β2 subunit-containing GABAA receptors in the cortex are thought to contribute to the sedative actions of several inhaled anesthetics. Some studies of tool drugs indicate the important role of GABA receptors and its subunits in the anesthetic effect. Tonic current in the thalamic VB neurons may contribute to the sedative action of 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP). Although THIP is not commonly used as an anesthetic, it promotes slow wave sleep and produces analgesic, sedative, hypnotic actions and ataxic properties. GABAA receptors that contain the α4 and δ subunit appear to contribute to the sedation effects of THIP. At low concentrations, THIP strongly potentiates the activity of GABAA receptors containing the δ subunit, and enhances a tonic conductance generated by α4δ GABAA receptors. Rotarod performance and spontaneous loco-motor activity were unimpaired by THIP in α4 subunit knock-out mice, which suggests that α4δ subunit containing GABAA receptors are necessary for the sedative and ataxic effects of THIP. THIP enhanced the tonic but not the phasic GABAA receptor currents in VB neurons, and had no effect on nRT neurons. Since the sedative actions of THIP were absent in α4 knock-out mice, it is likely that the tonic current mediated by α4β2δ GABAA receptors in VB neurons contributes to anesthetic sedation.
Actions of general anesthetics on GABAA receptors
The enhancement of GABA-activated chloride currents is the main effect of some intravenous general anesthetic such as propofol and etomidate, decreasing neuronal activity by producing hyperpolarization of the neuronal membrane. This is in agreement with the finding that etomidate-mediated sedation also depends on GABAA receptors containing the β2 subunit[9,10], although the specific contribution of thalamic β2 subunits to this effect is uncertain. Propofol and etomidate also enhance function of GABAA receptors to produce immobility[11-13]. In contrast, gaseous general anesthetics such as xenon, nitrous oxide, cyclopropane as well as ketamine have minimal or no effect on GABAA receptor subtypes[14-18].
Compared to other general anesthetics, volatile anesthetics show low potency to a variety of receptors at clinical concentrations. As a result, the determination of the specific sites of effect of volatile anesthetics is a challenge. In addition, behavioral evaluation with volatile anesthetics has some obvious practical difficulties. Even with these handicaps, it has been demonstrated by some carefully designed studies that isoflurane anesthesia is mediated by GABAA receptors. Volatile anesthetics at clinical concentrations could activate GABAA receptors both in vitro and in vivo, using heterologous expression systems and the postsynaptic membrane, respectively[20,21]. The depressive effects of isoflurane, enflurane and halothane on rat neocortical neuron activity were studied using in vivo recordings of spontaneous action-potential firing and in vitro recordings from isolated cortical networks. Sedative concentrations of isoflurane, enflurane and halothane similarly reduced the firing of spontaneous action potentials in vivo and in vitro by approximately 50%. This reduction in neuronal firing strongly correlated with an increase in GABAergic synaptic inhibition. Anesthetics prolonged the time course of GABAA receptor-mediated spontaneous IPSCs from pyramidal neurons in organ cortical cultures with no effect on their frequency or peak amplitude.
At the spinal level the role of inhibitory GABAA receptors on anesthetics actions has been extensively studied. With the evaluation of motor response, MAC of volatile anesthetics was more significantly affected by spinal injections of glycine receptor antagonists than GABAA receptor antagonists.
For many years, the binding site of GABAA receptor for volatile anesthetics is still unclear. The binding site for volatile anesthetics on the GABAA receptor was determined to be a binding pocket for volatile anesthetics, by complementary site directed mutagenesis, using general anesthetics of varying molecular size. With the finding of a binding pocket for general anesthetics, the long-held assumption that general anesthetics worked by a nonspecific mechanism was overturned. Dramatic progress has been made in dissecting the behavioral effects of general anesthetics, in particular the subunit combination of GABAA receptors, on anesthetic effect. GABAA receptors containing the α1β2γ2 subunits are enriched at synaptic sites throughout the brain. This suggests that the enhancement of synaptic activity within the cortex could be responsible for anesthetic sedation. The contribution of the cortex to the sedative properties of inhaled anesthetics was studied by Hentschke and colleagues. In recent studies, an anesthetic binding cavity for volatile anesthetics has been identified, critically involving in the α1 subunit[26,27]. Rudolph et al, reported that animal behavioral patterns induced by benzodiazepine were moderated by a point mutation on the mouse α1 GABAA subunit. At the same time, barbiturates directly activate and inhibit GABAA receptors by means of positive allosteric modulation depending on their concentration at the receptor. In addition, a mutation in the GABAA α subunit was identified that abolishes the action of barbiturates, although, the potentiating by etomidate on GABAA receptors was not affected. Furthermore, enhancement of GABAA mediated transmissions was also affected by alcohol, indicating an important role of alcohol in mediating its intoxicating effects.
The biophysical profile of GABA receptors and their sensitivity to general anesthetics can be dramatically altered by subunit composition. Using chimerical channel construction, Mihic and colleagues discovered a domain, relevant for mediating the effect of volatile anesthetics and etomidate, but not propofol. Two key amino acids in GABAA receptor subunits were found to be involved in their interaction with volatile anesthetics. These amino residues may contribute to the molecular binding pockets for general anesthetics. According to important studies, two amino acids in the α1 subunit are the most critical points for general anesthetic effect. Serine 270 is in the trans-membrane segment and while Alanine 291 is near the extracellular regions. For GABAA receptors, replacing Ser 270 with larger amino acid residues in the α1 subunit resulted in a decrease of sensitivity to volatile anesthetics[26,31], while replacement with smaller residues resulted in the opposite effect. Also, replacing the α1 Ser270 residue with histidine resulted in recombinant heteromeric GABAA receptors that were insensitive to isoflurane. However, an additional change to the GABAA receptors, introduced by the α1 (Ser270His) mutation, complicated the interpretation of receptor pharmacology. This problem was addressed by introducing an additional mutation into the α1 subunit, whereby the leucine residue at position 277 was replaced with alanine. This double knock-in mutation, α1 (Ser270His, Leu277Ala), restored normal sensitivity to GABA. These mutations laid the foundation for generating knock-in mice that were partially insensitive to isoflurane. Mice with a double knock-in mutation were used to explain the interaction between GABAA receptors containing α1 subunits to isoflurane anesthesia. Some studies demonstrated that double-mutant mice expressing the α1 (Ser270His, Leu277Ala) subunit was less sensitive to isoflurane, compared to wild-type controls, indicating the important role of α1 subunit in the hypnotic effect of isoflurane. Interestingly, according to the tail clamp test, the immobilizing effect of isoflurane was not affected in these double-mutant mice. Using cued and contextual fear conditioning, the amnesic effect of isoflurane was also unaffected in the α1 (Ser270His, Leu277Ala) mice, comparing to wild-type control, indicating that this subunit is not critical for amnesia induced by isoflurane. This last finding is in contrast to previous work using mouse mutants in which the α1 subunit was knocked out either globally or in the forebrain alone. In other studies with the α1 subunit knock-out mice, the amnesic effect induced by isoflurane was impaired, indicating the role of α1 subunit in isoflurane amnesia. At the same time, the β subunit of GABAA receptors is also important to the binding site of volatile anesthetics, as well as for the behavioral effects of volatile anesthetics[27,35]. In addition, on the β3 subunit when the asparagine residue at position 265 was replaced with methionine or the methionine at position 286 with tryptophan, GABA current potentiated by enflurane was reduced. With β3 (Asn265Met) knock-in mice, isoflurane is slightly less effect at inhibiting the righting reflex in β3 (Asn265Met) mice, suggesting the role of the β3 subunit in isoflurane hypnosis. The immobility induced by isoflurane, however, is significantly impaired in these knock-in mice, as measured by hind limb or tail clamp withdrawal reflex. Additionally, in β3 (Asn265Met) mice, heart rate and core temperature were decreased less by isoflurane, indicating the role of the β3 subunit in the effect of isoflurane on circulation. Therefore, neuronal depressive effect and cardiovascular effects induced by volatile anesthetics might be mediated by distinct GABAA receptor subunits.
Knock-in mutant mice have been used to determine the GABAA subunits responsible for the sedative and hypnotic actions of etomidate. Some studies indicate that amnesic effect induced by etomidate might contribute to the α5 GABAA receptors in hippocampal region, while the sedative effect of etomidate might be due to other GABAA receptor isoforms. GABAA receptors with some structural modifications (the asparagine at position 265 in the β2 or β3 subunits was replaced with serine or methionine, respectively) were insensitive to etomidate in vitro[9,10]. Etomidate showed low efficacy in reducing spontaneous loco-motor activity in β2 (Asn265Ser) knock-in mice, indicating that GABAA receptors with the β2 subunit were important for the sedative effect of etomidate.
In other studies, sedative property of diazepam has been demonstrated to be mediated by the α1 subunit of GABAA receptors. With some different features from general anesthetics, diazepam produces a sedative effect. GABAA receptors which contained a histidine to arginine mutation at position 101 of the α1 subunit were insensitive to diazepam in vitro. Behavioral tests indicated that the sedative effect induced by diazepam were eliminated in knock-in mice that expressed the α1 (His101Arg) mutation.
Some other types of GABAA receptors, such as the extra junction GABAA receptors, could be activated by GABA at very low concentrations. Junction GABAA receptors are widely expressed in important brain regions including the hippocampus, thalamus, cortex and cerebellum. Currents mediated by these junctions GABAA receptors are affected by volatile anesthetics at low concentrations.