GABAA receptor

The GABA_A receptor (GABA_AR) is an ionotropic receptor and ligand-gated ion channel. Its endogenous ligand is γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system. Accurate regulation of GABAergic transmission through appropriate developmental processes, specificity to neural cell types, and responsiveness to activity is crucial for the proper functioning of nearly all aspects of the central nervous system (CNS).^[1] Upon opening, the GABA_A receptor on the postsynaptic cell is selectively permeable to chloride ions (Cl⁻
) and, to a lesser extent, bicarbonate ions (HCO⁻
₃).^[2][3]

GABA_AR are members of the ligand-gated ion channel receptor superfamily, which is a chloride channel family with a dozen or more heterotetrametric subtypes and 19 distinct subunits. These subtypes have distinct brain regional and subcellular localization, age-dependent expression, and the ability to undergo plastic alterations in response to experience, including drug exposure.^[4]

GABA_AR is not just the target of agonist depressants and antagonist convulsants, but most GABA_AR medicines also act at additional (allosteric) binding sites on GABA_AR proteins. Some sedatives and anxiolytics, such as benzodiazepines and related medicines, act on GABA_AR subtype-dependent extracellular domain sites. Alcohols and neurosteroids, among other general anesthetics, act at GABA_AR subunit-interface transmembrane locations. High anesthetic dosages of ethanol act on GABA_AR subtype-dependent transmembrane domain locations. Ethanol acts at GABA_AR subtype-dependent extracellular domain locations at low intoxication concentrations. Thus, GABA_AR subtypes have pharmacologically distinct receptor binding sites for a diverse range of therapeutically significant neuropharmacological drugs.^[4]

Depending on the membrane potential and the ionic concentration difference, this can result in ionic fluxes across the pore. If the membrane potential is higher than the equilibrium potential (also known as the reversal potential) for chloride ions, when the receptor is activated Cl⁻
will flow into the cell.^[5] This causes an inhibitory effect on neurotransmission by diminishing the chance of a successful action potential occurring at the postsynaptic cell. The reversal potential of the GABA_A-mediated inhibitory postsynaptic potential (IPSP) in normal solution is −70 mV, contrasting the GABA_B IPSP (−100 mV).

The active site of the GABA_A receptor is the binding site for GABA and several drugs such as muscimol, gaboxadol, and bicuculline.^[6] The protein also contains a number of different allosteric binding sites which modulate the activity of the receptor indirectly. These allosteric sites are the targets of various other drugs, including the benzodiazepines, nonbenzodiazepines, neuroactive steroids, barbiturates, alcohol (ethanol),^[7] inhaled anaesthetics, kavalactones, cicutoxin, and picrotoxin, among others.^[8]

Much like the GABA_A receptor, the GABA_B receptor is an obligatory heterodimer consisting of GABA_B1 and GABA_B2 subunits. These subunits include an extracellular Venus Flytrap domain (VFT) and a transmembrane domain containing seven α-helices (7TM domain). These structural components play a vital role in intricately modulating neurotransmission and interactions with drugs. ^[9]

The ionotropic GABA_A receptor protein complex is also the molecular target of the benzodiazepine class of tranquilizer drugs. Benzodiazepines do not bind to the same receptor site on the protein complex as does the endogenous ligand GABA (whose binding site is located between α- and β-subunits), but bind to distinct benzodiazepine binding sites situated at the interface between the α- and γ-subunits of α- and γ-subunit containing GABA_A receptors.^[10][11] While the majority of GABA_A receptors (those containing α1-, α2-, α3-, or α5-subunits) are benzodiazepine sensitive, there exists a minority of GABA_A receptors (α4- or α6-subunit containing) which are insensitive to classical 1,4-benzodiazepines,^[12] but instead are sensitive to other classes of GABAergic drugs such as neurosteroids and alcohol. In addition peripheral benzodiazepine receptors exist which are not associated with GABA_A receptors. As a result, the IUPHAR has recommended that the terms "BZ receptor", "GABA/BZ receptor" and "omega receptor" no longer be used and that the term "benzodiazepine receptor" be replaced with "benzodiazepine site".^[13] Benzodiazepines like diazepam and midazolam act as positive allosteric modulators for GABA_A receptors. When these receptors are activated, there's a rise in intracellular chloride levels, resulting in cell membrane hyperpolarization and decreased excitation.^[14]

In order for GABA_A receptors to be sensitive to the action of benzodiazepines they need to contain an α and a γ subunit, between which the benzodiazepine binds. Once bound, the benzodiazepine locks the GABA_A receptor into a conformation where the neurotransmitter GABA has much higher affinity for the GABA_A receptor, increasing the frequency of opening of the associated chloride ion channel and hyperpolarising the membrane. This potentiates the inhibitory effect of the available GABA leading to sedative and anxiolytic effects.^[15]

Different benzodiazepines have different affinities for GABA_A receptors made up of different collection of subunits, and this means that their pharmacological profile varies with subtype selectivity. For instance, benzodiazepine receptor ligands with high activity at the α1 and/or α5 tend to be more associated with sedation, ataxia and amnesia, whereas those with higher activity at GABA_A receptors containing α2 and/or α3 subunits generally have greater anxiolytic activity.^[16] Anticonvulsant effects can be produced by agonists acting at any of the GABA_A subtypes, but current research in this area is focused mainly on producing α₂-selective agonists as anticonvulsants which lack the side effects of older drugs such as sedation and amnesia.

The binding site for benzodiazepines is distinct from the binding site for barbiturates and GABA on the GABA_A receptor, and also produces different effects on binding,^[17] with the benzodiazepines increasing the frequency of the chloride channel opening, while barbiturates increase the duration of chloride channel opening when GABA is bound.^[18] Since these are separate modulatory effects, they can both take place at the same time, and so the combination of benzodiazepines with barbiturates is strongly synergistic, and can be dangerous if dosage is not strictly controlled.^[19]

Also note that some GABA_A agonists such as muscimol and gaboxadol do bind to the same site on the GABA_A receptor complex as GABA itself, and consequently produce effects which are similar but not identical to those of positive allosteric modulators like benzodiazepines.

Structural understanding of the GABA_A receptor was initially based on homology models, obtained using crystal structures of homologous proteins like Acetylcholine binding protein (AChBP) and nicotinic acetylcholine (nACh) receptors as templates.^[22][23][24] The much sought structure of a GABA_A receptor was finally resolved, with the disclosure of the crystal structure of human β3 homopentameric GABA_A receptor.^[25] Whilst this was a major development, the majority of GABA_A receptors are heteromeric and the structure did not provide any details of the benzodiazepine binding site. This was finally elucidated in 2018 by the publication of a high resolution cryo-EM structure of rat α1β1γ2S receptor^[15] and human α1β2γ2 receptor bound with GABA and the neutral benzodiazepine flumazenil.^[26]

GABA_A receptors are pentameric transmembrane receptors which consist of five subunits arranged around a central pore. Each subunit comprises four transmembrane domains with both the N- and C-terminus located extracellularly. The receptor sits in the membrane of its neuron, usually localized at a synapse, postsynaptically. However, some isoforms may be found extrasynaptically.^[27] When vesicles of GABA are released presynaptically and activate the GABA receptors at the synapse, this is known as phasic inhibition. However, the GABA escaping from the synaptic cleft can activate receptors on presynaptic terminals or at neighbouring synapses on the same or adjacent neurons (a phenomenon termed 'spillover') in addition to the constant, low GABA concentrations in the extracellular space results in persistent activation of the GABA_A receptors known as tonic inhibition.^[28]

The ligand GABA is the endogenous compound that causes this receptor to open; once bound to GABA, the protein receptor changes conformation within the membrane, opening the pore in order to allow chloride anions (Cl⁻
) and, to a lesser extent, bicarbonate ions (HCO⁻
₃) to pass down their electrochemical gradient. The binding site to GABA is about 80Å away from the narrowest part of the ion channel. Recent computational studies have suggested an allosteric mechanism whereby GABA binding leads to ion channel opening.^[29] Because the reversal potential for chloride in most mature neurons is close to or more negative than the resting membrane potential, activation of GABA_A receptors tends to stabilize or hyperpolarise the resting potential, and can make it more difficult for excitatory neurotransmitters to depolarize the neuron and generate an action potential. The net effect therefore typically inhibitory, reducing the activity of the neuron, although depolarizing currents have been observed in response to GABA in immature neurons in early development. This effect during development is due to a modified Cl⁻
gradient wherein the anions leave the cells through the GABA_A receptors, since their intracellular chlorine concentration is higher than the extracellular.^[30] The difference in extracellular chlorine anion concentration is presumed to be due to the higher activity of chloride transporters, such as NKCC1, transporting chloride into cells which are present early in development, whereas, for instance, KCC2 transports chloride out of cells and is the dominant factor in establishing the chloride gradient later in development. These depolarization events have shown to be key in neuronal development.^[31] In the mature neuron, the GABA_A channel opens quickly and thus contributes to the early part of the inhibitory post-synaptic potential (IPSP).^[32][33] The endogenous ligand that binds to the benzodiazepine site is inosine.^[34]

Proper developmental, neuronal cell-type-specific, and activity-dependent GABAergic transmission control is required for nearly all aspects of CNS function.^[1]

It has been proposed that the GABAergic system is disrupted in numerous neurodevelopmental diseases, including fragile X syndrome, Rett syndrome, and Dravet syndrome, and that it is a crucial potential target for therapeutic intervention.^[35]

A number of ligands have been found to bind to various sites on the GABA_A receptor complex and modulate it besides GABA itself.^[which?] A ligand can possess one or more properties of the following types. Unfortunately the literature often does not distinguish these types properly.

Types

• Orthosteric agonists and antagonists: bind to the main receptor site (the site where GABA normally binds, also referred to as the "active" or "orthosteric" site). Agonists activate the receptor, resulting in increased Cl⁻
  conductance. Antagonists, though they have no effect on their own, compete with GABA for binding and thereby inhibit its action, resulting in decreased Cl⁻
  conductance.
• First order allosteric modulators: bind to allosteric sites on the receptor complex and affect it either in a positive (PAM), negative (NAM) or neutral/silent (SAM) manner, causing increased or decreased efficiency of the main site and therefore an indirect increase or decrease in Cl⁻
  conductance. SAMs do not affect the conductance, but occupy the binding site.
• Second order modulators: bind to an allosteric site on the receptor complex and modulate the effect of first order modulators.
• Open channel blockers: prolong ligand-receptor occupancy, activation kinetics and Cl ion flux in a subunit configuration-dependent and sensitization-state dependent manner.^[45]
• Non-competitive channel blockers: bind to or near the central pore of the receptor complex and directly block Cl⁻
  conductance through the ion channel.