Structure of theGABAA receptor (α1β1γ2S:PDB:6DW1). Top: side view of the GABAA receptor embedded in acell membrane. Bottom: view of the receptor from the extracellular face of the membrane. The subunits are labeled according to the GABAA nomenclature and the approximate locations of the GABA and benzodiazepine (BZ) binding sites are noted (between the α- and β-subunits and between the α- and γ-subunits respectively).Schematic structure of the GABAA receptor.Left: GABAA monomeric subunit embedded in alipid bilayer (yellow lines connected to blue spheres). The fourtransmembraneα-helices (1–4) are depicted as cylinders. The disulfide bond in the N-terminal extracellular domain which is characteristic of the family ofcys-loop receptors (which includes the GABAA receptor) is depicted as a yellow line.Right: Five subunits symmetrically arranged about the central chloride anion conduction pore. The extracellular loops are not depicted for the sake of clarity.
GABAAR 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]
GABAAR is not just the target of agonist depressants and antagonist convulsants, but most GABAAR medicines also act at additional (allosteric) binding sites on GABAAR proteins. Some sedatives and anxiolytics, such as benzodiazepines and related medicines, act on GABAAR subtype-dependent extracellular domain sites. Alcohols and neurosteroids, among other general anesthetics, act at GABAAR subunit-interface transmembrane locations. High anesthetic dosages of ethanol act on GABAAR subtype-dependent transmembrane domain locations. Ethanol acts at GABAAR subtype-dependent extracellular domain locations at low intoxication concentrations. Thus, GABAAR subtypes have pharmacologically distinct receptor binding sites for a diverse range of therapeutically significant neuropharmacological drugs.[4]
Depending on themembrane potential and the ionic concentration difference, this can result in ionic fluxes across the pore. If the membrane potential is higher than theequilibrium potential (also known as the reversal potential) for chloride ions, when the receptor is activatedCl− will flow into the cell.[5] This causes an inhibitory effect onneurotransmission by diminishing the chance of a successfulaction potential occurring at the postsynaptic cell. The reversal potential of the GABAA-mediatedinhibitory postsynaptic potential (IPSP) in normal solution is −70 mV, contrasting theGABAB IPSP (−100 mV).
Much like the GABAA receptor, the GABAB receptor is an obligatory heterodimer consisting of GABAB1 and GABAB2 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]
Theionotropic GABAA receptor protein complex is also the molecular target of thebenzodiazepine class of tranquilizer drugs. Benzodiazepines do not bind to the same receptorsite on the protein complex as does the endogenous ligandGABA (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 GABAA receptors.[10][11] While the majority of GABAA receptors (those containing α1-, α2-, α3-, or α5-subunits) are benzodiazepine sensitive, there exists a minority of GABAA 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 asneurosteroids and alcohol. In additionperipheral benzodiazepine receptors exist which are not associated with GABAA receptors. As a result, theIUPHAR 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 GABAA 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 GABAA 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 GABAA receptor into a conformation where the neurotransmitter GABA has much higher affinity for the GABAA 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 GABAA 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 withsedation,ataxia andamnesia, whereas those with higher activity at GABAA receptors containing α2 and/or α3 subunits generally have greateranxiolytic activity.[16]Anticonvulsant effects can be produced by agonists acting at any of the GABAA 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 forbarbiturates and GABA on the GABAA 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 GABAA agonists such asmuscimol andgaboxadol do bind to the same site on the GABAA receptor complex as GABA itself, and consequently produce effects which are similar but not identical to those of positive allosteric modulators like benzodiazepines.
Schematic diagram of a GABAA receptor protein ((α1)2(β2)2(γ2)) which illustrates the five combined subunits that form the protein, the chloride (Cl− ) ion channel pore, the two GABA active binding sites at the α1 and β2 interfaces, and the benzodiazepine (BZD) allosteric binding site[20]Side view of theEM structure of the α1β3γ2 GABAA receptor. GABA and the anaestheticetomidate are coloured magenta. Subunits in different colours. One alpha and one beta subunit is hidden. Green chloride ions illustrated in the channel pore.[21]
Structural understanding of the GABAA 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 GABAA receptor was finally resolved, with the disclosure of the crystal structure of human β3 homopentameric GABAA receptor.[25]Whilst this was a major development, the majority of GABAA 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 resolutioncryo-EM structure of rat α1β1γ2S receptor[15] and human α1β2γ2 receptor bound with GABA and the neutral benzodiazepine flumazenil.[26]
GABAA receptors arepentamerictransmembrane receptors which consist of five subunits arranged around a centralpore. Each subunit comprises four transmembrane domains with both the N- and C-terminus located extracellularly. The receptor sits in themembrane of itsneuron, usually localized at asynapse, postsynaptically. However, some isoforms may be found extrasynaptically.[27] Whenvesicles 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 GABAA receptors known as tonic inhibition.[28]
Theligand GABA is theendogenous compound that causes this receptor to open; once bound to GABA, theprotein receptor changes conformation within the membrane, opening the pore in order to allowchlorideanions (Cl− ) and, to a lesser extent,bicarbonate ions (HCO− 3) to pass down theirelectrochemical 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 thereversal potential for chloride in most mature neurons is close to or more negative than the restingmembrane potential, activation of GABAA receptors tends to stabilize or hyperpolarise the resting potential, and can make it more difficult for excitatoryneurotransmitters todepolarize the neuron and generate anaction 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 modifiedCl− gradient wherein the anions leave the cells through the GABAA 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 asNKCC1, 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 GABAA channel opens quickly and thus contributes to the early part of theinhibitory post-synaptic potential (IPSP).[32][33]The endogenous ligand that binds to the benzodiazepine site isinosine.[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]
GABAA receptors are members of the large pentameric ligand gated ion channel (previously referred to as "Cys-loop" receptors) super-family of evolutionarily related and structurally similarligand-gated ion channels that also includesnicotinic acetylcholine receptors,glycine receptors, and the5HT3 receptor. There are numerous subunitisoforms for the GABAA receptor, which determine the receptor's agonist affinity, chance of opening, conductance, and other properties.[36]
There are three ρ units (GABRR1,GABRR2,GABRR3); however, these do not coassemble with the classical GABAA units listed above,[37] but rather homooligomerize to formGABAA-ρ receptors (formerly classified as GABAC receptors but now thisnomenclature has been deprecated[38]).
Given the large number of GABAA receptors, a great diversity of final pentameric receptor subtypes is possible. Methods to produce cell-based laboratory access to a greater number of possible GABAA receptor subunit combinations allow teasing apart of the contribution of specific receptor subtypes and their physiological and pathophysiological function and role in the CNS and in disease.[39]
GABAA receptors are responsible for most of the physiological activities of GABA in the central nervous system, and the receptor subtypes vary significantly. Subunit composition can vary widely between regions and subtypes may be associated with specific functions. The minimal requirement to produce a GABA-gated ion channel is the inclusion of an α and a β subunit.[40] The most common GABAA receptor is a pentamer comprising two α's, two β's, and a γ (α2β2γ). In neurons themselves, the type of GABAA receptor subunits and their densities can vary betweencell bodies anddendrites.[41] Benzodiazepines and barbiturates amplify the inhibitory effects mediated by the GABAA receptor.[42]GABAA receptors can also be found in other tissues, includingleydig cells,placenta,immune cells,liver,bone growth plates and several otherendocrine tissues. Subunit expression varies between 'normal' tissue andmalignancies, as GABAA receptors can influencecell proliferation.[43]
A number ofligands have been found to bind to various sites on the GABAA 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.
GABA bound at its orthosteric site in the beta-alpha interface of an α1β2γ2 GABAA receptor. H-atoms hidden.
Orthosteric agonists andantagonists: 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 increasedCl− conductance. Antagonists, though they have no effect on their own, compete with GABA for binding and thereby inhibit its action, resulting in decreasedCl− 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 inCl− 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 blockCl− conductance through the ion channel.
Ligands which contribute to receptor activation typically haveanxiolytic,anticonvulsant,amnesic,sedative,hypnotic,euphoriant, andmuscle relaxant properties. Some such asmuscimol and thez-drugs may also behallucinogenic.[citation needed] Ligands which decrease receptor activation usually have opposite effects, includinganxiogenesis andconvulsion.[citation needed] Some of the subtype-selective negative allosteric modulators such asα5IA are being investigated for theirnootropic effects, as well as treatments for the unwanted side effects of other GABAergic drugs.[59] Advances in molecular pharmacology and genetic manipulation of rat genes have revealed that distinct subtypes of the GABAA receptor mediate certain parts of the anaesthetic behavioral repertoire.[60]
A useful property of the many benzodiazepine site allosteric modulators is that they may display selective binding to particular subsets of receptors comprising specific subunits. This allows one to determine which GABAA receptor subunit combinations are prevalent in particular brain areas and provides a clue as to which subunit combinations may be responsible for behavioral effects of drugs acting at GABAA receptors. These selective ligands may have pharmacological advantages in that they may allow dissociation of desired therapeutic effects from undesirable side effects.[61] Few subtype selective ligands have gone into clinical use as yet, with the exception ofzolpidem which is reasonably selective for α1, but several more selective compounds are in development such as the α3-selective drugadipiplon. There are many examples of subtype-selective compounds which are widely used in scientific research, including:
There are multiple indications thatparadoxical reactions upon — for example — benzodiazepines, barbiturates,inhalational anesthetics,propofol,neurosteroids, andalcohol are associated with structural deviations of GABAA receptors. The combination of the five subunits of the receptor (see images above) can be altered in such a way that for example the receptor's response to GABA remains unchanged but the response to one of the named substances is dramatically different from the normal one.
There are estimates that about 2–3% of the general population may suffer from serious emotional disorders due to such receptor deviations, with up to 20% suffering from moderate disorders of this kind. It is generally assumed that the receptor alterations are, at least partly, due togenetic and alsoepigenetic deviations. There are indication that the latter may be triggered by, among other factors,social stress oroccupational burnout.[63][64][65][66]
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