Candidate Review:

Assembly and Heterogeneity of GABAA Receptors

Katharine N. Gurba

Neuroscience Graduate Program, Vanderbilt University School of Medicine, U1205 Medical Center North, Nashville, TN 37232, USA.
Correspondence e-mail: kate.gurba@vanderbilt.edu

Abstract | Full Text | PDF

ABSTRACT | GABAA receptors (GABAARs)are pentameric, ligand-gated chloride channels that mediate the majority offast inhibitory synaptic neurotransmission in the brain.  The receptors areassembled from a repertoire of 19 subunits (α1-6, β1-3, γ1-3,δ, ε, π, and ρ1-3), providing the possibility for vastisoform heterogeneity.  Because the subunit subtypes included in a receptordetermine its physiological and pharmacological properties, identification ofreceptor isoforms has clear clinical relevance.  A large body of literatureindicates that GABAARs do not assemble randomly; rather,incorporation of specific subunits into a receptor is regulated at manylevels.  Each subunit has a characteristic temporal and spatial expressionpattern; however, most neurons express many GABAAR subunits atonce.  Consequently, certain “rules” of assembly must exist to limit receptorheterogeneity.  In this review, we discuss the regulation of GABAARbiogenesis, including limitation of heterogeneity, as well as the specificreceptor isoforms that have been identified in vivo.

The vast majority of inhibitoryneurotransmission in the brain is mediated by γ-aminobutyric acid (GABA). It has been detected in approximately 30% of all synapses1 and acts via ionotropic GABAAreceptors, which mediate fast inhibitory neurotransmission2, and metabotropic GABAB receptors,which mediate slower inhibitory effects3.  GABAA receptors (GABAARs)are chloride channels belonging to the Cys-loop receptor superfamily ofligand-gated ion channels (LGIC), which also includes nicotinic acetylcholinereceptors (nAChR), 5-hydroxytryptamine type 3 receptors (5-HT3), and glycine receptors(GlyR)4.  Like most members of thissuperfamily, GABAARs are pentamers that are assembled from an arrayof homologous subunits.  All subunits share a common structure: each contains alarge, extracellular N-terminal domain, which contains the ligand-binding siteand the eponymous Cys-loop; four α-helical transmembrane domains (M1-4); alarge intracellular loop between the third and fourth transmembrane helices(M3-M4 loop); and a very short, extracellular C-terminal domain5 (Figure 1a).

Nineteen subunits, grouped by sequencehomology into eight subunit families, have been identified for the GABAAreceptor: α1-6, β1-3, γ1-3, δ, ε, π, andρ1-36.  Several of these subunit subtypesalso undergo alternative splicing and/or RNA editing, further increasing thepotential diversity of GABAA receptor isoforms.  Each subunitexhibits a characteristic expression pattern in the brain; however, thesepatterns overlap extensively.  Indeed, a single neuron can express manysubunits simultaneously.  Consequently, many but not all of themathematically-possible GABAAR isoforms could exist somewhere in thebrain.  The most common isoforms, however, are thought to comprise two αsubunits, two β subunits, and one γ or δ subunit7-9 (Figure 1b), though this remains a subject ofvigorous debate.

The large variety of GABAARisoforms exhibit a concomitant variety of physiological properties2.  For instance, most receptorscontaining a γ subunit are located in the synapse, where they mediatephasic inhibition in response to presynaptically-released GABA10.  These receptors have a relativelylow affinity for GABA, activate quickly, desensitize extensively, anddeactivate slowly.  Conversely, receptors containing a δ subunit arelocated outside the synapse, where they mediate tonic inhibition in response tolow concentrations of ambient GABA.  Unsurprisingly, δ-subunit-containingreceptors also differ physiologically; they have a relatively high affinity forGABA, activate slowly, desensitize minimally, and deactivate rapidly11.

Additionally, GABAARs havebeen linked to many diseases and disorders, including epilepsy12-14, insomnia15, anxiety16, depression16, schizophrenia17, alcoholism18, and autism19.  Predictably, then, GABAARsare targeted by numerous drugs, particularly sedatives, anxiolytics, andanticonvulsants; examples include benzodiazepines, zolpidem, etomidate, andpropofol20, 21.  Both the pathology and thepharmacology of GABAARs depend highly upon receptor subunitcomposition – for instance, epilepsy-associated mutations have been identifiedonly in the α1, β3, γ2, and δ subunits, and benzodiazepinesact only at receptor isoforms containing both a γ subunit and certain αsubunits.

Figure 1 |GABAA receptor morphology. a | Structure of a GABAAR subunit.  Cys-loop cysteines marked in orange; transmembrane domains enclosed in cylinders and numbered 1-4. b | Schematic view of most common GABAA isoform  (putative) from the synaptic cleft.  G = GABA binding site; BZ = benzodiazepine binding site.

Given the prevalence of GABAARexpression, the pathology resulting from receptor malfunction, and thepharmacological dependence upon isoform identity, it is clearly important tounderstand the process of receptor assembly.  Therefore, in this review, wewill examine the generation of GABAAR diversity.  First, we willreview the general processes of receptor biogenesis, after which we willdiscuss the selective oligomerization of GABAAR subunits.  Finally,we will examine the ultimate product of these processes: native GABAAreceptor isoforms.

BIOGENESIS OF GABAARECEPTORS

As with other LGICs, GABAAreceptor subunits are inserted co-translationally into the membrane of theendoplasmic reticulum (ER).  There, they fold and oligomerize in a process thatdepends heavily upon ER-resident chaperones.  The process of receptoroligomerization is slow and inefficient; studies suggest that approximately 70%of subunits are degraded without being incorporated into a pentameric receptor,and receptors do not appear on the cell surface for several hours followingtransfection22.  While in the ER, GABAAreceptor subunits also undergo typical protein modifications, including theearly stages of N-linked glycosylation.  Interestingly, however, N-linkedglycosylation is not required for subsequent forward trafficking, althoughmultiple glycosylation sites have been identified on all subunits23 and glycosylation is necessary forproper assembly and trafficking of other Cys-loop receptors24, 25.  Properly folded and assembledsubunits proceed to the Golgi apparatus, where they undergo furthermodification such as palmitoylation and glycan trimming26.  With the assistance of multipleGABAAR-associated proteins, receptors are then trafficked to theneuronal surface.  They may be inserted directly into their final subcellularlocation (i.e. post-, peri-, or extrasynaptic), or they may diffuse intothat location after membrane insertion27.  Finally, GABAARs undergoconstitutive and activity-dependent endocytosis (both clathrin-dependent andclathrin-independent)28, after which they are recycled to thecell surface or targeted for lysosomal degradation.  Every step of GABAAreceptor assembly and trafficking is regulated by signals within the subunits29 as well as by various associatedproteins30.

SELECTIVE OLIGOMERIZATION OF GABAARECEPTOR SUBUNITS

After temporal andspatial regulation of subunit expression, the first (and, arguably, the mostimportant) opportunity for a neuron to control what GABAA receptorisoforms it will produce is the process of selective subunit oligomerization. Presumably, a neuron expressing many GABAAR subunit subtypes wouldhave a hierarchical yet flexible assembly mechanism that favors associationbetween certain subunits and, ultimately, directs the incorporation of assemblyintermediates (e.g. dimers, trimers) into full receptors.  Indeed, severalstudies have indicated that, though all subunit combinations can formoligomers, only a subset can form pentamers23.  This is a key distinction becausepentamers are trafficked to the cell surface, but oligomers of lower molecularweight are retained in the ER and subsequently degraded23, 31.  Importantly, some disease-causingmutations appeared to reduce surface expression and function by disrupting theprocess of oligomerization14.

Expression of recombinant subunits inheterologous cells has provided insight into the “rules” governing assembly ofthe most prevalent subunit subtypes.  When expressed individually, α1,β2, and γ2 subunits formed primarily monomers and dimers, as didcombinations of γ2 with either α1 or β2/3.  Conversely,co-expression of α1 and β2/3 subunits, with or without γ2subunits, predominantly yielded pentamers, indicating that the combination ofα and β subunits is necessary and sufficient for complete receptorassembly31, 32.  Interestingly, however, receptorsincluding a third (non-α/β) subunit appear to assemble moreefficiently.  When α, β, and a third subunit (either γ, δ,ε, or π) were co-expressed in heterologous systems, the kineticsignature of αβ receptors could not be detected33-35; furthermore, that signature has beendetected in very few neurons36, 37.  Clearly, both neurons andheterologous cells are capable of selective oligomerization, suggesting theexistence of assembly signals within the subunits themselves. 

Several studies have, in fact,isolated amino acid sequences and individual residues that are important forspecific subunit interactions29, 38. These sequences have beenidentified in the α139-43, α639, β342-45, γ242, 46, and γ347 subunits, primarily in the largeN-terminal domain, though there were some reports of assembly sequences in theM3-M4 loop48, 49.  Although homology modeling based onthe nAChR50 and AChBP51 has provided some insight into thestructural basis of these interactions, it is important to note that thesesequences might not directly contact adjacent subunits; rather, they mightsimply facilitate oligomerization by encouraging proper protein folding.

HETEROGENEITY IN VIVO: NATIVEGABAA RECEPTOR ISOFORMS

Most studies mentioned thus far havebeen conducted in heterologous expression systems or in cultured neurons. Because of the great potential for GABAAR heterogeneity, it isnecessary to use such systems to investigate properties of specific subunits (i.e.assembly sequences) and isoforms (i.e. kinetic and pharmacologicalproperties).  Unfortunately, these studies cannot answer a crucial question:what GABAA receptor isoforms actually exist in the brain?  In anattempt to construct a standardized response to that question, theInternational Union of Pharmacology recently established a list of potentialnative GABAAR oligomers6.  These receptor isoforms weredivided into three categories (“identified”, “existence with high probability”,and “tentative”) based on multiple types of evidence.  The authors alsospecified a logical strategy, summarized below, for determining whether or nota receptor isoform exists in vivo.  First, the long list of potentialisoforms can be narrowed based on subunit co-expression patterns, which can beascertained by in situ hybridization and immunoreactivity.  If subunitsare indeed co-expressed in a specific cell type, evidence for association ofthose subunits should then be sought, primarily throughco-immunoprecipitation.  Subunits that associate should be co-expressed inheterologous systems, where electrophysiology can be performed andcharacteristic kinetics and pharmacology can be assessed.  These characteristicproperties can then be sought in neurons.  Finally, knockout animals can becreated and studied for the absence of characteristic physiology andpharmacology associated with isoforms containing the deleted subunit.  The listof “identified” and “high probability” isoforms, along with their localization(regional and subcellular) and basic forms of inhibition (phasic or tonic), ispresented in Table 1

Isoforms that have been unequivocallyidentified

Given the widespread distribution ofthe α1β2γ2 GABAAR isoform, it is perhaps unsurprisingthat this isoform is thought to account for up to 60% of all GABAAreceptors in the brain20. Mice lacking either the α1 orβ2 subunit have been generated; in both lines, total GABAARexpression in the brain was reduced by more than 50%52.  A γ2 knockout mouse was foundto lack 94% of all benzodiazepine binding sites53 (recall that the BZ binding site islocated at the interface of an α and a γ subunit; consequently, thisresult indicates that receptors including the γ1 or γ3 subunit mightmake up only 6% of all αβγ receptors).  As indicated in Table1, the other five α subunits can likewise co-assemble with β andγ2 subunits.  Strong evidence for the existence of theseαxβxγ2 receptors is provided by isoform-specific pharmacologyfrom benzodiazepine (BZ) site ligands.  Such ligands include classicbenzodiazepines (i.e. diazepam); imidazobenzodiazepines (i.e.flumazenil and Ro15-4513); and the so-called “Z-drugs” (i.e. zolpidemand zaleplon).

Classic benzodiazepines cannot bindreceptors containing α4 or α6 subunits, and they have much loweraffinity for receptors containing γ1 or γ3 subunits than forreceptors containing γ2 subunits.  Furthermore, through the use oftransgenic mice, the various actions of benzodiazepines have been attributed tospecific α subunit subtypes.  Point mutations conferring diazepaminsensitivity were introduced into the genes of individual α subunits andthe resulting mice were subjected to behavioral tests with and withoutadministration of diazepam54, 74, 75.  Results indicated that the α1subunit mediated the sedative, anterograde amnestic, and some of theanticonvulsant effects of diazepam74, 76; the α2 and α3 subunitsmediated the anxiolytic and muscle-relaxant effects54, 75 and the α5 subunit was involvedin amnestic effects as well as other aspects of learning and memory. Imidazobenzodiazepines, however, bind without regard to α subunitsubtype.  Therefore, receptors that are benzodiazepine-insensitive but imidazobenzodiazepine-sensitivecan be identified as α4βγ2 or α6βγ2 isoforms. Conversely, Z-drugs act with differing potency at BZ-sensitive isoformscontaining α1,2,3, or 5; specifically, they display high potency atα1βγ2 isoforms, lower potency at α2βγ2 andα3βγ2 isoforms, and no action at α5βγ277.  Taken together, thesepharmacological properties allow positive identification ofα1βγ2 and α5βγ2 receptors, as well as tentativeidentification of α(2,3)βγ2 and α(4,6)βγ2receptors; however, expression patterns can differentiate these latter twopairs of isoforms.  Consequently, all αβγ2 isoforms areconsidered to have been identified in vivo.

The aforementioned evidence accountsfor six of the 11 identified native isoforms.  Four of the remaining fiveisoforms contain the δ subunit, which possesses many unusual propertiesthat help to identify δ-subunit-containing isoforms in vivo. First, the δ subunit has been found exclusively in extrasynapticmembranes, where it is incorporated into receptors that have a high affinityfor GABA and mediate a constant, “tonic” current with low amplitude and littledesensitization11, 78.  The pharmacology ofδ-subunit-containing receptors is also very different from that ofγ-subunit-containing receptors.  Though GABA binds to δ-containingisoforms with high affinity, its efficacy is relatively low.  Conversely,ethanol79 and neuroactive steroids80 act strongly atδ-subunit-containing receptors.  Demonstration of these properties invivo56, combined with co-localization,co-immunoprecipitation, and gene deletion studies81, have allowed identification of theδ-subunit-containing receptors listed in Table 155.

Table 1 | GABAAR isoforms likely to exist in vivo.

List of isoforms from reference 6, which also identifies “tentative” isoforms that assembled in heterologous systems (ρ1-3, αβγ1, αβγ3, αβε, αβθ, αβπ, and αxαyβγ2). Also see the following general references: in situ hybridization70; immunohistochemistry71, 72; reviews20, 73.

The last isoform that has beenidentified unequivocally in vivo comprises ρ subunits alone.  Thesereceptors, previously classified as GABAC receptors due to theirunique pharmacology, are expressed predominantly in retinal bipolar cells63; however, low levels of ρsubunit transcripts have also been detected in hippocampus82, cerebellum83, amygdala84, and certain brain areas importantfor visual signal processing (superior colliculus, lateral geniculate nucleus,and visual cortex)62, 83 .  Evidence for both homomeric andheteromeric ρ isoforms has been reported85, 86; consequently, the subunit subtypespresent in these receptors remain undefined.

Isoforms that exist with highprobability

Finally, we will briefly discuss theevidence supporting the “existence with high probability” of certain key GABAARisoforms listed in Table 1.  Each of these isoforms assemblesefficiently and has been studied extensively in heterologous systems11, 31, 33, 35, 80, 87-89; moreover, the subunits areco-expressed in vivo70-72.  Indeed, most were not classified as“identified” simply because few animal studies have been conducted.  First,although α1 and γ2 subunits seem to partner most frequently with theβ2 subunit, expression patterns indicate that this cannot always be thecase, because certain areas expressing the α1 and γ2 subunits do notexpress the β2 subunit71.  In these areas, it is quite likelythat α1β3γ2 receptors are formed, as indicated by variouspharmacological properties64.  The evidence supporting theexistence of α5β3γ2 is also extensive; the only reason that itis not considered to be unequivocally identified is that, to date, α5 andβ3 have not been co-immunoprecipitated6.  However, these three subunits havebeen co-localized71, α5 and β3 subunits wereco-depleted in knockout mice6, α5-selective etomidate effectshave been identified90, and electrophysiology indicates thatthis isoform mediates tonic inhibition in the hippocampus66.  Another widely-accepted isoform,α1βδ, clearly assembled in heterologous systems and responded toknown modulators of δ-subunit-containing receptors.  Furthermore, onerecent report identified this isoform in molecular layer interneurons of thehippocampus65. Finally, as previously mentioned,two different αβ isoforms have been identified in rat brain viasequential co-immunoprecipitation37 and electrophysiology36.

CONCLUDING REMARKS

GABAA receptors in thebrain are ubiquitous, implicated in many diseases, and highly heterogeneous. Each receptor isoform exhibits unique physiological and pharmacologicalproperties and a characteristic expression pattern.  Consequently, a thoroughunderstanding of GABAAR assembly, trafficking, and function couldyield significant therapeutic advantages, such as isoform-specific drugs thatminimize unwanted side effects.  Currently, only 11 GABAAR isoformshave been conclusively identified in vivo, and the existence of anothersix is considered to be highly probable.  Further study of the assembly,trafficking, and function of these receptors may improve clinical practice, aswill attempts to identify other GABAAR isoforms that occur in thebrain.

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