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GABAA receptors are the main class of inhibitory ligand-gated ion channels in the mammalian CNS (receptor nomenclature follows Alexander et al., 2013). They are hetero-pentameric complexes forming a central anion-conducting channel. To date, eight classes of GABAA receptor subunits have been identified, with half of these exhibiting multiple isoforms: α(1–6), β(1–3), γ(1–3), δ, ε, θ, π and ρ(1–3). Although GABAA receptors exhibit distinct regional and developmental expression patterns in the CNS, γ2-containing receptors are considered the dominant subtype found at GABAergic inhibitory synapses (Somogyi et al., 1996). By contrast, δ-containing receptors are thought to be exclusively found at extrasynaptic sites, where they play an important role in mediating tonic inhibition (Farrant and Nusser, 2005). Extrasynaptic α4βδ receptors have been identified in several neuronal cell types, including dentate gyrus granule cells and thalamic relay neurons (Sur et al., 1999; Peng et al., 2002). In addition, extrasynaptic α6δ and α1δ pairs have been identified in cerebellar granule cells (Jones et al., 1997) and hippocampal interneurons (Glykys et al., 2007), respectively, together with extrasynaptic α5βγ and α1β assemblies identified in the hippocampus (Mortensen and Smart, 2006; Glykys et al., 2008).
The subunit composition of GABAA receptors is an important determinant of their functional properties as demonstrated by the type of α subunit and presence of the γ2 subunit affecting, for example, receptor sensitivity to benzodiazepines (Korpi et al., 2002). Given that orthosteric and many allosteric binding sites on GABAA receptors are interfacial (Sieghart et al., 2012), it becomes important to understand whether there are preferred subunit stoichiometries that will critically define the nature of these subunit interfaces and thus the receptor's response to ligand binding. Compared with αβγ receptors, we know least about δ subunit-containing GABAA receptors, which play an important role mediating tonic inhibition in several brain regions (Brickley et al., 2001; Porcello et al., 2003; Farrant and Nusser, 2005; Santhakumar et al., 2010). To address this deficit, we have employed a pharmacological analysis, in combination with a reporter mutation, to better understand the structural properties of δ-containing receptors using heterologous expression systems (e.g. HEK-293 and Xenopus laevis oocytes). Previous reports note that some functional discrepancies have been observed for αβδ receptors, such as EC50 values for GABA and ethanol sensitivity (Wallner et al., 2003), which have been postulated to arise, in part, from differences in subunit stoichiometry (Borghese et al., 2006; Wagoner and Czajkowski, 2010). Although the stoichiometry of major synaptic αβγ GABAA receptor isoforms has broad consensus support for 2α:2β:1γ (Backus et al., 1993; Chang et al., 1996; Tretter et al., 1997), an unequivocal view of the stoichiometry for extrasynaptic δ-containing receptors remains elusive. Although atomic force microscopy of recombinant α4β3δ receptors has suggested a stoichiometry of 2α:2β:1δ (Barrera et al., 2008), biochemical analysis of recombinant α4β2δ receptors indicates that more than one δ can be incorporated into the receptor complex (Wagoner and Czajkowski, 2010). Moreover, it was recently demonstrated on the basis of using α1β3δ (Kaur et al., 2009) and α6β3δ concatemers (Baur et al., 2009) that more than one δ subunit can be incorporated into functional channels, although for the former subtype, a constrained conformation of 2α:2β:1δ most closely resembled the pharmacological profile of unconstrained recombinant α1β3δ receptors (Kaur et al., 2009).
In this study, we have examined the subunit stoichiometry of functional recombinant α4β3δ receptors, utilizing polar substitutions of a highly conserved leucine residue within the second transmembrane region (M2) of GABAA receptors. This residue exchange acts as a reporter mutation causing a profound increase in agonist potency consequently displacing the agonist dose–response curve (Chang et al., 1996; Chang and Weiss, 1999), as also observed for nicotinic ACh receptors (nAChRs) (Filatov and White, 1995; Labarca et al., 1995) and 5-HT3 receptors (Yakel et al., 1993). The extent of the curve shift is correlated with the number of polar substitutions per ion channel complex, and this has been used to deduce the subunit stoichiometry of recombinant α1β2γ2 GABAA receptors (Chang et al., 1996). By inserting this highly characterized 9′ serine to leucine (L9′S) mutation into α4, β3 and δ subunits, we derive a subunit stoichiometry of 2α:2β:1δ for functional α4β3δ GABAA receptors. Furthermore, our data indicate that for three different, but commonly used, cDNA transfection ratios, the number of incorporated δ subunits seemingly remains fixed at one.
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Although the stoichiometry of synaptic α1β2γ2 subunit-containing GABAA receptors has consensus support for 2α:2β:1γ (Backus et al., 1993; Chang et al., 1996; Tretter et al., 1997), the stoichiometry of extrasynaptic δ-containing receptors remains unclear and potentially variable with a dependence on experimental conditions. This may reflect a difference in the co-assembly properties of δ , with different α and β subunits (Baur et al., 2009; Kaur et al., 2009; Wagoner and Czajkowski, 2010). We used an alternative approach to probe α4β3δ stoichiometry by introducing a well-characterized 9′ leucine-to-serine mutation into the M2 domains of α4, β3 and δ subunits. Each polar substitution increased the GABA sensitivity of mutant subunit-containing receptors (by approximately fourfold) in relative proportion with the number of mutant subunits assembled in the receptor. This, in conjunction with data derived from cells co-expressing mutant and respective WT subunits, revealed a relatively consistent subunit stoichiometry, by these methods, of 2α, 2β and 1δ.
Assumptions and limitations
Our deductions regarding α4β3δ GABAA receptor stoichiometry are predicated on the assumption that the L9′S mutations do not perturb the ‘normal’ subunit stoichiometry of these receptors. Because N-terminal motifs have been established as the key determinants of GABAA receptor subunit assembly (Connolly et al., 1996; Taylor et al., 1999; Klausberger et al., 2001), it seemed unlikely that a point mutation within the ion channel-lining M2 region would alter receptor subunit stoichiometry. However, it is intriguing that for most αβδδm-expressing cells, the component that was attributable to αβδm receptors was larger than that for αβδ receptors (∼75 and 24%, respectively), suggesting that δm might be more efficiently incorporated into functional receptors than δ.
Given the M2 location of the point mutation, a more likely explanation for the disproportionate percentage components is that the mutation may affect the gating kinetics of the receptor. For nAChRs (Filatov and White, 1995) and GABAA α1β3γ2L receptors (Bianchi and Macdonald, 2001), it has been demonstrated that 9′ mutant-containing receptors can exhibit altered single-channel conductances and/or open probabilities. This could cause the apparent percentage components of αβδ and αβδm to vary (Chang et al., 1996). Nevertheless, because our conclusions rely on the number of observable components in the dose–response curves and not on the relative contribution of each individual component, our conclusion that α4β3δ receptors contain only one δ subunit still remains valid.
Comparison with previous studies
To date, only two studies have investigated the subunit stoichiometry of unconstrained recombinant α4β2/3δ receptors. Although atomic force microscopy has revealed a subunit stoichiometry of 2α4:2β3:1δ (Barrera et al., 2008), the immunopurification of cell surface α4β2δ receptors suggested that by increasing the relative abundance of δ, more than one δ can be incorporated into the receptor complex (Wagoner and Czajkowski, 2010). Moreover, a study using receptor expression in oocytes reported that increasing relative amounts of δ cRNA increased the GABA EC50 and decreased the Hill slopes for α4β3δ GABA dose–response curves (You and Dunn, 2007).
Although a change in stoichiometry may account for altered receptor function, it is also equally plausible, from studies using concatemers and thus constrained subunit positions, that δ subunits may assume various locations within a functional receptor pentamer, and also potentially contribute to an agonist-binding site (Kaur et al., 2009; Sigel et al., 2009). Thus the previously described effects on receptor function may also have arisen from the rearrangement of subunits within the receptor. Indeed, for concatemeric α1β3δ receptors, those with an βαβαδ (anticlockwise) subunit arrangement appear to be ∼26-fold less sensitive to GABA than receptors with the βαβδα (anticlockwise) subunit arrangement (Kaur et al., 2009), demonstrating the functional importance of subunit location within a receptor pentamer.
Our data indicate that, at least for three commonly used α : β : δ transfection ratios 1:1:1, 1:1:10 or 10:1:10 (Borghese et al., 2006; Stórustovu and Ebert, 2006; Barrera et al., 2008; Hoestgaard-Jensen et al., 2010), the number of incorporated δ subunits seemingly remains fixed at one. Moreover, we found no significant effect of altering cDNA transfection ratio on α4β3δ receptor function. In accordance with our findings, another oocyte study had demonstrated no significant effect of altering cRNA transfection ratio on the sensitivity of WT α4β3δ receptors to GABA or Zn2+ (Borghese and Harris, 2007).
Although the discrepancy between our observations and those previously reported remain unclear, one difference may be the use of different expression systems. Alternatively, the use of different β isoforms may also give rise to these discrepancies. Given that β2 and β3 subunits have been demonstrated to have distinctive assembly properties (Taylor et al., 1999), this might have important implications for their oligomerization with δ subunits.
Although we demonstrate a stoichiometry of 2α:2β:1δ for α4β3δ receptors, our data give little indication of subunit arrangement, which could be an important determinant of αβδ receptor function (Baur et al., 2009; Kaur et al., 2009). The subunit positional arrangement of α1βγ2 receptors is widely accepted to be βαβαγ (anticlockwise) (Baumann et al., 2001; 2002; Baur et al., 2006; Smart and Paoletti, 2012). Given the conflicting evidence regarding the number of incorporated δ subunits, it is unsurprising that the subunit arrangement of recombinant αβδ remains undefined. For α4β3δ receptors with a stoichiometry of 2α:2β:1δ, structural microscopic analysis has revealed a predominant βαβαδ anticlockwise arrangement (Barrera et al., 2008), suggesting δ can assume the position of the γ2 subunit in an αβγ receptor. However, in the same study, a minority of receptors (∼21%) were found to have an alternative βαβδα subunit arrangement, indicating more than one arrangement may be possible (Barrera et al., 2008). Indeed it has been recently demonstrated that δ can assume multiple positions when constrained within αβδ concatemers (Baur et al., 2009; Kaur et al., 2009). Intriguingly, concatemeric α4β2δ receptors with the βαβαδ conformation (Shu et al., 2012) form functional receptors with similar pharmacological profiles to unconstrained recombinant α4β2δ receptors (Stórustovu and Ebert, 2006), whereas α1β3δ receptors formed from the alternative βαβδα anticlockwise arrangement exhibit similar GABA and Zn2+ sensitivities to non-concatenated receptors (Kaur et al., 2009). These findings suggest that the arrangement of recombinant and native δ-containing receptors is still open to question.
To conclude, we demonstrate that the subunit stoichiometry of heterologously expressed α4β3δ receptors is 2α:2β:1δ. This stoichiometry remains unchanged even with varying cDNA transfection ratios, which may reflect that this is the preferred, dominant subunit assembly for this important extrasynaptic GABAA receptor subtype that underlies tonic inhibition in some areas of the brain.