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We have expressed the α4β3δ and α4β3γ2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the α4β3δ receptor (EC50 ≈ 1.4 μmol/L) than of the α4β3γ2L subtype (EC50 ≈ 27.6 μmol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric δ/γ2L subunits with α4 and β3 subunits. A stretch of amino acids in the δ subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the δ and γ2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.
γ-Aminobutyric acid type A (GABAA) receptors are the major inhibitory neurotransmitter receptors in the mammalian brain. These receptors are members of the cys-loop family of ligand-gated ion channels (LGICs) that includes the nicotinic, serotonin type 3, and glycine receptors (Sieghart et al. 1999). Each member of the LGIC family is likely to be a pentamer in which homologous transmembrane (TM) subunits are arranged in a rosette conformation to form a central ion channel pore (Nayeem et al. 1994). Nineteen mammalian GABAA receptor subunits have been identified i.e. α1-6, β1-3, γ1-3, ρ1-3, δ, ε, θ, and π (McKernan and Whiting 1996; Barnard et al. 1998). The most common subtype of GABAA receptor in the mammalian CNS is the α1β2γ2 combination (reviewed by Sieghart et al. 1999; Whiting 2003), where the likely stoichiometry is 2α : 2β : 1γ (Farrar et al. 1999). However, the inclusion of other subunits within the pentamer results in differences in physiological function and sensitivity to the large number of pharmacological agents that target these receptors (Sieghart 1995).
Recently, it has been suggested that there are two inhibitory neuronal signaling pathways which are mediated by GABAA receptors, namely phasic inhibition and tonic inhibition (Brickley et al. 2001; Mody 2001). Phasic inhibition is evoked by the action potential-dependent release of high concentrations of GABA (0.5–1 mmol/L) at the synapse. These high local concentrations of GABA are transient and induce rapid inhibitory responses. In contrast, tonic inhibition is mediated by the persistent but low concentrations of GABA that either overspill from the synapse or are released from glial cells. The extrasynaptic receptors that are responsible for these functional differences are likely to include the α4, α5, α6, γ2/3 or δ subunits, with the most likely combinations in the brain being α4βxδ, α5β3γ2/3, and α6β2/3δ (see Farrant and Nusser 2005). Sur et al. (1999), using immunoprecipitation techniques, suggested that approximately one-third of the α4-containing GABAA receptor subtypes in the hippocampus and thalamus include the γ2 subunit while two-thirds co-associate with the δ subunit. Another study, using a similar approach and brain membrane extracts, suggested a higher ratio of the α4γ2 containing subtypes compared to α4δ (Bencsits et al. 1999). Overall, the available data suggest that a heterogeneous population of GABAA receptors containing the α4 subunit (mainly α4βxγ2 and α4βxδ subtypes) exists in the same regions of the brain.
Extrasynaptic receptor subtypes appear to have a higher sensitivity to GABA than those that mediate synaptic responses (Saxena and Macdonald 1994). This is not unexpected as these receptors respond to low ambient concentrations of GABA. The major aim of the present study was to identify the molecular basis for the higher potency of GABA at the α4δ-containing receptors. There is considerable evidence to demonstrate that binding sites within the LGIC family lie at subunit-subunit interfaces and agonist binding sites that are involved in GABAA receptor activation have been localized to the β–α subunit interfaces (see Amin and Weiss 1993). However, structural determinants lying outside the putative agonist binding domains have also been shown to affect agonist sensitivity. These include motifs in the α subunit that confer differential sensitivities between α6- or α1-containing receptors (Korpi and Luddens 1993) or between α3 and other α subunits (Bohme et al. 2004). The current structural and functional evidence suggests that, following formation of the receptor–agonist complex, a concerted movement of all subunits may be required to induce channel opening (see Unwin 2005). Thus, all subunits may contribute to agonist affinity and efficacy.
In this study, we explore the role of structural determinants within the δ and/or γ2L subunit that are important for conferring the differential potencies of agonists. We have generated δ/γ2L chimeric subunits and co-expressed these with α4 and β3 subunits in Xenopus oocytes for functional studies. The results demonstrate that a domain within the GABAA receptor δ subunit (S238-V264) confers high agonist sensitivity to the α4β3δ subtype.
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The importance of tonic inhibitory conductances mediated by extrasynaptic GABAA receptors has become clear over the last few years (see Farrant and Nusser 2005). At least in some cases, the extrasynaptic GABAA receptors have subunit compositions that are distinct from their synaptic counterparts and this confers their unique activation and pharmacological properties. Thus far, the δ subunit has been localized exclusively to extrasynaptic and perisynaptic membranes in a variety of cell types, including cerebellar granule cells (Nusser et al. 1998) and the dentate gyrus of the hippocampus (Wei et al. 2003). This subunit appears to be preferentially expressed with the α6 or α4 subunit. The α4 and δ subunits are co-localized in restricted regions of the brain, especially the thalamus and hippocampus (Bencsits et al. 1999; Sur et al. 1999). Furthermore, parallel changes in the expression levels of these subunits in response to various physiological and pharmacological challenges (see e.g. Sundstrom-Poromaa et al. 2002; Lovick et al. 2005) suggest that the α4βxδ subtype is likely to be a native receptor combination.
The presence of the γ2 subunit in the GABAA receptor has been implicated in clustering of receptor subtypes at the post-synaptic synapse (Essrich et al. 1998) suggesting that the α4β3γ2 receptor is located predominantly in this region. In this study, we have, therefore, used the rat α4β3δ and α4β3γ2 receptors as representatives of putative extrasynaptic and synaptic receptors, respectively.
The human forms of these GABAA receptors were characterized previously in stably transfected cell lines (Brown et al. 2002) and it was shown that the α4β3δ receptor was more sensitive to GABA than the α4β3γ2 subtype. In the present study, we found a similar difference in potency (approximately 20-fold) for the rat receptor subtypes expressed in Xenopus oocytes. It is thus clear that inclusion of either the δ or γ2 subunit in the complex has a significant effect on the potency of GABA activation. However, neither of these subunits has been implicated directly in the binding of GABA or other receptor agonists. Site-directed mutagenesis studies have been used to localize the agonist activation sites to the interfaces between the β and α subunits (Amin and Weiss 1993). These sites appear to be formed by distinct loops of amino acids contributed by the primary (β) and secondary (α) subunits, in homologous positions to neurotransmitter binding sites in other members of the receptor family.
The aim of the present study was to identify structural determinants within the GABAA receptor δ subunit that contributes to the higher potency of δ-containing receptors for GABAergic agonists. We used a random chimeragenesis approach to form δ/γ2L subunits and expressed these with native α4 and β3 subunits in Xenopus oocytes. Similar approaches have been used to investigate structural requirements for the binding of benzodiazepine site ligands to other GABAA receptor subtypes (e.g. Boileau et al. 1998; Derry et al. 2004).
Our major finding is that a domain in the GABAA receptor δ subunit (S238-V264) confers high agonist sensitivity to the α4β3δ subtype. For all agonists studied (GABA, muscimol, THIP, and I4AA) inclusion of the χ277 chimeric subunit (containing the first 277 residues of δ) imparted δ-like characteristics to the receptor activation properties. In contrast, inclusion of the χ237-subunit led to receptors whose activation characteristics were not significantly different from the wild-type α4β3γ2L receptor. These results suggest that a specific region of the δ subunit (residues 238–277) plays a major role in conferring higher agonist potency. Inspection of the sequence homologies of the rat δ and γ2 subunits (Fig. 1c) further restricts this domain. Based on the recent receptor model and alignments of Ernst et al. (2005), the sequences of these subunits are identical between residues 265 and 277 (δ subunit numbering); thus the domain that confers high agonist potency can be narrowed further to residues 238–264. In the case of activation by either THIP or I4AA, the receptor that included the χ255 chimeric subunit displayed similar potency to the α4β3δ subtype. Again inspection of the sequence homology shows that there is conservation between residues 249 and 255 (δ subunit numbering) suggesting that the sequence S238-M248 of the δ subunit, lying in the N-terminal segment of TM1 may be of particular importance in conferring high affinity for these agonists.
Of the 27 amino acid residues lying in the S238-V264 domain of the δ subunit, 16 are identical to those in the equivalent positions of γ2 (see Fig. 1c). Where divergences occur, homologous amino acids are found in five positions (S238T, M240I, L245I, M248L, Q257K) leaving only six semi- or non-conservative substitutions (S242C, V243T, A247V, S256N, A258D, V264T), five of which lie within the putative TM1 domain (see Fig. 1c). In terms of overall structure, this domain appears to be highly conserved among all subunits of the cys-loop LGIC family (see Ernst et al. 2005) but, despite this homology, there is increasing evidence for its influence on the binding–gating coupling properties of different receptor subtypes (see below).
The structural determinants within the δ and γ2L subunits that underlie differences in agonist efficacy are less clear. In the α4β3δ receptor, THIP has been suggested to act as a ‘superagonist’ (see above and Brown et al. 2002). No clear patterns for determining agonist efficacy have emerged from the present study. This is perhaps not surprising as many factors lying in the pathway from ligand recognition to channel opening may dictate differences in transduction efficiency.
In agreement with the earlier results of Brown et al. (2002) who studied the human GABAA receptor subtypes, we found that bicuculline, SR95531, and picrotoxin did not differentiate between the wild-type rat α4β3δ and α4β3γ2 receptors. Apart from a small effect (approximately threefold) of bicuculline on the χ237 containing receptor, none of the expressed receptors showed any significant differences in their responses to these ligands. These results suggest that the influence of the δ/γ2L subunits on agonist potency does not involve radical changes in the architecture of the binding sites. Similarly, the lack of influence of the chimeric subunit on inhibition by the channel blocker, picrotoxin, suggests that the open channel characteristics are not dramatically affected. These results are consistent with the involvement of the δ/γ2L subunits, in particular the TM1 domain, in the transduction mechanism leading from agonist binding to channel opening rather than agonist recognition per se.
As both the α4 and δ subunits have been reported to be difficult to express in recombinant systems, the actual subunit composition of the receptors investigated in different laboratories has been questioned (see Borghese et al. 2006). In the present study, we have compared the expression of different combinations of α4, β3, δ, and γ2L subunits. Only the α4β3, α4β3δ, and α4β3γ2L combinations resulted in the expression of functional GABAA receptors. The expressed α4β3 and α4β3δ receptors displayed distinct properties with respect to the times required for expression, block by Zn2+, sensitivity to THIP and desensitization characteristics (unpublished results). However, increasing the relative amount of cRNA encoding the δ subunit affected both the EC50 for GABA activation and the apparent Hill coefficient of the response. Control experiments revealed that using higher than stoichiometric amounts of the δ subunit cDNA does not force the aberrant expression of other functional receptors containing only one or two subunit isoforms. These results are difficult to rationalize in terms of expression of a heterogeneous population of receptors. It seems more likely that the effects of changing cRNA ratios reflect changes in subunit stoichiometry within the pentameric α4β3δ complex. Further analysis of receptor composition under different expression conditions using biophysical approaches (see e.g. Barrera et al. 2005) will be required to interpret these observations. In the present study, although we cannot exclude the possibility that a heterogeneous population of receptors existed, the results obtained for the native receptor subtypes combinations are similar to those reported by others (see Brown et al. 2002; Storustova and Ebert 2006). Furthermore, the differential responses obtained for receptors incorporating the chimeric δ/γ2L subunits suggest that these subunits were also efficiently incorporated into the receptors examined.
A major issue in studying GABAA receptor properties is how the binding of an agonist to its extracellular sites that are predicted to lie approximately 30 Å above the membrane surface is communicated to the channel gate lying deep within the TM domains (see Unwin 2005). A number of previous studies have used chimeric/mutagenesis strategies to investigate the roles of the δ/γ2 subunits in signal transduction. Using a chimeragenesis approach, Jones-Davis et al. (2005), demonstrated that the TM1 domain of the γ2 subunit was important for benzodiazepine potentiation of recombinant α1β2γ/δ receptors. Haas and Macdonald (1999) reported that the kinetics of desensitization of the α1β3δ receptor were slower than those of the α1β3γ2L subtype and Bianchi and Macdonald (2002) subsequently demonstrated that these results could be attributed to determinants within the extracellular N-terminal domain and two residues in the TM1 domain (V233, Y234). In studies of general anesthetic action, it has been shown that determinants for modulation of GABA currents by propofol and pentobarbital lie within the N-terminal and TM1 domains of these subunits (Feng and Macdonald 2004; Feng et al. 2004). Together with our current results, the available data suggest that the TM1 domain of the δ subunit plays a significant role in conferring the unique functional and pharmacological properties of δ-containing receptors.
In conclusion, we have identified a structural domain within the GABAA receptor δ subunit (S238-V264) that confers high agonist sensitivity to the α4β3δ subtype. Further, we have shown that these effects are agonist-dependent and are likely to involve changes in the transduction mechanism that links agonist binding to channel activation.