The promiscuous role of the epsilon subunit in GABA_A receptor biogenesis

Abstract

The formation of α1β2γ2ε receptors suggests that the ε subunit does not displace the single γ2 subunit in α1β2γ2 receptors. Thus, ε must replace α and/or β subunit(s) if the pentameric receptor structure is to be preserved. To assess the potential for which subunit is replaced in αβε and αβγε receptors we analyzed the assembly and functional expression of the ε subunit with respect to α1, β2 and γ2 subunits. Using concatenated subunits, we have determined that ε is capable of substituting for either (but not both) of the α subunits, one of the β subunits, and possibly the γ2 subunit. However, the most likely sites at which the ε subunit may contribute to receptor function appears to be at position 1 (replaces α1) in αβγε (ɛ–β2–α1–β2–γ2) receptors, or at position 4 (replaces β2) in αβε (α1–β2–α1–ɛ–β2) receptors. In both cases, it appears that only a single GABA binding site is present.

Introduction

The rapid inhibitory actions of GABA are exerted through the activation of ionotropic GABA_A receptors. The importance of GABA_A receptors is exemplified by their therapeutic targeting in the treatment of anxiety, sleep disorders and epilepsy (Foster and Kemp, 2006). In addition, a number of mutations in GABA_A receptor subunits, each responsible for a reduction in GABAergic inhibition, have been associated with epilepsy (Kang and Macdonald, 2004, Sancar and Czajkowski, 2004, Hales et al., 2005, Kang et al., 2006, Feng et al., 2006, Mizielinska et al., 2006, Gallagher et al., 2007). This is in keeping with the observation that receptor negative allosteric modulators promote epileptic seizures, whereas positive allosteric modulators are anticonvulsants.

In mammals, GABA_A receptors are constructed as pentameric structures from multiple subunits selected predominantly from the following distinct classes: α(1–6), β(1–3), γ(1–3), δ, ɛ, θ and π, creating an incredible (16⁵/5 ɛ 210,000) potential for structural diversity. However, GABA_A receptor assembly appears to be strictly controlled and there is a general agreement that the co-expression of α and β subunits is sufficient for the production of GABA-gated chloride currents (Moss and Smart 2001). However, native receptors are believed to include an additional subunit, typically γ, δ or ɛ, which affects, or imparts, modulatory functions to benzodiazepines, neurosteroids and anesthetics (Whiting, 2003). Unlike most known GABA_A receptor subtypes, those containing the ɛ subunit exhibit spontaneous, agonist-independent, channel activity (Neelands et al., 1999, Davies et al., 2001, Maksay et al., 2003, Wagner et al., 2005, Ranna et al., 2006) with slowed deactivation and recovery (Wagner et al., 2005).

That receptor assembly occurs via defined pathways is supported by a number of independent studies that have identified that recombinant receptors are expressed with a fixed stoichiometry of 2α, 2β and 1γ (Connolly and Wafford, 2004, Sigel et al., 2006) with a subunit arrangement (positions 1–5) αβαβγ (Baumann et al., 2001, Baumann et al., 2002). Moreover, GABA_A receptor assembly signals have been identified in the α1, β2/3 and γ3 subunits (Taylor et al., 1999, Taylor et al., 2000, Klausberger et al., 2000, Klausberger et al., 2001a, Klausberger et al., 2001b, Sarto et al., 2002, Bollan et al., 2003a, Bollan et al., 2003b). The existence of defined pathways is supported by the observations that each identified assembly signal exhibits the ability to interact with only a subset of receptor subunits.

Given the relatively recent cloning of the ɛ subunit, little information regarding its assembly into GABA_A receptors is known. The ɛ subunit shares most amino acid sequence identity with the γ subunits (38–47% homology), followed by the α subunits (28–30% homology) and shows < 25% homology to all other subunits. In support of a γ-like role, the ɛ, when expressed alone or in combination with either an α or a β subunit does not exhibit any significant ligand-binding or ligand-induced channel activity. However, upon the co-expression of α β and ɛ subunits, the formation of GABA-activated currents and ligand binding is observed (Davies et al., 1997a, Whiting et al., 1997). From these findings, the ɛ subunit’s requirements for receptor function appear to mirror those of the γ2L subunit for surface expression (Connolly et al., 1996a). Therefore, the failure of ɛα and ɛβ (as also observed for γ2Lα1 and γ2Lβ2) to produce functional receptors may reflect an inability to correctly assemble and reach the cell surface (for ɛβ), or to generate an agonist binding site (for ɛβ). However, given that sequence homology within a class is ɛ 70% and between classes is ɛ 30%, the ɛ subunit is in a class of its own and may therefore have different assembly requirements to those of the γ subunits. Indeed, the ɛ subunit does not appear to simply replace the single γ subunit, instead, the formation of αβγɛ receptors has been proposed (Davies et al., 2001).

A recent study suggests that the ɛ subunit may replace a β or γ2 subunit in the construction of ɛ subunit-containing receptors (Jones and Henderson, 2007). In this study, we identify that the ɛ subunit exhibits assembly characteristics akin to the β subunits, in that it is able to co-traffic to the cell surface with α1 (albeit non-functionally), but not β2 or γ2 subunits. Moreover, we determined the assembly profile of the ɛ subunit to be most similar to that of the β3 subunit. However, the failure of αɛ (unlike that of α1β2), α1ɛγ2L (unlike that of α1β2γ2L) and ɛγ2L (unlike that of β3γ2L) subunit combinations to assemble into functional receptors reveals a unique assembly profile for the ɛ subunit that is distinct from that of a β subunit. Our results, using concatenated receptor constructs to study receptor architecture (Minier and Sigel, 2004a, Minier and Sigel, 2004b), suggest that the ɛ subunit is promiscuous in its ability to be incorporated into GABA_A receptors. However, the functionally most relevant isoforms appear to be restricted to (at positions 1–5) ɛ–β2–α1–β2–γ2 (αβγɛ receptors) compared to α1–β2–α1–β2–γ2 (αβγ receptors) and α1–β2–α1–ɛ–β2 (αβɛ receptors) compared to α1–β2–α1–β2–β2 (αβ receptors). Thus, the ɛ subunit is able to replace the α1 subunit at position 1 in αβγɛ receptors or the β2 subunit at position 4 in αβɛ receptors.