Research ReportRegulation of the surface expression of α4β2δ GABAA receptors by high efficacy states
Highlights
► Surface expression of α4FLAGβ2δ is increased by high efficacy states. ► Internalized α4FLAGβ2δ is decreased by high efficacy states. ► α4FLAGβ2δ expression is not dependent on the polarity of GABAergic current. ► α4FLAGβ2δ expression is dependent upon PKC-δ. ► THP increases α4FLAGβ2δ via increased receptor insertion rather than endocytosis.
Introduction
The ligand-gated GABAA receptor (GABAR) is responsible for most inhibition in the CNS (Olsen and Sieghart, 2009). This receptor is a pentameric membrane protein which gates a Cl–conductance, and is generally of the forms 2α, 2β and 1γ (Chang et al., 1990) although many subtypes exist from a pool of 6α, 3β, 3γ, δ, ε, π, ρ and θ. The remarkable diversity of the properties associated with the various receptor subtypes provides a basis for the selective expression of particular subtypes in a region or function-specific manner. In particular, receptors containing the δ subunit have relatively low expression in the CNS (Pirker et al., 2000, Wisden et al., 1992) but display a high degree of plasticity (Shen and Smith, 2009). These receptors express at extrasynaptic locations (Wei et al., 2003) and generate a tonic current (Stell and Mody, 2002) in response to ambient levels of GABA (~ 1 μM) (Wu et al., 2001). The tonic inhibitory current has been shown to generate more current (i.e., total charge transfer) than the phasic inhibitory synaptic current (Bai et al., 2000), suggesting that regulating the tonic inhibitory current may be an efficient mechanism to reduce neuronal excitability.
One trigger for altered expression of α4βδ GABAR is exposure to the neurosteroid THP (3α-OH-5[α]β-pregnan-20-one or [allo]pregnanolone), a metabolite of the ovarian steroid progesterone (Compagnone and Mellon, 2000) which can also be formed directly in hippocampal pyramidal cells from cholesterol (Agis-Balboa et al., 2006). Levels of this steroid fluctuate across the ovarian cycle, pregnancy and at the onset of puberty (Compagnone and Mellon, 2000, Shen et al., 2007) and are also increased by sustained stress (Girdler et al., 2001, Higashi et al., 2005, Purdy et al., 1991). In vivo administration of this steroid to female rats increases hippocampal expression of α4 and δ subunits by 2 to 3-fold above control levels after 48 h (Shen et al., 2005), accompanied by increases in the tonic inhibitory current. The naturally occurring fluctuations in this steroid also alter expression of α4βδ GABAR in a number of CNS sites, including CA1 hippocampus, dentate gyrus and the midbrain central grey (Lovick et al., 2005, Maguire and Mody, 2009, Maguire et al., 2005, Sanna et al., 2009, Shen et al., 2007, Shen et al., 2010). Alterations in expression of this receptor by fluctuating steroid levels, either endogenous or exogenously administered (Smith et al., 2006), can be associated with alterations in anxiety behavior, panic responses and seizure susceptibility, suggesting that this receptor may play a role in certain neuropsychological pathologies. Despite this in vivo evidence of neurosteroid regulation of α4βδ GABAR expression, however, little is known of the cellular mechanisms which underlie these changes in expression. Although recent studies have shown that brain-derived neurotrophic factor (BDNF) (Joshi and Kapur, 2009) and protein kinase C (PKC)-induced phosphorylation (Abramian et al., 2010) increase δ and α4 expression, respectively, the mechanism by which THP alters surface expression of these receptors is not known.
Recent studies have shown that GABA can increase trafficking of α1β2γ2 GABARs to the cell membrane (Eshaq et al., 2010). α4βδ GABARs have a unique pharmacological profile, however, different from α1β2γ2 GABARs. Although they have a high sensitivity to GABA (EC50 = 0.5 μM) (Brown et al., 2002), GABA is a partial agonist at these receptors (Bianchi and Macdonald, 2003, Zheleznova et al., 2008), unlike its effect at α1β2γ2 where it acts as a full agonist. However, δ-containing GABARs are the most sensitive target for THP (Belelli et al., 2002) and the related steroid THDOC (3α,21-dihydroxy-5α-pregnan-20-one) (Brown et al., 2002, Wohlfarth et al., 2002), which are positive modulators at physiological concentrations. These steroids increase receptor efficacy (Bianchi and Macdonald, 2003, Zheleznova et al., 2008), producing current greater than the maximal GABA-gated current by increasing long duration receptor channel openings. A number of high efficacy agonists for α4βδ GABARs have been reported, which include both synthetic (THIP or gaboxadol) (Brown et al., 2002) and endogenous (β-alanine (Bianchi and Macdonald, 2003) and taurine (Jia et al., 2008)) compounds. Thus, we initially tested the effect of THP in combination with GABA on cell surface expression of a FLAG-tagged α4 construct transfected with β2 and δ in HEK-293 cells and cultured hippocampal neurons. We assessed receptor trafficking by employing a high expression CMV promoter and assessed surface receptor expression under non-permeabilized conditions following expression of intracellular protein (Eshaq et al., 2010). This 3XFLAG tag on the C-terminus of α4 produces a highly visible signal when targeted with monoclonal anti-FLAG antibodies and a fluorescent secondary antibody (Hernan et al., 2000). Functional receptor expression was assessed with whole cell patch clamp recordings from transfected cells. These findings were compared with those obtained with high efficacy agonists and GABA itself in their effect on trafficking of α4βδ GABARs to the cell surface in order to determine whether steroid effects on expression of this receptor are due to increases in receptor efficacy.
Regulation of cell surface expression of α4βδ GABAR protein may either be due to an increase in receptor insertion or a reduction in receptor internalization and degradation. Recent studies have suggested that δ-containing GABARs have a greater stability in the membrane than γ2-containing GABARs, with a τ1/2 for internalization of hours versus minutes, respectively (Joshi and Kapur, 2009). Thus, regulation of receptor insertion rate may be a more likely mechanism for increasing cell surface expression. Our findings suggest that conditions which increase receptor efficacy increase expression of α4βδ GABARs, regardless of whether the steroid was present. These increases in receptor expression appear to be due to increased receptor insertion.
Section snippets
The α4(3XFLAG)β2δ GABAR displays functional expression in HEK-293 cells
We initially characterized our novel 3XFLAG-tagged α4 (α4F) subunit via electrophysiological techniques comparing the GABA responses of α4F to untagged α4 co-transfected with β2 and δ cDNAs (Fig. 1A). To this end, whole cell voltage clamp recordings were used to determine responses to GABA (0.01–100 μM) of the two constructs expressed in HEK-293 cells in the presence of 1 μM ZnCl which inhibits current from binary receptors (Meera et al., 2011). In fact, concentration–response curves for α4β2δ
Discussion
This study shows that cell surface expression of α4Fβδ GABARs is increased by GABAR modulators and agonists which increase the efficacy of this receptor. 48 h exposure of HEK-293 cells or neurons to the neurosteroid THP along with GABA or to the GABA agonists gaboxadol and β-alanine produced significant increases in expression of α4Fβ2δ GABARs, which were tightly correlated with increases in current gated by GABA agonists. Expression of native α4 and δ GABAR subunits was also increased by
HEK-293 cells
Human embryonic kidney (HEK) 293 cells (ATCC, Manassas, VA) were grown in Dulbecco's Modified Eagle's Medium (DMEM/F-12, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO), penicillin (100 IU/ml) and streptomycin (100 μg/ml) (Invitrogen, Carlsbad, CA) on MatTek glass bottom dishes (MatTek Corp, Ashland, MA) at 37 °C in a humidified incubation chamber (5% CO2, 95% O2).
Neurons
Embryonic day 18 (E18) dissociated rat hippocampal cells were removed from timed
Acknowledgments
The authors thank NL Leidenheimer for the immunocytochemistry protocol, DH Smith for helpful discussions as well as Q H Gong and Y Deng (Microscopy Core of NYU Langone Medical Center, NY, NY) for helpful technical assistance. This work was supported by NIH grants DA09618 and AA12958 to SSS.
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