Stoichiometry of Expressed α₄β₂δ γ-Aminobutyric Acid Type A Receptors Depends on the Ratio of Subunit cDNA Transfected

Abstract

The γ-aminobutyric acid type A receptor (GABA_AR) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent αβγ GABA_AR subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing δ subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of δ-containing GABA_ARs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed δ-containing GABA_ARs, we inserted the α-bungarotoxin binding site tag in the α₄, β₂, and δ subunit N termini. An enhanced green fluorescent protein tag was also inserted into the β₂ subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged α₄β₂δ GABA_ARs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent α-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA_ARs. The results demonstrate that the subunit stoichiometry of α₄β₂δ GABA_ARs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of δ subunit cDNA transfected increased δ subunit incorporation into surface receptors with a concomitant decrease in β₂ subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA_AR pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed α₄β₂δ GABA_ARs. Successful bungarotoxin binding site tagging of GABA_AR subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA_AR trafficking in live cells.

Introduction

The γ-aminobutyric acid type A receptor (GABA_AR)² is the main inhibitory ligand-gated ion channel in the brain and is the target for a wide range of drugs, including benzodiazepines, anesthetics, neurosteroids, and barbiturates. The actions of these drugs are dependent on the GABA_AR subunit isoforms present, 16 of which have been identified, including α1–6, β1–3, γ1–3, δ, ϵ, θ, and π. Although the highly prevalent GABA_AR subtype comprised of α, β, and γ subunits mediates the majority of fast synaptic inhibition in the brain, αβδ GABA_ARs also play a key role, mediating tonic inhibition and the molecular actions of endogenous neuroactive steroids, alcohol, and several anesthetic agents (1).

Heterologous expression of GABA_AR subtypes in cell lines (e.g. HEK293 and Chinese hamster ovary cells) and Xenopus laevis oocytes has been used extensively to study their structural, electrophysiological, and pharmacological properties. Although the subunit stoichiometry of αβγ GABA_ARs has been established as 2α:2β:1γ using a variety of methods, including using a reporter mutation (2), quantifying antibody bound per subunit (3), fluorescence resonance energy transfer (4), and using concatenated subunits (5, 6), the stoichiometry of δ-containing receptors is not as well established. Using atomic force microscopy, the stoichiometry of α₄β₃δ GABA_ARs expressed in tsA 201 cells, was determined to be 2α:2β:1δ (7), suggesting that the δ subunit simply replaces the γ subunit in a pentameric receptor. However, in recent studies using concatenated subunits to express α₁β₃δ GABA_ARs in X. laevis oocytes, functional receptors with more than one δ subunit were observed, and when the subunit stoichiometry was constrained to 2α:2β:1δ, multiple subunit arrangements were capable of forming functional receptors (8, 9).

Generally, cDNA or cRNA ratios of 1:1:5 to 1:1:10 are used to heterologously express αβδ receptors (8, 10, 11). Interestingly, in oocytes, when the ratio of δ cRNA was increased from 1α:1β:1δ to 1α:1β:5δ and 1α:1β:10δ, the GABA EC₅₀ values increased, and the Hill slope decreased, suggesting changes in receptor subunit stoichiometry (12). If different ratios of cDNA/cRNA alter subunit stoichiometry of surface-expressed αβδ GABA_ARs, this may contribute to the discrepancies in the functional properties (e.g. alcohol sensitivity, GABA EC₅₀ values, maximal GABA currents) of expressed αβδ GABA_ARs reported in the literature (13–16). In heteromeric neuronal nicotinic acetylcholine receptors, varying the transfection ratios of α₄ and β₂ subunits results in receptors with alternate stoichiometries of 3α:2β or 2α:3β (17). Similarly, the stoichiometry of recombinant heteromeric P2X receptors is regulated by subunit transfection ratios (18).

Here, we examined whether the ratio of subunit cDNA in a transfection influences the subunit stoichiometry of cell surface α₄β₂δ GABA_ARs expressed in HEK293T cells. We inserted a 13-amino acid sequence that encodes for a high affinity α-bungarotoxin binding site (BBS) into the α₄, β₂, and δ subunits (see Fig. 1). This sequence comes from the nicotinic acetylcholine receptor, where α-bungarotoxin (α-BTX) is a native antagonist. The BBS tag has been successfully engineered into GABA_A receptors (19) and other proteins, including GABA_B receptors (20) and ionotropic glutamate receptor, (21) and used to monitor receptor trafficking. BBS-tagged α₄β₂δ GABA_ARs were heterologously expressed in HEK293T cells using a variety of subunit cDNA ratios. Receptor subunit stoichiometry was determined by immunopurifying surface tagged α₄β₂δ GABA_ARs and quantitating fluorophore-conjugated α-BTX binding to each subunit. Our data demonstrate that the subunit stoichiometry of cell surface α₄β₂δ receptors is highly influenced by the ratio of subunit cDNA used in a transfection.

FIGURE 1.

Insertion of BBS and EGFP tags in GABA_AR subunits is tolerated. A, schematic illustrating the insertion sites for BBS in the α₄, β₂, and δ subunits and EGFP in the β₂ subunit. B, radioligand binding curves depicting the displacement of [³H]muscimol binding by muscimol for WT α₄β₂δ receptors (●) and tagged α₄^BBSβ₂^BBS::EGFPδ^BBS receptors (■). Each point is the mean ± S.E. of triplicate measurements compiled from two (WT) or three (tagged) experiments. The data were fit by nonlinear regression as described under “Experimental Procedures.” For WT receptors, K_I = 32 ± 2 nm; for tagged receptors, K_I = 21 ± 3 nm.

EXPERIMENTAL PROCEDURES

GABA_A Receptor cDNA Constructs

cDNAs encoding rat α₁, α₄, β₂, and δ subunit polypeptides including their signal peptides were inserted into the pUNIV vector as described previously (22). The cDNA encoding the BBS tag, WRYYESSLEPYPD, was introduced between the third and fourth amino acids of each mature subunit using recombinant PCR. The cDNA encoding enhanced green fluorescent protein (EGFP) (Clontech) was inserted between the fourth and fifth amino acids similarly in the mature β₂ subunit. All of the cDNA constructs were verified by double-stranded DNA sequencing.

Cell Culture and Transfection

Human embryonic kidney cells (HEK293T), a gift from Vsevolod V. Gurevich (Vanderbilt University), were incubated at 37 °C in humidified 5% CO₂, 95% air and grown in minimum essential medium with Earle's salts and l-glutamine (Mediatech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum (Hyclone Laboratories, New Brunswick, NJ) and 50 μg/ml gentamicin (Invitrogen). In general, the cells were transfected with a total of 21 μg of cDNA/100-mm dish with a variety of different subunit cDNA ratios (as noted in text) using a standard CaHPO₄ precipitation method (23) to heterologously express wild type α₄β₂δ receptors, tagged α₄^BBSβ₂^BBS::EGFPδ^BBS receptors, tagged α₄^BBSβ₂^BBS::EGFP receptors, or tagged α₁^BBSβ₂^BBS::EGFP receptors. For nontransfected controls, the cells were treated similarly to transfected cells (e.g. medium changes, washes, and incubations), but no CaHPO₄ solution or cDNA was added to the cells. For mock transfected controls, the cells were treated similarly to transfected cells, and CaHPO₄ solution was added, but the solution contained no cDNA.

Radioligand Binding Assays

Approximately 60 h post-transfection, HEK293T cells transfected with either wild type or tagged subunit cDNAs at a 2:1:4 ratio (α:β:δ) were harvested, and membrane homogenates were prepared as described previously (24). 100 μg of membrane protein was incubated at room temperature for 40 min with a sub-K_d concentration of [³H]muscimol (30 Ci/mmol; PerkinElmer Life Sciences) in the absence or presence of seven different concentrations of nonradioactive muscimol in a final volume of 250 μl. The data were fit using a nonlinear least squares method to a one-site competition curve defined by the equation y = B_max/[1 + (x/IC₅₀)], where y is the total bound [³H]muscimol in disintegrations/min, B_max is maximal binding, and x is the concentration of displacing drug (Prism v5.02; GraphPad Software, San Diego, CA). Equilibrium dissociation constant values for unlabeled muscimol (K_i) were calculated according to the Cheng-Prusoff-Chou equation: K_i = IC₅₀/[1 + L/K_d], where K_d is the equilibrium dissociation constant of the radioligand, and L is the concentration of radioligand (25, 26).

Determining GABA EC₅₀ and Expression of Tagged Receptors in X. laevis Oocytes

Capped cRNA encoding wild type or tagged α₄, β₂, and δ subunits in the pUNIV vector was prepared as described previously (27). The oocytes were harvested from X. laevis and prepared as described in Ref. 28 before injection with 27 nl of cRNA mixture (370 pg/nl) in the ratio 1:1:10 (α:β:δ). The oocytes were incubated 5–7 days after injection with cRNA before being used for two-electrode voltage clamp electrophysiological recordings to determine GABA EC₅₀ values for either wild type or tagged receptors. For detailed methods regarding oocyte storage, two-electrode voltage clamp and concentration response analysis, see Ref. 27. GABA concentration response data were fit by the following equation: I = I_max/[1 + (EC₅₀/[A]ⁿ)], where I is the peak response to a given drug concentration, I_max is the maximum current amplitude, EC₅₀ is the drug concentration that produces a half-maximal response, [A] is drug concentration, and n is the Hill coefficient using Prism v5.02 (GraphPad Software).

Immunopurification of Surface Receptors

30–60 h post-transfection, intact cells expressing GABA_ARs were washed with ice-cold PBS (2.7 mm KCl, 1.5 mm KH₂PO₄, 0.5 mm MgCl₂, 137 mm NaCl, and 14 mm Na₂HPO₄, pH 7.1) and incubated with 1 μl/ml rabbit polyclonal anti-δ subunit antibody (Millipore, Billerica, MA) or 0.75 μg/ml mouse monoclonal anti-GFP antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS for 1 h at 4 °C. The cells were washed twice in ice-cold PBS and incubated in 10 mm N-ethylmaleimide in PBS for 15 min at 4 °C. The cells were washed three more times in ice-cold PBS and were then solubilized with 1 ml of lysis buffer (1% Triton X-100, 50 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA, pH 7.5, 10 mm N-ethylmaleimide) supplemented with Complete Protease Inhibitor tablets (Roche Applied Science), scraped into a fresh tube, and passed through a 25-gauge needle three times. The solubilized cells were incubated at 4 °C for 3–6 h and were then centrifuged at 10,000 × g for 10 min at 4 °C. A 25-μl aliquot of lysate supernatant was removed for Western blotting, and the remaining cell lysate labeled with anti-δ or anti-GFP antibodies was incubated with 120 μl of 20% slurry of protein A-Sepharose or protein G-Sepharose (Sigma-Aldrich), respectively. The samples were incubated at 4 °C while rotating overnight and then centrifuged at 16,000 × g for 5 min at 4 °C. The supernatant was discarded, and the beads were washed six times with 1 ml of buffer (0.5% Triton X-100, 50 mm Tris-Cl, 150 mm NaCl, and 5 mm EDTA, pH 7.5). The wash buffer was completely removed, and surface GABA_AR protein was eluted with 30 μl of 2× Laemmli sample buffer (6% SDS, 20% glycerol, 125 mm Tris-Cl, pH 6.8) at room temperature for 1 h with rotation. Dithiothreitol was added to a final concentration of 55 mm as well as 1 μl of 50× bromphenol blue prior to SDS-PAGE.

Western Blotting

The proteins were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (0.45 μ NitroPure; GE Water and Process Technologies) for 1 h at 100 V. The blots were incubated overnight at 4 °C in blocking buffer containing 2% nonfat milk in PBST (PBS + 0.1% Tween 20, pH 7.1). The blots were washed in PBST once for 5 min at room temperature and were then incubated in 1 μg/ml α-BTX-Alexa680 (molecular weight = 8,800; Molecular Probes) in PBST, unless noted differently, for 1 h at room temperature in the dark. The blots were kept in the dark for all of the subsequent steps until scanned. The blots were washed with PBST three times for 5 min and then dried on gel blot paper (Whatman, Sanford, ME) and stored at room temperature.

Determining α-BTX-Alexa680 Affinity for BBS-tagged Subunits

The cells were transfected to express GABA_A receptors that were tagged with BBS on a single subunit (α₄^BBSβ₂δ, α₄β₂^BBS::EGFPδ, or α₄β₂δ^BBS). Approximately 45 h after transfection, the cells were lysed with buffer (1% Triton X-100, 150 mm NaCl, 5 mm glucose, 20 mm NaEDTA, 10 mm NaEGTA, and 25 mm Tris-HCl, pH 7.4) supplemented with Complete Protease Inhibitor (Roche Applied Science). The cell lysate was passed through a 25-gauge needle three times and then incubated overnight at 4 °C with rotation. The cell lysates were spun down at 1,000 × g for 5 min at 4 °C, and the supernatant was collected. 150 μl of cell lysate supernatant was diluted with 150 μl of 2× Laemmli sample buffer and bromphenol blue and dithiothreitol were added, as described above. Eight 30-μl aliquots of each lysate were run on two 10% SDS-PAGE gels and transferred to nitrocellulose as described above. Each lane on the blot was cut out, washed, and blocked (as described above) and then incubated in varying concentrations of α-BTX-Alexa680 in PBST (ranging from 0.1 to 25 μg/ml) for 1 h at room temperature, rotating. All of the blots were kept in the dark during fluorophore labeling and for the remainder of the experiment. For a loading control, each blot below 30 kDa was excised prior to labeling with α-BTX-Alexa680 and was incubated in 1 μg/ml rabbit polyclonal anti-cyclophilin B antibody (Abcam Inc., Cambridge, MA), washed in PBST three times for 5 min, and then incubated in 0.01 μg/ml goat anti-rabbit IRDye 800CW IgG (LI-COR Biosciences). After labeling with either α-BTX-Alexa680 or IRDye 800, the blots were washed with PBST 30 min or 1.5 h in the dark and dried on gel blot paper before scanning with an Odyssey infrared imaging system (LI-COR Sciences, Lincoln, Nebraska). The different wash times had no significant effect on the K_D values calculated. Using Odyssey imaging software (version 1.2), boxes were manually drawn around each tagged subunit band, and the fluorescence intensity of the band was measured (in arbitrary fluorescence units) and corrected with right-left background subtraction as per the manufacturer's recommendations. Fluorescence intensity for each tagged subunit band was divided by the fluorescence intensity of the corresponding cyclophilin B band in that lane. These corrected fluorescence intensity values were then plotted as a function of [α-BTX-Alexa680] using a one-site hyperbola equation: Y = [(B_max * X)/(K_d + X)], where X is the concentration of the ligand, and Y is the specific binding. The data were fit by nonlinear regression (Prism v5.02) to determine the apparent affinity (K_D) of α-BTX-Alexa680 binding to each tagged subunit. The K_D values are the means ± S.E. from fits of each individual experiment (n ≥ 3). The curves in Fig. 3 are fits of normalized data compiled from all of the experiments for each tagged subunit.

FIGURE 3.

α-BTX-Alexa680 binds with similar affinity to the α-BTX binding site tag on α₄, β₂, and δ subunits. A, representative Western blot of whole cell lysates from cells expressing α₄^BBSβ₂δ receptors. Individual lanes containing equal amounts of cell lysate were incubated in different concentrations of α-BTX-Alexa680, ranging from 0.1 to 25 μg/ml. The endogenous protein cyclophilin B was used as a loading control. B, normalized α-BTX-Alexa680 fluorescence was fit by nonlinear regression as described under “Experimental Procedures.” K_D values for α-BTX-Alexa680 binding to each BBS-tagged subunit are the means ± S.E. from fits of individual experiments (n ≥ 3). The curves shown are fits of normalized data compiled from all of the experiments for each tagged subunit.

Calculating Surface GABA_A Receptor Subunit Stoichiometry

Western blots of surface immunopurified α₄^BBSβ₂^BBS::EGFPδ^BBS receptors were blocked (as described above) and then incubated in PBST with 1 μg/ml α-BTX-Alexa680 for 1 h at room temperature in the dark. In a few experiments, 5 μg/ml α-BTX-Alexa680 was used to label blots, which had no effect on the receptor subunit stoichiometry calculated. The blots were washed three times for 5 min in PBST and dried between filter papers before scanning with the Odyssey infrared imaging system. The fluorescence intensity of each tagged subunit band was measured as described above. The fluorescence intensities of the α₄^BBS, β₂^BBS::EGFP, and δ^BBS subunit bands in each lane were summed to determine total fluorescence signal associated with the purified receptors in that lane. Because GABA_ARs are pentamers, the total fluorescence signal in a lane was divided by 5 to calculate the amount of fluorescence/subunit. The experimentally determined fluorescence intensity for each subunit was divided by the calculated fluorescence/subunit to determine the average subunit stoichiometry of surface GABA_ARs. For example, if the measured fluorescence intensity for α₄ = 200, β₂ = 200, and δ = 600, then the total fluoresence signal = 1000, and the calculated fluorescence signal per subunit is 200. Thus, the average subunit stoichiometry = 1α₄:1β₂:3δ.

RESULTS

Characterization of BBS-tagged and EGFP-tagged GABA_AR Subunits

We inserted a BBS tag in the extracellular N terminus of the α₄, β₂, and δ subunits (Fig. 1A) to allow us to measure fluorophore-conjugated α-BTX binding to each of the subunits on a Western blot. We also inserted EGFP (27 kDa) into the N terminus of the β₂ subunit (Fig. 1A) to distinguish it from the δ subunit, because β₂ and δ subunits have approximately the same molecular mass (∼50 kDa). Initially, we measured the binding of [³H]muscimol, a GABA binding site agonist, to WT α₄β₂δ and tagged α₄^BBSβ₂^BBS::EGFPδ^BBS GABA_ARs to examine whether inserting the BBS tag and EGFP altered α₄β₂δ receptor expression or function. Muscimol bound with similar affinity to WT α₄β₂δ receptors (K_I = 32 ± 2 nm) and tagged α₄^BBS β₂^BBS::EGFPδ^BBS receptors (K_I = 21 ± 3 nm) (Fig. 1B). In addition, the maximal number of [³H]muscimol binding sites (B_max) was not significantly different for wild type α₄β₂δ receptors and tagged α₄^BBS β₂^BBS::EGFPδ^BBS receptors (WT, B_max = 0.90 ± 0.6 pmol/mg; tagged, B_max = 0.41 ± 0.3 pmol/mg). The tags also had no effect on the GABA EC₅₀ values or the maximum GABA-elicited currents (I_max) as determined using two-electrode voltage clamping of oocytes expressing wild type α₄β₂δ or tagged α₄^BBS β₂^BBS::EGFPδ^BBS GABA_ARs (supplemental Fig. S1; WT, GABA EC₅₀ = 1.3 ± 0.10 μm (n = 3); tagged, GABA EC₅₀ = 1.1 ± 0.23 μm (n = 7); means ± S.E.). At 7 days post-injection, I_max ranged from 2.6 to 3.7 μA for wild type receptors and from 2.0 to 3.6 μA for tagged receptors. The above data indicate that insertion of the BBS tags and EGFP is tolerated and has little effect on α₄β₂δ GABA_AR expression, muscimol binding, and GABA-activated functional responses.

α-BTX-Alexa680 Binding to BBS-tagged GABA_AR Subunits

Western blots of lysates prepared from nontransfected cells and cells expressing α₄β₂δ GABA_ARs, in which only one subunit was BSS-tagged, were incubated with α-BTX-Alexa680 to test the specificity of its binding. As shown in Fig. 2, α-BTX-Alexa680 only labeled the subunits containing the BBS tag, demonstrating that α-BTX-Alexa680 binding was specific. The tagged subunits ran at their predicted molecular masses (α₄^BBS ≈ 64 kDa, β₂^BBS::EGFP ≈ 77 kDa, δ^BBS ≈ 51 kDa) and were easily distinguished from one another. The δ subunit signal contained multiple bands, which reflect varying amounts of glycosylation (data not shown).

FIGURE 2.

α-BTX-Alexa680 binding to BBS-tagged GABA_AR subunits. Representative Western blots of whole cell lysates from nontransfected cells (NT) and from cells expressing α₄β₂δ GABA_ARs where different subunits contain the BBS tag. The blots were incubated in α-BTX-Alexa680 to visualize BBS-tagged subunits as described under “Experimental Procedures.” Each subunit can be distinguished by its apparent molecular mass (β₂^BBS::EGFP = 77 kDa, α₄^BBS = 64 kDa, δ^BBS = 51 kDa). Multiple bands for the δ subunit reflect varying amounts of glycosylation.

Our method for calculating subunit stoichiometry is dependent on α-BTX-Alexa680 binding to each subunit with a similar affinity. Western blots of lysates prepared from cells expressing α₄β₂δ receptors in which only one subunit was tagged (e.g. α₄^BBSβ₂δ) were incubated in increasing concentrations of α-BTX-Alexa680 (Fig. 3A). The fluorescence signals associated with the BBS-tagged subunit were quantitated and fit by nonlinear regression to determine the α-BTX-Alexa680 binding affinity (K_d) to each tagged subunit (Fig. 3B). The α-BTX-Alexa680 binding affinities were not significantly different, demonstrating that α-BTX-Alexa680 bound to each BBS-tagged subunit with a similar affinity (α₄^BBSβ₂δ receptors, K_d = 250 ± 130 nm (n = 3); α₄β₂^BBS::EGFPδ receptors, K_d = 190 ± 100 nm (n = 4); α₄β₂δ^BBS receptors, K_d = 390 ± 110 nm (n = 3); means ± S.E.).

Analysis of α₄β₂δ GABA_AR Subunit Stoichiometry

When calculating receptor stoichiometry, we needed to avoid including unassembled receptor subunits and/or partially assembled receptors. Thus, we determined the subunit stoichiometry of cell surface GABA_ARs. Moreover, we purified cell surface α₄β₂δ GABA_ARs using two different antibodies, either a GFP antibody or a δ subunit antibody. If high levels of α₄β₂ receptors were contaminating our α₄β₂δ receptor population or if significant amounts of the δ subunit trafficked to the cell surface alone or as a homo-oligomeric receptor, we would expect to see different subunit stoichiometries for receptors purified using the different antibodies. Initially, we assessed the subunit stoichiometry of surface α₄^BBSβ₂^BBS::EGFPδ^BBS GABA_ARs using a transfection ratio of 1:1:5 (α:β:δ), because this is a commonly reported ratio used in the field (8, 10, 11). For α₄^BBSβ₂^BBS::EGFPδ^BBS GABA_ARs immunoprecipitated using a δ subunit antibody, the average subunit stoichiometry was calculated to be 0.4α₄: 0.5β₂: 4.1δ (Fig. 4A and Table 1). The stoichiometry of anti-GFP-purified receptors was similar: 0.5α₄:0.5β₂:3.9δ (Fig. 4A and Table 1). The calculated subunit stoichiometries are the average values for the entire pool of surface GABA_ARs, which were expressed and purified. Noninteger values likely reflect surface receptors with mixed stoichiometries, because a single receptor cannot contain half of a subunit.

FIGURE 4.

Varying the subunit cDNA ratios used to transfect cells changes the subunit stoichiometry of surface-expressed α₄β₂δ GABA_ARs. Representative Western blots of anti-GFP or anti-δ surface immunoprecipitations (IP) from mock transfected cells (mock) and from cells transfected with α₄^BBS, β₂^BBS::EGFP, and δ^BBS at a 1α:1β:5δ ratio (A) and 2α:1β:0.5δ ratio (B). The subunits were visualized using α-BTX-Alexa680. Subunit stoichiometry of surface GABA_ARs was determined by quantitating the amount of α-BTX-Alexa680 bound to each tagged subunit normalized to the total fluorescence signal in each lane as described under “Experimental Procedures.” The average receptor subunit stoichiometries for each condition are shown and reported in Table 1. Increasing the ratio of δ subunit cDNA transfected increased δ subunit incorporation into surface receptors. Similar results were obtained regardless of how surface GABA_ARs were immunopurified.

TABLE 1.

Average subunit stoichiometry of tagged α₁β₂, α₄β₂, and α₄β₂δ GABA_ARs

Subunit stoichiometry for surface receptors purified with either anti-GFP or anti-δ subunit antibody was determined for various GABA_AR subtypes expressed using different cDNA transfection ratios. The values reported are the means ± S.E., when n > 1. IP, immunoprecipitation.

cDNA ratio transfected	Surface receptor subunit stoichiometry
	Anti-GFP IP				Anti-δ IP
	α	β	δ	n	α	β	δ	n
1α1:1β2	3.2 ± 0.2	1.9 ± 0.2		3
2α4:1β2	2.0 ± 0.3	3.1 ± 0.3		3
2α4:1β2:0.1δ	1.5 ± 0.2	2.9 ± 0.1	0.6 ± 0.2	3
2α4:1β2:0.25δ	1.7 ± 0.2	2.2 ± 0.2	1.1 ± 0.2	3	1.5 ± 0.1	2.3 ± 0.0	1.2 ± 0.0	3
2α4:1β2:0.5δ	1.5 ± 0.1	2.3 ± 0.1	1.3 ± 0.0	2	1.4 ± 0.1	2.2 ± 0.2	1.4 ± 0.2	2
2α4:1β2:1δ	1.4 ± 0.1	1.7 ± 0.2	1.9 ± 0.2	4
2α4:1β2:4δ	1.5 ± 0.1	1.0 ± 0.1	2.5 ± 0.1	7	1.1 ± 0.2	0.8 ± 0.2	3.2 ± 0.0	2
2α4:1β2:5δ	0.9	0.9	3.2	1
1α4:1β2:0.1δ	1.6	3.4	0	1
1α4:1β2:5δ	0.5 ± 0.1	0.5 ± 0.0	3.9 ± 0.1	4	0.4 ± 0.1	0.5 ± 0.0	4.1 ± 0.1	4

Regardless of the antibody used to immunoprecipitate the surface receptors, the calculated subunit stoichiometries were not significantly different, indicating that there were few to no α₄β₂ receptors in our receptor preparations. In addition, the data suggest that overexpression of the δ subunit using a 1:1:5 transfection ratio does not result in detectable amounts of the δ subunit being trafficked to the cell surface alone or as a homo-oligomeric protein because the same average stoichiometry was calculated for receptors purified with either an antibody to the β₂ or δ subunit. When we altered the cDNA transfection ratio to 2:1:0.5 (α:β:δ), the subunit stoichiometry changed to 1.5α:2.3β:1.3δ for anti-GFP-purified receptors and 1.4α:2.2β:1.4δ for anti-δ-purified receptors (Fig. 4B and Table 1), indicating that changing the cDNA transfection ratio alters the subunit stoichiometry of surface-expressed GABA_ARs.

Dependence of Subunit Stoichiometry on the Ratio of Transfected cDNA

To extend our analysis further, we determined the subunit stoichiometry of GABA_ARs that were expressed using nine different cDNA transfection ratios ranging from 2:1:0 to 2:1:5 (α:β:δ). As seen in Fig. 5, increasing the ratio of δ cDNA increased the incorporation of the δ subunit into surface α₄β₂δ GABA_ARs almost exclusively at the expense of the β₂ subunit. Again, regardless of the antibody used to immunoprecipitate the surface receptors, the calculated subunit stoichiometries were not significantly different (Fig. 5 and Table 1). Moreover, the total amount of cDNA used in a transfection did not influence subunit stoichiometry. HEK293T cells were transfected with either 21 or 10.5 μg of total cDNA using ratios of 2:1:0.1 and 2:1:0.5 (α:β:δ). Regardless of the amount of cDNA transfected, the calculated subunit stoichiometries of surface α₄β₂δ GABA_ARs were not significantly different (supplemental Fig. S2).

FIGURE 5.

Increasing the ratio of δ cDNA increased δ incorporation into surface GABA_ARs with a concomitant decrease in β₂ subunit incorporation. Scatter plots of the subunit stoichiometry of surface α₄^BBSβ₂^BBS::EGFPδ^BBS receptors expressed using various cDNA transfection ratios for anti-GFP-immunopurified (A) and anti-δ-immunopurified (B) receptors. The number of each α₄ (*), β₂ (■), and δ (◇) subunit per surface GABA_AR are plotted for each cDNA ratio. Aside from the transfection ratio on the far right, the amount of α₄ and β₂ cDNA remained the same, and the ratio of δ cDNA was increased. The bars are the means ± S.E. C, the average number of subunits/anti-GFP immunopurified surface receptor were plotted versus the ratio of δ cDNA transfected for 2:1:X (α:β:δ) cDNA transfections. The data were fit using a one-phase association (δ) or decay (α₄ and β₂) equation using Prism v5.02 (GraphPad Software). The data points are the means ± S.E. from ≥3 experiments, except for 2:1:5 cDNA ratio, where n = 1, and for the 2:1:0.5 cDNA ratio, where n = 2. IP, immunoprecipitation.

Subunit Stoichiometry of α₄β₂ and α₁β₂ Receptors

We also determined the subunit stoichiometry of α₄β₂ and α₁β₂ GABA_ARs. The stoichiometry of α₄β₂ receptors was 2.0α₄:3.1β₂, using cells transfected with a 2:1 (α:β) cDNA ratio (Fig. 5 and Table 1). Surprisingly, the subunit stoichiometry is reversed in αβ receptors containing the α₁ subunit. In cells transfected with a 1:1 (α:β) cDNA ratio, the subunit stoichiometry was determined to be 3.2α₁:1.9β₂ (Table 1), indicating that the stoichiometry of αβ GABA_ARs is influenced by the subunit isoforms present.

DISCUSSION

Here, we demonstrate that the subunit stoichiometry of heterologously expressed α₄β₂δ GABA_ARs is highly influenced by the ratio of subunit cDNAs used in a transfection. A transfection cDNA ratio of 2:1:0.25 (α:β:δ) yielded GABA_ARs with an average subunit stoichiometry of 2α:2β:1δ, whereas a 2:1:5 cDNA ratio yielded receptors with an average stoichiometry of 1α:1β:3δ (Table 1). As the amount of δ cDNA transfected increased, δ subunit incorporation into surface GABA_ARs increased with a concomitant decrease in β₂ subunit incorporation (Fig. 5C). In addition, we determined that the subunit stoichiometry of recombinant αβ GABA_ARs was dependent on the α subunit isoform, such that α₄β₂ GABA_ARs had a stoichiometry of 2α:3β, whereas the stoichiometry α₁β₂ GABA_ARs was 3α:2β (Table 1). The ability to alter receptor stoichiometry by subunit transfection ratios has been observed in other related pentameric ligand-gated ion channels including neuronal nicotinic acetylcholine receptors and P2X receptors (17, 18).

Varying the amount and availability of the δ subunit during GABA_ARs assembly resulted in surface α₄β₂δ GABA_ARs with different amounts of δ subunit incorporated. One possible reason for this is that the δ subunit possesses promiscuous assembly properties, similar to the ϵ subunit (29). Recently, it has been shown for concatenated α₆β₃δ receptors that the δ subunit can assemble in either of the two β subunit positions, in the γ subunit position, or in the position of the α subunit between the β subunits (9, 30). Our data suggest that for recombinant α₄β₂δ GABA_ARs, the δ subunit preferentially replaces the β₂ subunit (Fig. 5C). Phylogenetic analysis of GABA_AR subunits suggests that the δ subunit is more related to β subunits than α subunits (31), consistent with its substitution for the β₂ subunit in our studies.

Our data have important implications when interpreting data from recombinant αβδ GABA_ARs because the transfection protocols used to express these receptors vary between lab groups. For example, alcohol potentiation of GABA-mediated tonic current from αβδ GABA_ARs is routinely observed in whole cell recordings of hippocampal granule cells and cerebellar granule cells (32–34), but it is not reproducibly observed in recombinant systems. Although some studies have reported that GABA-activated currents from recombinant αβδ GABA_ARs are potentiated by low concentrations of alcohol (1–30 mm) (13, 14), others have reported that recombinant αβδ receptors are not responsive to alcohol (15, 16).

Numerous groups expressing recombinant αβδ GABA_ARs use a greater ratio of δ cDNA or cRNA to ensure adequate δ subunit expression and assembly (8, 10, 11, 35). However, we show that increasing the δ cDNA transfection ratio can dramatically change receptor stoichiometry. Thus, differences in subunit cDNA/cRNA ratios used for transient expression, coupled with the effects of using cDNA/cRNA from different species, vectors, and cell types, likely result in expression of GABA_ARs with varying subunit stoichiometries and arrangements, each of which can have different pharmacological and functional properties. This could be one reason for the variability in alcohol-sensitive GABA_AR data between independent groups, particularly if only αβδ GABA_ARs with a specific subunit stoichiometry and arrangement are responsive to low concentrations of alcohol.

Functional studies of α₄β₂δ GABA_ARs expressed in cells using a variety of cDNA ratios are needed to determine which subunit stoichiometries form functional GABA-activated channels and to tease apart the pharmacological and kinetic properties of α₄β₂δ GABA_ARs with different subunit stoichiometries. Two studies in oocytes have examined the effects of varying the subunit cRNA ratio used to express α₄β₃δ receptors. In one study, when the ratio of δ cRNA was increased from 1α:1β:1δ to 1α:1β:5δ and 1α:1β:10δ, the GABA EC₅₀ values increased, and the Hill slope decreased, suggesting changes in receptor subunit stoichiometry (12). However, in another study, no changes in GABA EC₅₀ values or the sensitivity to zinc block were seen for α₄β₃δ GABA_ARs expressed using cRNA ratios of 1α:1β:0.1δ, 1α:1β:1δ, 1α:1β:3δ, or 1α:1β:10δ (36). A recent functional study examining α₁β₂δ GABA_ARs in HEK293 cells reported that maximal GABA-activated peak currents from α₁β₂δ GABA_ARs were obtained by decreasing the ratio of δ subunit cDNA transfected to 1:1:0.1 (α₁:β₂:δ), suggesting that this transfection ratio was optimum for maximal expression of functional α₁β₂δ receptors (37).

The results of our study provide guidelines for heterologously expressing α₄β₂δ GABA_ARs with a particular average stoichiometry. It is important to note that the use of different vectors, cell types, or especially subunit isoforms, may change the receptor subunit composition. Here, we show that the stoichiometry of αβ GABA_ARs is affected by the subunit isoforms present. The stoichiometry of recombinant α₄β₂ GABA_ARs was 2α₄:3β₂, whereas the α₁β₂ GABA_AR stoichiometry was 3α₁:2β₂ (Table 1). Our previous work, using tandem subunit constructs, also indicated that α₁β₂ GABA_ARs stoichiometry was 3α₁:2β₂ (6), but others have reported a 2α₁:3β₂ stoichiometry (38, 39), suggesting that both stoichiometries are possible. Studies examining α₆β₂ GABA_AR subtypes report that the subunit stoichiomety is 3α₆:2β₂ (40), whereas for α₁β₃ GABA_ARs the stoichiometry is 2α₁:3β₃ (3). Taken together, the data indicate that the subunit composition of recombinant GABA_ARs is influenced by the subunit isoforms present.

An important question that arises from our results is which of the different α₄β₂δ GABA_AR stoichiometries that are obtained in recombinant systems correspond to those found in native neuronal α₄β₂δ GABA_ARs. The subunit stoichiometry and subunit arrangement of native αβδ GABA_ARs are currently unknown. It is possible that a native alcohol-sensitive αβδ GABA_AR subtype may have more than one δ subunit incorporated in a receptor pentamer. The subunit composition, subunit stoichiometry and arrangement of native GABA_ARs likely depend on subunit availability as well as a variety of factors, including subunit coassembly affinities and the accessory proteins present. Thus, it can be difficult to replicate expression of native type receptors using recombinant expression systems. Nonetheless, recombinant expression remains an invaluable tool for studying GABA_ARs and other ligand-gated ion channels. The technique has distinct advantages, including the ability to express receptors in isolation and with mutations that can be probed with a variety of compounds and labels to study the structure and function of a particular residue or region of the receptor. These unique capabilities make recombinant expression a powerful and essential tool. Thus, despite its limitations, improving and understanding the recombinant expression of αβδ GABA_ARs remains critical.

We studied α₄β₂δ GABA_ARs in our experiments, which are expressed predominately in thalamic relay neurons and dentate gyrus granule cells (41–44). Although it is hypothesized and often assumed that the stoichiometry of these receptors is 2α:2β:1δ with a similar arrangement to αβγ GABA_ARs, additional studies are needed to confirm this idea. Given the extrasynaptic localization of δ-containing GABA_ARs and their role in mediating tonic inhibition and the effects of volatile anesthetics, neurosteroids, and ethanol, characterizing their structural, physiological, and pharmacological properties remains an important task. Moreover, recent studies have shown that the actions of neurosteroids likely alter the trafficking and expression of δ-containing receptors (45–47). Successful BBS tagging of GABA_AR subunits is a novel tool with which to accurately quantitate subunit/receptor expression and receptor subunit stoichiometry that is suitable for any multisubunit membrane protein. The method will also be useful for monitoring the trafficking of GABA_ARs in live cells.