High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ subunits

The inhibitory γ-aminobutyric acid type A receptors are implicated in numerous physiological processes, including cognition and inhibition of neurotransmission, rendering them important molecular targets for many classes of drugs. Functionally, the entire GABAAR family of receptors can be subdivided into phasic, fast acting synaptic receptors, composed of α-, β- and γ-subunits, and tonic extrasynaptic receptors, many of which contain the δ-subunit in addition to α- and β-subunits. Whereas the subunit arrangement of the former group is agreed upon, that of the αβδ GABAARs remains unresolved by electrophysiological and pharmacological research. To resolve such issues will require biophysical techniques that demand quantities of receptor that have been previously unavailable. Therefore, we have engineered a stable cell line with tetracycline inducible expression of human α4-, β3- and N-terminally Flag-tagged δ-subunits. This cell line achieved a specific activity between 15 and 20 pmol [3H]muscimol sites/mg of membrane protein, making it possible to obtain 1 nmole of purified α4β3δ GABAAR from sixty 15–cm culture dishes. When induced, these cells exhibited agonist–induced currents with characteristics comparable to those previously reported for this receptor and a pharmacology that included strong modulation by etomidate and the δ-subunit-specific ligand, DS2. Immunoaffinity purification and reconstitution in CHAPS/asolectin micelles resulted in the retention of equilibrium allosteric interactions between the separate agonist, anesthetic and DS2 sites. Moreover, all three subunits retained glycosylation. The establishment of this well–characterized cell line will allow molecular level studies of tonic receptors to be undertaken

All subunits of the α4β3N-Flag-δ GABA_A receptor are glycosylated.

<p>Purified receptors were resolved by Western blotting with antibodies for α4- and β3-subunits or Flag, as shown on the left, middle and right panels, respectively. In each of the three panels, reading left to right the lanes are: purified receptor; purified receptor after deglycosylation with PNGase F, and PNGase F alone. The band below the 43 kDa marker was nonspecific and associated with PNGase F. Numbers on the left side indicate MW in kDa. The α4 and β3 antibodies are polyclonal. Brightness and contrast were uniformly adjusted for each panel.</p

Stability of the α4-subunit.

<p>(<b>A</b>) Western blot depicting fragmentation of α4-subunit seen as two bands in N-Flag-α4β3 and α4β3N-Flag-δ receptors reconstituted into CHAPS/asolectin micelles is presented. Both α4 bands are identified by polyclonal anti-α4 and monoclonal anti-Flag antibodies in the former receptor, confirming identity of the band. Numbers on the side indicate the position of the molecular weight markers (kDa). (<b>B</b>) Cells induced to express indicated GABA_ARs were prepared for Western blotting by either 1. suspending cells in suspension buffer; 2. suspending and sonicating; 3. leaving in a monolayer (untreated); or 4. lysing directly in the well with suspension buffer supplemented with 10 mM DDM; as indicated, prior to lysing cells with a 4x Laemmli sample buffer with 10% β-mercaptoethanol. Suspension buffer was supplemented with Protease Inhibitor Cocktail (Sigma) at 1:100 dilution. (<b>C</b>) Representative Western blot of samples obtained during α4β3N-Flag-δ receptor purification, as described in the materials and methods section. Numbers under each lane indicate the fraction the lower band comprises of the higher band, expressed as percentile points. (<b>D</b>) Membrane fraction from the α4β3N-Flag-δ was incubated for 1 hour at indicated temperatures prior to analysis by Western blotting. All blots are presented as grayscale and were uniformly adjusted for brightness and contrast to facilitate analysis. Full immunoblots used to make panels A-D are presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191583#pone.0191583.s004" target="_blank">S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191583#pone.0191583.s007" target="_blank">S7</a> Figs.</p

The binding isotherm of the agonist [³H]muscimol to the α4β3N-Flag-δ GABA_A receptor in native membranes and reconstituted into CHAPS/lipid micelles.

<p>Binding curves of [³H]muscimol to α4β3N–Flag–δ GABA_ΑRs, both, in cell membranes (pmol/mg membrane protein) and after purification and reconstitution into micelles of 5 mM CHAPS and 200 μM DOPC:DOPS:Cholesterol in mole ratio 52:15:33 (pmol/mL). Displaceable binding was determined as the difference between binding in the presence and absence of 1 mM GABA using a filtration assay in triplicate. The displaceable binding and its standard deviation was determined by subtracting these two values and propagating errors at each total muscimol concentration. The curves were fitted by nonlinear least squares with weighting by standard deviation. These yielded apparent dissociation constants of 9.2 ± 0.6 and 35 ± 12 nM, respectively. The B_max of the membranes was 22.6 ± 0.5 pmol/mg and for reconstituted receptors in micelles was 19 ± 3 pmol/mL. The Hill coefficients differed little from one (1.07 ± 0.3 and 0.90 ± 0.05 respectively).</p