The gating properties of α4β3N–Flag–δ GABA_ARs.

<p>(<b>A</b>) GABA concentration-response curve for α4β3N–Flag–δ receptor mediated currents. Currents were elicited by application of varying concentrations of GABA (0.1–10 mM). Peak current amplitudes in each cell were normalized to that obtained with 10 mM GABA. (<b>B</b>) Representative current trace obtained by application of an 8.5 ms pulse of 100 μM GABA to measure the deactivation rate. (<b>C</b>) α4β3N–Flag–δ receptors are spontaneously open. (Left panel) Representative trace of outward currents observed by application of 2 mM Picrotoxin to inhibit the spontaneously open receptors. (Right panel) The estimate of the maximum inward currents obtained by co-application of 10 mM GABA with 10 μM Etomidate to gate all available receptors. The gray lines are drawn by eye to represent the baseline. At least three cells per concentration were used throughout experiments.</p

DS2 enhances agonist binding in δ-subunit containing receptors.

<p>The δ-subunit specific modulator, DS2, modulates [³H]muscimol binding (2 nM) in α4β3N-Flag-δ GABA_ARs. For the membranes, the data are the mean and standard deviation of two experiments, and for micelle reconstituted receptors for a single experiment in triplicate. The curves were fitted by nonlinear least squares to the Hill equation. The EC₅₀ (μM), Hill coefficient and maximum modulation were: for membranes, 2.0 ± 0.7 μM, 0.9 ± 0.2, 139 ± 4%; for reconstituted 2.3 ± 0.8 μM, 1.2 ± 0.5, 132 ± 4%.</p

Representative yield of α4β3N-Flag-δ GABA_AR purification by anti-Flag affinity chromatography.

<p>Representative yield of α4β3N-Flag-δ GABA_AR purification by anti-Flag affinity chromatography.</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

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

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

The δ-subunit is expressed in the α4β3N–Flag–δ GABA_AR stable cell line.

<p>Representative current traces show the effect of DS2 (<b>A</b>) and Etomidate (<b>B</b>) on 10 mM GABA–elicited currents on α4β3N–Flag–δ (upper panel) compared to N–Flag–α4β3 (lower panel). Currents were elicited in a notch protocol by an eight second pulse of GABA, during which drug was co-applied for 4 seconds 1 second after the GABA perfusion started. Concentrations are indicated in the figure.</p

Mapping General Anesthetic Binding Site(s) in Human α1β3 γ-Aminobutyric Acid Type A Receptors with [³H]TDBzl-Etomidate, a Photoreactive Etomidate Analogue

The γ-aminobutyric acid type A receptor (GABA_AR) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA_ARs purified from bovine brain, [³H]­azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA_AR transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) <i>J. Neurosci. 26</i>, 11599–11605]. To further define GABA_AR etomidate binding sites, we now use [³H]­TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA_ARs and more extensively identify photolabeled GABA_AR amino acids. [³H]­TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [³H]­azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA_AR homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [³H]­TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA_AR intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [³H]­TDBzl-etomidate and [³H]­azietomidate also provides evidence of a homologous etomidate binding site at the β3−β3 subunit interface in the α1β3 GABA_AR

Ligand-induced changes in the vestibule and grotto region of luciferase.

<p>The “active” DSLA bound (left, 2D1S.pdb) and ATP bound (right, 2D1Q.pdb) structures are compared from the same viewing points. Upper panels show a cross section through the vestibule and grotto region beyond the luciferin binding pocket (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g007" target="_blank">Fig. 7</a>). The lower panels show the vestibule as viewed from the luciferin pocket; the left panel shows the O10 of DLSA for orientation. Water molecules are shown in red in the +ATP structure and in gold in the DSLA structure (one of the eight water molecules is partially hidden in panel C and is indicated with a circle). The cross-section surface capping is semitransparent. Surface coloring is grey for carbon, red for oxygen and blue for nitrogen. DSLA (gold carbons in A) is a surrogate for luciferin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g001" target="_blank">Fig. 1B</a>). Similar changes are seen in American luciferase (3IES.pdb and 3IEP.pdb).</p

The 3-Azioctanol inhibits ATP-induced luciferase activity.

<p>Fresh American firefly luciferase pre-incubated with luciferin and increasing concentrations of the alkanol were rapidly mixed with ATP (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#s3" target="_blank">Results</a>). <b>A.</b> Increasing concentrations of 3-azioctanol inhibited the initial phase of light emission. <b>B.</b> The slope of the linear portion of the initial peak of each curve was normalized (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#s3" target="_blank">Results</a>) and plotted against the 3-azioctanol concentrations. Nonlinear least squares fitting yielded an IC₅₀ value of 220±47 µM and a Hill coefficient of 1.1±0.2.</p