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BACKGROUND AND PURPOSEGABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, a4 and d subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant d subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACHWe engineered concatemeric (fused) subunits to ensure d and a4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (a1 + b2 + g2L), and with free-subunit a4/d receptors, expressed in Xenopus oocytes. KEY RESULTSd-b2 -a4 + b2-a4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of a4/d-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a] pyridine-3-yl benzamide; a d-selective positive modulator), furosemide, and Zn 2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, d-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations.

BackgroundPropofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the “+” of the β subunit, in the β-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the “-” side of the β subunit, in the α-β interface (or β-β interface, in the case of homomeric β receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the β(H267) residue lines a cavity that docks propofol with favorable interaction energy.MethodWe used two-electrode voltage clamp to determine the functional effects of mutations to the 
“+” and “-” sides of the β subunit on activation of the α1β3 GABAA receptor by propofol.ResultsWe found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-β interface leads to strong reduction in gating efficacy for propofol.ConclusionWe conclude that α1β3 GABAA receptors can be activated by propofol interactions with the β-β, α-β, and β-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.

Native g-aminobutyric acid (GABA) A receptors consisting of a4, b1-3, and d subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed a4bd receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of a4b2d receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct b2-d and a free a4 subunit exhibited large steroid responses. We propose that free a4, b2, and d subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with a4 and the picrotoxin-resistant d(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that a4bd receptors may assemble in a similar configuration in neurons and oocytes.

Background The γ-aminobutyric acid type A receptor is target for several anesthetics, anticonvulsants, anxiolytics and sedatives. Neurosteroids, barbiturates and etomidate both potentiate responses to γ-aminobutyric acid (GABA) and allosterically activate the receptor. We examined the ability of a benzodiazepine, diazepam, to potentiate responses to allosteric agonists. Methods The γ-aminobutyric acid type A receptors were expressed in human embryonic kidney 293 cells, and studied using whole-cell and single-channel patch clamp. The receptors were activated by the orthosteric agonist GABA, and allosteric agonists pentobarbital, etomidate and alfaxalone. Results Diazepam is equally potent at enhancing responses to orthosteric and allosteric agonists. Diazepam EC50s were 25±4, 26±6, 33±6, and 26±3 nM for receptors activated by GABA, pentobarbital, etomidate, and alfaxalone, respectively (mean±S.D., 5–6 cells at each condition). Mutations to the benzodiazepine-binding site (α1(H101C), γ2(R144C), γ2(R197C)) reduced or removed potentiation for all agonists, and an inverse agonist at the benzodiazepine site reduced responses to all agonists. Single-channel data elicited by GABA demonstrate that in the presence of 1 μM diazepam the prevalence of the longest open-time component is increased from 13±7 (mean±S.D., n=5 patches) to 27±8 % (n=3 patches) and the rate of channel closing is decreased from 129±28 s−1 to 47±6 s−1 (mean±S.D.) Conclusions We conclude that benzodiazepines do not act by enhancing affinity of the orthosteric site for GABA but rather by increasing channel gating efficacy. The results also demonstrate the presence of significant interactions between allosteric activators and potentiators, raising a possibility of effects on dosage requirements or changes in side effects.