Che Has AT , Absalom N , van Nieuwenhuijzen PS , Clarkson AN , Ahring PK , Chebib M .

	Figure 1. GABA-evoked responses at α1β3 and α1β3γ2 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage-clamp electrophysiology as described in the methods. For experimentation, oocytes were clamped at −60 mV and full GABA concentration response relationships were obtained on each oocyte. (A) Representative GABA-evoked traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate application periods and GABA concentrations and “/” a wash period. Dotted lines indicate a 0 nA baseline and holding currents were −150 ± 93 nA, n = 6 for α1 + β3 (1:1) and −13 ± 10 nA, n = 9 for α1 + β3 (30:1). (B,C) Baseline subtracted peak current amplitudes for full GABA concentration-response curves at oocytes injected with the indicated cRNA mixtures using free subunits (B) or a concatenated β3-α1 construct (C) were fitted to the Hill equation using non-linear regression (fixed bottom of 0 and slope of 1) and normalized to the maximal fitted value (IGABA_max_fit). Averaged normalized data points are depicted as means ± S.E. as a function of the GABA concentration, fitted to the Hill equation and regression results are presented in Table 1. Each data point represents experiments from n = 5–9 oocytes from ≥2 batches. (D) α1β3 GABAA receptors can express in two stoichiometries of 2α1:3β3 (left) and 3α1:2β3 (right). The two binding sites for GABA at the β3(+)-α1(−) subunit interface are indicated by red arrowheads.
	Figure 2. Zn2+ inhibition of GABA-evoked currents from α1β3 and α1β3γ2 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. Control currents (Icontrol) were evoked using a GABA concentration corresponding to ~EC50 and inhibition by Zn2+ was evaluated by co-applications with GABAcontrol. (A) Representative GABA-evoked current traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate GABA and Zn2+ application periods. Dotted lines indicate the peak current amplitude by GABAcontrol and “/” a wash period. (B,C) Concentration-response relationships of Zn2+ inhibition of GABAcontrol-evoked currents at α1β3 or α1β3γ2 receptors stemming from injecting the indicated cRNA mixtures using free subunits (B) or a concatenated β3-α1 construct (C). Averaged Zn2+ inhibition values were depicted as means ± S.E.M as a function of the Zn2+ concentration and fitted to the Hill equation by non-linear regression. Regression results for α1 + β3 (1:1) were IC50 = 0.84 (95% CI: 0.53–1.3), nH = −0.5 ± 0.32 and for β3-α1 + β3 (1:2) were IC50 = 1.6 (95% CI: 1.1–2.4), nH = −0.6 ± 0.06. For the remaining cRNA mixtures, Zn2+ inhibition at the maximal tested concentration was too low to allow for meaningful fitting. Each data point represent experiments from n = 5–8 oocytes of ≥2 batches. (D) The depiction of α1β3 GABAA receptor stoichiometries from Fig. 1D was modified to indicate Zn2+ binding in the β3(+)-β3(−) subunit interface (red/orange arrowhead).
	Figure 3. Zolpidem modulation of GABA-evoked currents from α1β3 and α1β3γ2 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. Control currents (Icontrol) were evoked using a GABA concentration corresponding to ~EC5–10 and modulation by zolpidem was evaluated by co-applications with GABAcontrol. (A–C) Representative GABA-evoked current traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate GABA, zolpidem (Zolp) and flumazenil (Flu) concentrations and application periods. Dotted lines indicate the peak current amplitude by GABAcontrol and “/” a wash period. For the specific traces, zolpidem had no robust effects at receptors from α1 + β3 (1:1) injection (A), but showed 130% modulation at receptors from α1 + β3 (30:1) injection which was inhibited 95% by co-application of flumazenil (B). Zolpidem likewise modulated receptors from injection of α1 + β3 + γ2 (1:1:5) by 160% which could be inhibited 85% by flumazenil (C). (D,E) Concentration-response relationships of zolpidem modulation of GABAcontrol-evoked currents at α1β3 or α1β3γ2 receptors stemming from injecting the indicated cRNA mixtures using free subunits (D) or a concatenated β3-α1 construct (E). Average modulatory values were depicted as means ± S.E.M as a function of the zolpidem concentration and fitted to the Hill equation by non-linear regression. Each data point represents experiments from n = 5–7 oocytes from ≥2 batches and regression results are presented in Table 1.
	Figure 4. Mechanism of zolpidem modulatory actions at α1β3 and α1β3γ2 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. (A–C) Full GABA concentration-response relationships were obtained in presence of the indicated concentrations of zolpidem (Zolp) at receptors stemming from injection of cRNA of free subunits (A,C) or a concatenated construct (B). Baseline subtracted GABA + zolpidem peak current amplitudes were normalized to a maximal GABA control response (3 mM) in the same oocytes. Averaged normalized data points are depicted as means ± S.E.M. as a function of the GABA concentration and fitted to the Hill equation with regression results are presented in Table 1. Each data point represents experiments from n = 5–7 oocytes from ≥2 batches. GABA concentration response relationships in absence of zolpidem (from Fig. 1) are included for comparison. (D) The depiction of α1β3 GABAA receptor stoichiometries from Fig. 2D was modified to indicate zolpidem binding in the α1(+)-α1(−) subunit interface (red/green arrowhead).
	Figure 5. Diazepam modulation of GABA-evoked currents from α1β3 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. Control currents (Icontrol) were evoked using a GABA concentration corresponding to ~EC5–10 and modulation by diazepam was evaluated by co-applications with GABAcontrol. (A,B) Representative GABA-evoked current traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate GABA, diazepam (Diaz) and flumazenil (Flu) concentrations and application periods. Dotted lines indicate the peak current amplitude by GABAcontrol and “/” a wash period. (C) Concentration-response relationships of diazepam modulation of GABAcontrol-evoked currents at α1β3 receptors stemming from injecting the indicated cRNA mixtures using free subunits or a concatenated β3-α1 construct. Averaged modulatory values were depicted as means ± S.E as a function of the diazepam concentration and fitted to the Hill equation by non-linear regression. Each data point represents experiments from n = 6 oocytes from ≥2 batches. Regression results with 95% confidence intervals for α1 + β3 (30:1) were: EC50 value of 0.040 μM (0.010–0.12) and Emax value of 40% (33–48) whereas results for β3-α1 + α1 (1:2) were: EC50 value of 0.020 μM (0.010–0.04) and Emax value of 51% (46–56).

Ahring, Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface. 2015, Pubmed, Xenbase

Ahring, Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface. 2015, Pubmed , Xenbase
Baumann, Subunit arrangement of gamma-aminobutyric acid type A receptors. 2001, Pubmed , Xenbase
Baur, Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs. 2008, Pubmed , Xenbase
Baur, Replacement of histidine in position 105 in the α₅ subunit by cysteine stimulates zolpidem sensitivity of α₅β₂γ₂ GABA(A) receptors. 2007, Pubmed , Xenbase
Bencsits, A significant part of native gamma-aminobutyric AcidA receptors containing alpha4 subunits do not contain gamma or delta subunits. 1999, Pubmed
Boileau, Tandem subunits effectively constrain GABAA receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAA receptor-associated protein. 2005, Pubmed
Brefel-Courbon, Clinical and imaging evidence of zolpidem effect in hypoxic encephalopathy. 2007, Pubmed
Brickley, Single-channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes. 1999, Pubmed
Buhr, A point mutation in the gamma2 subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity. 1997, Pubmed , Xenbase
Chang, Trial of Zolpidem, Eszopiclone, and Other GABA Agonists in a Patient with Progressive Supranuclear Palsy. 2014, Pubmed
Chebib, GABA-Activated ligand gated ion channels: medicinal chemistry and molecular biology. 2000, Pubmed
Chen, Zolpidem improves akinesia, dystonia and dyskinesia in advanced Parkinson's disease. 2008, Pubmed
Chua, The Direct Actions of GABA, 2'-Methoxy-6-Methylflavone and General Anaesthetics at β3γ2L GABAA Receptors: Evidence for Receptors with Different Subunit Stoichiometries. 2015, Pubmed , Xenbase
Clauss, Cerebral blood perfusion after treatment with zolpidem and flumazenil in the baboon. 2002, Pubmed
Clauss, Transient improvement of spinocerebellar ataxia with zolpidem. 2004, Pubmed
Cohen, Transient improvement of aphasia with zolpidem. 2004, Pubmed
Cope, Abolition of zolpidem sensitivity in mice with a point mutation in the GABAA receptor gamma2 subunit. 2004, Pubmed
Cope, Enhanced tonic GABAA inhibition in typical absence epilepsy. 2009, Pubmed
Cope, Loss of zolpidem efficacy in the hippocampus of mice with the GABAA receptor gamma2 F77I point mutation. 2005, Pubmed
Ernst, Comparative modeling of GABA(A) receptors: limits, insights, future developments. 2003, Pubmed
Farrant, Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. 2005, Pubmed
Farrar, Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer. 1999, Pubmed
Frederickson, Synaptic release of zinc from brain slices: factors governing release, imaging, and accurate calculation of concentration. 2006, Pubmed
Frederickson, Concentrations of extracellular free zinc (pZn)e in the central nervous system during simple anesthetization, ischemia and reperfusion. 2006, Pubmed
Gao, Zolpidem modulation of phasic and tonic GABA currents in the rat dorsal motor nucleus of the vagus. 2010, Pubmed
Hall, GABA-mediated changes in inter-hemispheric beta frequency activity in early-stage Parkinson's disease. 2014, Pubmed
Hanson, Structural requirements for eszopiclone and zolpidem binding to the gamma-aminobutyric acid type-A (GABAA) receptor are different. 2008, Pubmed
Harpsøe, Unraveling the high- and low-sensitivity agonist responses of nicotinic acetylcholine receptors. 2011, Pubmed
Hiu, Enhanced phasic GABA inhibition during the repair phase of stroke: a novel therapeutic target. 2016, Pubmed
Hoerbelt, Mutagenesis and computational docking studies support the existence of a histamine binding site at the extracellular β3+β3- interface of homooligomeric β3 GABAA receptors. 2016, Pubmed , Xenbase
Hosie, Zinc-mediated inhibition of GABA(A) receptors: discrete binding sites underlie subtype specificity. 2003, Pubmed
Karim, 2'-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA(A) receptors. 2012, Pubmed , Xenbase
Karim, 3-Hydroxy-2'-methoxy-6-methylflavone: a potent anxiolytic with a unique selectivity profile at GABA(A) receptor subtypes. 2011, Pubmed , Xenbase
Kaur, Unanticipated structural and functional properties of delta-subunit-containing GABAA receptors. 2009, Pubmed , Xenbase
Kharlamov, Expression of GABA A receptor alpha1 subunit mRNA and protein in rat neocortex following photothrombotic infarction. 2008, Pubmed
Khom, Pharmacological properties of GABAA receptors containing gamma1 subunits. 2006, Pubmed , Xenbase
Liu, Quantitative reverse transcription-polymerase chain reaction of GABA(A) alpha1, beta1 and gamma2S subunits in epileptic rats following photothrombotic infarction of neocortex. 2002, Pubmed
May, Benzodiazepine-site pharmacology on GABAA receptors in histaminergic neurons. 2013, Pubmed
Mortensen, Extrasynaptic alphabeta subunit GABAA receptors on rat hippocampal pyramidal neurons. 2006, Pubmed
Patat, Flumazenil antagonizes the central effects of zolpidem, an imidazopyridine hypnotic. 1994, Pubmed
Patel, Context-Dependent Modulation of GABAAR-Mediated Tonic Currents. 2016, Pubmed
Pritchett, Gamma-aminobutyric acidA receptor alpha 5-subunit creates novel type II benzodiazepine receptor pharmacology. 1990, Pubmed
Prokic, Cortical oscillatory dynamics and benzodiazepine-site modulation of tonic inhibition in fast spiking interneurons. 2015, Pubmed
Qian, Visualization of transmitter release with zinc fluorescence detection at the mouse hippocampal mossy fibre synapse. 2005, Pubmed
Ramerstorfer, The point mutation gamma 2F77I changes the potency and efficacy of benzodiazepine site ligands in different GABAA receptor subtypes. 2010, Pubmed
Ramerstorfer, The GABAA receptor alpha+beta- interface: a novel target for subtype selective drugs. 2011, Pubmed , Xenbase
Raol, Increased GABA(A)-receptor alpha1-subunit expression in hippocampal dentate gyrus after early-life status epilepticus. 2006, Pubmed
Raol, Enhancing GABA(A) receptor alpha 1 subunit levels in hippocampal dentate gyrus inhibits epilepsy development in an animal model of temporal lobe epilepsy. 2006, Pubmed
Sancar, Structural determinants for high-affinity zolpidem binding to GABA-A receptors. 2007, Pubmed
Sanna, Comparison of the effects of zaleplon, zolpidem, and triazolam at various GABA(A) receptor subtypes. 2002, Pubmed , Xenbase
Saras, Histamine action on vertebrate GABAA receptors: direct channel gating and potentiation of GABA responses. 2008, Pubmed , Xenbase
Sieghart, Structure, pharmacology, and function of GABAA receptor subtypes. 2006, Pubmed
Sieghart, Subunit composition, distribution and function of GABA(A) receptor subtypes. 2002, Pubmed
Sieghart, Allosteric modulation of GABAA receptors via multiple drug-binding sites. 2015, Pubmed
Sigel, Mapping of the benzodiazepine recognition site on GABA(A) receptors. 2002, Pubmed
Sinkkonen, Autoradiographic imaging of altered synaptic alphabetagamma2 and extrasynaptic alphabeta GABAA receptors in a genetic mouse model of anxiety. 2004, Pubmed
Smart, GABAA receptors are differentially sensitive to zinc: dependence on subunit composition. 1991, Pubmed
Tretter, Stoichiometry and assembly of a recombinant GABAA receptor subtype. 1997, Pubmed
Vogt, The actions of synaptically released zinc at hippocampal mossy fiber synapses. 2000, Pubmed
Walters, Benzodiazepines act on GABAA receptors via two distinct and separable mechanisms. 2000, Pubmed , Xenbase
Wieland, Four amino acid exchanges convert a diazepam-insensitive, inverse agonist-preferring GABAA receptor into a diazepam-preferring GABAA receptor. 1994, Pubmed