GABAA receptors (GABAARs) are members of the Cys-loop ligand-gated ion channel (LGIC) superfamily, which includes nicotinic acetylcholine, glycine, and serotonin (5HT3) receptors [1,2,3,4]. LGICs typically mediate fast synaptic transmission via the movement of ions through channels gated by neurotransmitters, such as acetylcholine for nicotinic receptors and GABA for GABAARs [5]. The term Cys-loop receptors originates from the presence of a conserved disulphide bond (or bridge) which holds together two cysteine amino acids of the loop that forms from the structure of polypeptides in the extracellular domain of the receptor's subunit [6]. GABAARs are pentameric transmembrane protein complexes consisting of five subunits from a variety of polypeptide subunits [7,8]. All of these subunits are pseudo-symmetrically organized in the plane of the membrane, with a Cl--selective channel in the middle of the complex. To date, nineteen GABAAR subunits have been identified and categorized into eight classes, α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3, but their variety is further broadened by the existence of several splice forms for certain subunits (e.g., α6, β2 and γ2) [9,10,11,12]. The subunits within each class have an amino acid sequence homology of 70% or more, whereas those with a sequence homology of 30% or less are grouped into different classes [13,14]. A subunit consists of four transmembrane domains (TM1-TM4), each forming an α-helix; a large extracellular N-terminal domain that incorporates part of the orthosteric agonist/antagonist binding site; a large intracellular loop between the TM3 and TM4; a small intracellular loop between TM1 and TM2; a small extracellular loop between TM2 and TM3; and a C-terminal extracellular domain [15,16]. Each subunit is arranged in such a way as to create principal and complementary interfaces with the other subunits, and in a position such that the TM2 of each subunit forms the wall of the channel pore [17,18,19]. The major subunit combination found in the brain comprises α1, β2 and γ2 subunits with the stoichiometry 2α1:2β2:1γ2 [18,20]. For the GABAA α1β2γ2 receptors, the subunits form a specific arrangement in which α1 and β2 subunits alternate with each other and are connected by a γ2 subunit (Figure A) [16,20,21]. For minor subtypes, different α and β subunits have been detected to co-exist as proven by the existence of GABAARs containing α1α2, α1α3, α1α5, α2α3, α3α5, α4α1, α4α2 and α4α3 in the central nervous system [22,23]. Meanwhile, the same pattern has been detected with β and γ subunits, at least the co-occurrence of β in the same GABAAR as well as γ2 with γ3, indicating that these subunits have the capacity to co-exist with each other [24,25,26]. Since different subunits can actually occur in one receptor, GABAARs are considered to exist in a multi-subunit arrangement, leading to ambiguity in the determination of a receptor's stoichiometry. In this review, we first briefly discuss the different subunit arrangements of α1 and β3 subunits in the binary α1β3 receptors. Then we review the GABAA ε-containing receptors predominantly in terms of the ability of ε subunit to present in different position in the ternary α1β3ε receptors, which introduce distinct populations of receptor.

To intoxicated patients in the emergency room, toxicological analysis can be considerably helpful for identifying the involved toxicants. In order to develop a urine multi-drug screening (UmDS) method, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) were used to determine targeted and unknown toxicants in urine. A GC-MS method in scan mode was validated for selectivity, limit of detection (LOD) and recovery. An LC-MS-MS multiple reaction monitoring (MRM) method was validated for lower LOD, recovery and matrix effect. The results of the screening analysis were compared with patient medical records to check the reliability of the screen. Urine samples collected from an emergency room were extracted through a combination of salting-out assisted liquid-liquid extraction (SALLE) and hybrid protein precipitation/solid phase extraction (hybrid PPT/SPE) plates and examined by GC-MS and LC-MS-MS. GC-MS analysis was performed as unknown drug screen and LC-MS-MS analysis was conducted as targeted drug screen. After analysis by GC-MS, a library search was conducted using an in-house library established with the automated mass spectral deconvolution and identification system (AMDISTM). LC-MS-MS used Cliquid®2.0 software for data processing and acquisition in MRM mode. An UmDS method by GC-MS and LC-MS-MS was developed by using a SALLE-hybrid PPT/SPE and in-house library. The results of UmDS by GC-MS and LC-MS-MS showed that toxicants could be identified from 185 emergency room patient samples containing unknown toxicants. Zolpidem, acetaminophen and citalopram were the most frequently encountered drugs in emergency room patients. The UmDS analysis developed in this study can be used effectively to detect toxic substances in a short time. Hence, it could be utilized in clinical and forensic toxicology practices.

Zolpidem (Ambien®) is one of the "Z" drugs often used to improve sleep in older patients and those suffering from insomnia. Schwope, D.M., DePriest, A., Black, D.L., Caplan, Y.H., Cone, E.J., Heltsley, R. (2014) Determing zolpidem compliance: urinary metabolite detection and prevalence in chronic pain patients . Journal of Analytical Toxicology, 38, 513-518 reported that zolpidem in urine is not very prevalent being present <23% of the time in patient urine while the major metabolite, zolpidem 4-phenyl carboxylic acid (ZCA), is much more prevalent in urine with positive rates as high as 50% of the patient samples reviewed. Results from patient testing over a year's time are in agreement with the reported zolpidem results. However, the data observed herein for ZCA are not consistent with the earlier report. These data suggest that monitoring ZCA may result in even higher levels of positivity. Further, while the Food and Drug Administration has pointed out that female dosing should be half that given to males, results of this population testing indicate that the majority of patients (83% male and 73% female) receive 10 mg/day or 12.5 mg/day for Ambien CR® with females demonstrating statistically significantly higher levels of ZCA albeit zolpidem levels are not statistically significantly different between men and women. Estimates of patient positivity are dependent upon the value of the limit of quantification (LOQ) as demonstrated by the zolpidem results herein (LOQ = 50 ng/mL vs. 4 ng/mL). However, even with a much higher LOQ of 50 ng/mL for ZCA in this work, the positivity from ZCA results is significantly higher (e.g., 64.8%) than reported earlier (50.3%). Nevertheless, these data support the addition of ZCA for monitoring zolpidem in urine.

Using non-human primates, we introduced a new set of behavioral categories for observable sedative effects based on pediatric anesthesiology classifications. We examined the effects of alprazolam and diazepam (non-selective benzodiazepines), zolpidem (preferential binding to α1 subunit-containing GABAA receptors), HZ-166 (8-ethynyl-6-(2'-pyridine)-4H-2,5,10b-triaza-benzo[e]azulene-3-carboxylic acid ethyl ester; functionally selective for α2 and α3 subunit-containing GABAA receptors), MRK-696 (7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-2-ylmethoxy)-3-(2-flurophenyl)-1,2,4-triazolo(4,3-b)pyridazine; no selectivity but partial intrinsic activity), and TPA023B (6,2'-difluro-5'-(3-(1-hydroxy-1-methylethyl)imidazo(1,2-b((1,2,4)triazin-7-yl)(1,1'-biphenyl)-2-carbonitrile; selectivity for α2, α3, α5 subunit-containing GABAA receptors) using quantitative behavioral observation techniques in rhesus monkeys. We further examined the role of α1 subunit-containing GABAA receptors in benzodiazepine-induced sedative effects by pretreating animals with the α1 subunit-preferring antagonist β-carboline-3-carboxylate-t-butyl ester (βCCT). Increasing doses of alprazolam and diazepam resulted in the emergence of observable ataxia, rest/sleep posture, moderate and deep sedation. In contrast, zolpidem engendered dose-dependent observable ataxia and deep sedation but not rest/sleep posture or moderate sedation, while HZ-166 and TPA023 induced primarily rest/sleep posture. MRK-696 induced rest/sleep posture and observable ataxia. Zolpidem, but no other compounds, significantly increased tactile/oral exploration. The sedative effects engendered by alprazolam, diazepam, and zolpidem generally were attenuated by βCCT pretreatments, whereas rest/sleep posture and suppression of tactile/oral exploration were insensitive to βCCT administration. These data suggest that α2/3-containing GABAA receptor subtypes unexpectedly may mediate a mild form of sedation (rest/sleep posture), whereas α1-containing GABAA receptors may play a role in moderate/deep sedation.

In our previous studies, we have demonstrated that marine polyphenol phlorotannins promote sleep through the benzodiazepine site of the gamma-aminobutyric acid type A (GABAA) receptors. In this follow-up study, the sleep-promoting effects of triphlorethol A, one of the major phlorotannin constituents, were investigated. The effect of triphlorethol A on sleep-wake architecture and profiles was evaluated based on electroencephalogram and electromyogram data from C57BL/6N mice and compared with the well-known hypnotic drug zolpidem. Oral administration of triphlorethol A (5, 10, 25, and 50 mg/kg) dose-dependently decreased sleep latency and increased sleep duration during pentobarbital-induced sleep in imprinting control region mice. Triphlorethol A (50 mg/kg) significantly decreased sleep latency and increased the amount of non-rapid eye movement sleep (NREMS) in C57BL/6N mice, without affecting rapid eye movement sleep (REMS). There was no significant difference between the effects of triphlorethol A at 50 mg/kg and zolpidem at 10 mg/kg. Triphlorethol A had no effect on delta activity (0.5⁻4 Hz) of NREMS, whereas zolpidem significantly decreased it. These results not only support the sleep-promoting effects of marine polyphenol phlorotannins, but also suggest that the marine polyphenol compound triphlorethol A is a promising structure for developing novel sedative hypnotics.