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Purpose The efficacy of neoadjuvant chemoradiotherapy (NCRT) plus surgery for locally advanced esophageal squamous cell carcinoma (ESCC) remains controversial. In this trial, we compared the survival and safety of NCRT plus surgery with surgery alone in patients with locally advanced ESCC. Patients and Methods From June 2007 to December 2014, 451 patients with potentially resectable thoracic ESCC, clinically staged as T1-4N1M0/T4N0M0, were randomly allocated to NCRT plus surgery (group CRT; n = 224) and surgery alone (group S; n = 227). In group CRT, patients received vinorelbine 25 mg/m 2 intravenously (IV) on days 1 and 8 and cisplatin 75 mg/m 2 IV day 1, or 25 mg/m 2 IV on days 1 to 4 every 3 weeks for two cycles, with a total concurrent radiation dose of 40.0 Gy administered in 20 fractions of 2.0 Gy on 5 days per week. In both groups, patients underwent McKeown or Ivor Lewis esophagectomy. The primary end point was overall survival. Results The pathologic complete response rate was 43.2% in group CRT. Compared with group S, group CRT had a higher R0 resection rate (98.4% v 91.2%; P = .002), a better median overall survival (100.1 months v 66.5 months; hazard ratio, 0.71; 95% CI, 0.53 to 0.96; P = .025), and a prolonged disease-free survival (100.1 months v 41.7 months; hazard ratio, 0.58; 95% CI, 0.43 to 0.78; P < .001). Leukopenia (48.9%) and neutropenia (45.7%) were the most common grade 3 or 4 adverse events during chemoradiotherapy. Incidences of postoperative complications were similar between groups, with the exception of arrhythmia (group CRT: 13% v group S: 4.0%; P = .001). Peritreatment mortality was 2.2% in group CRT versus 0.4% in group S ( P = .212). Conclusion This trial shows that NCRT plus surgery improves survival over surgery alone among patients with locally advanced ESCC, with acceptable and manageable adverse events.

The disrupted in schizophrenia 1 (DISC1) gene is a candidate susceptibility factor for schizophrenia, but its mechanistic role in the disorder is unknown. Here we report that the gene encoding phosphodiesterase 4B (PDE4B) is disrupted by a balanced translocation in a subject diagnosed with schizophrenia and a relative with chronic psychiatric illness. The PDEs inactivate adenosine 3',5'-monophosphate (cAMP), a second messenger implicated in learning, memory, and mood. We show that DISC1 interacts with the UCR2 domain of PDE4B and that elevation of cellular cAMP leads to dissociation of PDE4B from DISC1 and an increase in PDE4B activity. We propose a mechanistic model whereby DISC1 sequesters PDE4B in resting cells and releases it in an activated state in response to elevated cAMP.

The neuroprotective effects of MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors, were evaluated in models of cerebral ischemia using Mongolian gerbils. Bilateral occlusion of the carotid arteries for a period of 5 min resulted in a consistent pattern of degeneration of hippocampal CA1 and CA2 pyramidal neurons, which was quantified using an image analyzer. Systemic administration of MK-801 (0.01-10 mg/kg, i.p.) 1 hr prior to the occlusion caused a dose-dependent protection of the CA1 and CA2 neurons. The ED50 value for neuroprotection by MK-801 was calculated to be 0.3 mg/kg, and at doses greater than or equal to 3 mg/kg the majority of animals were completely protected against the ischemic insult. Systemic administration of MK-801 (1 or 10 mg/kg, i.p.) 1 hr prior to unilateral occlusion of the right carotid artery resulted in significant protection against hippocampal neurodegeneration following 10 min of occlusion, and increased the survival rate after 30 min of occlusion. The potent neuroprotective effects of MK-801 in these cerebral ischemia models add further weight to the evidence that NMDA receptors are involved in the mechanism of ischemia-induced neuronal degeneration.

The alpha5 subunit of the GABA(A) receptor is localized mainly to the hippocampus of the mammalian brain. The significance of this rather distinct localization and the function of alpha5-containing GABA(A) receptors has been explored by targeted disruption of the alpha5 gene in mice. The alpha5 -/- mice showed a significantly improved performance in a water maze model of spatial learning, whereas the performance in non-hippocampal-dependent learning and in anxiety tasks were unaltered in comparison with wild-type controls. In the CA1 region of hippocampal brain slices from alpha5 -/- mice, the amplitude of the IPSCs was decreased, and paired-pulse facilitation of field EPSP (fEPSP) amplitudes was enhanced. These data suggest that alpha5-containing GABA(A) receptors play a key role in cognitive processes by controlling a component of synaptic transmission in the CA1 region of the hippocampus.

1: The pharmacology of the stable cell line expressing human alpha(4)beta(3)delta GABA(A) receptor was investigated using whole-cell patch-clamp techniques. 2: alpha(4)beta(3)delta receptors exhibited increased sensitivity to GABA when compared to alpha(4)beta(3)gamma(2) receptors, with EC(50)'s of 0.50 (0.46, 0.53) microM and 2.6 (2.5, 2.6) microM respectively. Additionally, the GABA partial agonists piperidine-4-sulphonate (P4S) and 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) displayed markedly higher efficacy at alpha(4)beta(3)delta receptors, indeed THIP demonstrated greater efficacy than GABA at these receptors. 3: The delta subunit conferred slow desensitization to GABA, with rate constants of 4.8+/-0.5 s for alpha(4)beta(3)delta and 2.5+/-0.2 s for alpha(4)beta(3)gamma(2). However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this effect is agonist specific. 4: alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2) demonstrated equal sensitivity to inhibition by the cation zinc (2-3 microM IC(50)). However, alpha(4)beta(3)delta receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC(50) for GABA antagonists SR-95531 and picrotoxin, was similar for alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2). Likewise, inhibition was observed on both subtypes at high and low pH. 5: alpha(4)beta(3)delta receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15-4513 and bretazenil potentiated GABA responses on alpha(4)beta(3)gamma(2) cells, and the inverse agonist DMCM showed allosteric inhibition of alpha(4)beta(3)gamma(2) receptors. 6: The efficacy of neurosteroids at alpha(4)beta(3)delta receptors was greatly enhanced over that observed at alpha(4)beta(3)gamma(2) receptors. The greatest effect was observed using THDOC with 524+/-71.6% potentiation at alpha(4)beta(3)delta and 297.9+/-49.7% at alpha(4)beta(3)gamma(2) receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The efficacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at alpha(4)beta(3)delta receptors versus alpha(4)beta(3)gamma(2) receptors. 7: We show that the alpha(4)beta(3)delta receptor has a distinct pharmacology and kinetic profile. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics. British Journal of Pharmacology (2002) 136, 965-974

Glycine markedly potentiates N-methyl-D-aspartate (N-Me-D-Asp) responses in mammalian neurons by an action at a modulatory site on the N-Me-D-Asp receptor-ionophore complex. Here we present evidence that 7-chlorokynurenic acid (7-Cl KYNA) inhibits N-Me-D-Asp responses by a selective antagonism of glycine at this modulatory site. In rat cortical slices 7-Cl KYNA (10-100 microM) noncompetitively inhibited N-Me-D-Asp responses, and this effect could be reversed by the addition of glycine (100 microM) or D-serine (100 microM). Radioligand binding experiments showed that 7-Cl KYNA had a much higher affinity for the strychnine-insensitive [3H]glycine binding site (IC50 = 0.56 microM) than for the N-Me-D-Asp (IC50 169 microM), quisqualate (IC50 = 153 microM), or kainate (IC50 greater than 1000 microM) recognition sites. In whole-cell patch-clamp recordings from rat cortical neurones in culture, the inhibitory effects of 7-Cl KYNA on N-Me-D-Asp-induced currents could not be overcome by increasing the N-Me-D-Asp concentration but could be reversed by increasing the glycine concentration. 7-Cl KYNA could completely abolish N-Me-D-Asp responses, including basal responses in the absence of added glycine, suggesting that it may possess negative modulatory effects at the glycine site. These findings indicate that the glycine modulatory site is functional in intact adult tissue and that 7-Cl KYNA should prove to be a selective tool for elucidating the involvement of this site in physiological and pathological events mediated by N-Me-D-Asp receptors.

Alzheimer's disease is the leading cause of dementia. However, neither Alzheimer's disease nor Alzheimer's dementia are an inevitable consequence of aging. This review provides an overview of the issues involved in a diagnosis of Alzheimer's disease before an individual meets the criteria for Alzheimer's dementia. It examines how Alzheimer's disease diagnosis rates can be improved, the implications of an early diagnosis for the individual, carer and society, and the importance of risk reduction to prevent or delay progression. Although no disease-modifying agents capable of reversing the initial pathological changes are currently available, it may be possible to prevent or delay the development of dementia in a proportion of the population by modifying exposure to common risk factors. In other individuals, diagnosing the disease or risk of disease early is still valuable so that the individual and their carers have time to make choices and plan for the future, and to allow access to treatments that can help manage symptoms. Primary healthcare professionals play a pivotal role in recognising individuals at risk, recommending lifestyle changes in mid-adult life that can prevent or slow down the disease, and in timely diagnosis. Early intervention is the optimal strategy, because the patient's level of function is preserved for longer.

The primary cause of hypoglycaemia in Type 2 diabetes is diabetes medication-in particular, those which raise insulin levels independently of blood glucose, such as sulphonylureas (SUs) and exogenous insulin. The risk of hypoglycaemia is increased in older patients, those with longer diabetes duration, lesser insulin reserve and perhaps in the drive for strict glycaemic control. Differing definitions, data collection methods, drug type/regimen and patient populations make comparing rates of hypoglycaemia difficult. It is clear that patients taking insulin have the highest rates of self-reported severe hypoglycaemia (25% in patients who have been taking insulin for > 5 years). SUs are associated with significantly lower rates of severe hypoglycaemia. However, large numbers of patients take SUs in the UK, and it is estimated that each year > 5000 patients will experience a severe event caused by their SU therapy which will require emergency intervention. Hypoglycaemia has substantial clinical impact, in terms of mortality, morbidity and quality of life. The cost implications of severe episodes-both direct hospital costs and indirect costs-are considerable: it is estimated that each hospital admission for severe hypoglycaemia costs around pound1000. Hypoglycaemia and fear of hypoglycaemia limit the ability of current diabetes medications to achieve and maintain optimal levels of glycaemic control. Newer therapies, which focus on the incretin axis, may carry a lower risk of hypoglycaemia. Their use, and more prudent use of older therapies with low risk of hypoglycaemia, may help patients achieve improved glucose control for longer, and reduce the risk of diabetic complications.

BACKGROUND: Acid suppression with famotidine, a histamine H2-receptor antagonist, provides protection against gastric injury in normal subjects receiving short courses of aspirin or naproxen. The efficacy of famotidine in preventing peptic ulcers in patients receiving long-term therapy with nonsteroidal antiinflammatory drugs (NSAIDs) is not known. METHODS: We studied the efficacy of two doses of famotidine (20 mg and 40 mg, each given orally twice daily), as compared with placebo, in preventing peptic ulcers in 285 patients without peptic ulcers who were receiving long-term NSAID therapy for rheumatoid arthritis (82 percent) or osteoarthritis (18 percent). The patients were evaluated clinically and by endoscopy at base line and after 4, 12, and 24 weeks of treatment. The evaluators were unaware of the treatment assignment. The primary end point was the cumulative incidence of gastric or duodenal ulceration at 24 weeks. RESULTS: The cumulative incidence of gastric ulcers was 20 percent in the placebo group, 13 percent in the group of patients receiving 20 mg of famotidine twice daily (P = 0.24 for the comparison with placebo), and 8 percent in the group receiving 40 mg of famotidine twice daily (P = 0.03 for the comparison with placebo). The proportion of patients in whom duodenal ulcers developed was significantly lower with both doses of famotidine than with placebo (13 percent in the placebo group, 4 percent in the low-dose famotidine group [P = 0.04], and 2 percent in the high-dose famotidine group [P = 0.01]). Both doses of famotidine were well tolerated. CONCLUSIONS: Treatment with high-dose famotidine significantly reduces the cumulative incidence of both gastric and duodenal ulcers in patients with arthritis receiving long-term NSAID therapy.

It is becoming clear that receptors that initiate signal transduction by interacting with G-proteins do not function as monomers, but often require accessory proteins for function. Some of these accessory proteins are chaperones, required for correct transport of the receptor to the cell surface, but the function of many accessory proteins remains unknown. We determined the role of an accessory protein for the receptor for calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide. We have previously shown that this accessory protein, the CGRP-receptor component protein (RCP), is expressed in CGRP responsive tissues and that RCP protein expression correlates with the biological efficacy of CGRP in vivo. However, the function of RCP has remained elusive. In this study stable cell lines were made that express antisense RCP RNA, and CGRP- and adrenomedullin-mediated signal transduction were greatly reduced. However, the loss of RCP did not effect CGRP binding or receptor density, indicating that RCP did not behave as a chaperone but was instead coupling the CGRP receptor to downstream effectors. A candidate CGRP receptor named calcitonin receptor-like receptor (CRLR) has been identified, and in this study RCP co-immunoprecipitated with CRLR indicating that these two proteins interact directly. Since CGRP and adrenomedullin can both signal through CRLR, which has been previously shown to require a chaperone protein for function, we now propose that a functional CGRP or adrenomedullin receptor consists of at least three proteins: the receptor (CRLR), the chaperone protein (RAMP), and RCP that couples the receptor to the cellular signal transduction pathway.

Fast inhibitory neurotransmission in the mammalian CNS is mediated primarily by the neurotransmitter gamma-aminobutyric acid (GABA), which, upon binding to its receptor, leads to opening of the intrinsic ion channel, allowing chloride to enter the cell. Over the past 10 years it has become clear that a family of GABA-A receptor subtypes exists, generated through the coassembly of polypeptides selected from alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta, epsilon, and pie to form what is most likely a pentomeric macromolecule. The gene transcripts, and indeed the polypeptides, show distinct patterns of temporal and spatial expression, such that the GABA-A receptor subtypes have a defined localization that presumably reflects their physiological role. A picture is beginning to emerge of the properties conferred to receptor subtypes by the different subunits; these include different functional properties, differential modulation by protein kinases, and the targeting to different membrane compartments. These properties presumably underlie the different physiological roles of the various receptor subtypes. Recently we have identified a further member of the GABA-A receptor gene family, which we have termed theta, which appears to be most closely related to the beta subunits. The structure, function, and distribution of theta-containing receptors, and receptors containing the recently reported epsilon subunit, are described.

The decrease in plasticity that occurs in the central nervous system during postnatal development is accompanied by the appearance of perineuronal nets (PNNs) around the cell body and dendrites of many classes of neuron. These structures are composed of extracellular matrix molecules, such as chondroitin sulfate proteoglycans (CSPGs), hyaluronan (HA), tenascin-R, and link proteins. To elucidate the role played by neurons and glial cells in constructing PNNs, we studied the expression of PNN components in the adult rat cerebellum by immunohistochemistry and in situ hybridization. In the deep cerebellar nuclei, only large excitatory neurons were surrounded by nets, which contained the CSPGs aggrecan, neurocan, brevican, versican, and phosphacan, along with tenascin-R and HA. Whereas both net-bearing neurons and glial cells were the sources of CSPGs and tenascin-R, only the neurons expressed the mRNA for HA synthases (HASs), cartilage link protein, and link protein Bral2. In the cerebellar cortex, Golgi neurons possessed PNNs and also synthesized HASs, cartilage link protein, and Bral2 mRNAs. To see whether HA might link PNNs to the neuronal cell surface by binding to a receptor, we investigated the expression of the HA receptors CD44, RHAMM, and LYVE-1. No immunolabelling for HA receptors on the membrane of net-bearing neurons was found. We therefore propose that HASs, which can retain HA on the cell surface, may act as a link between PNNs and neurons. Thus, HAS and link proteins might be key molecules for PNN formation and stability.

Cerebellar granule cells express six GABAA receptor subunits abundantly (alpha1, alpha6, beta2, beta3, gamma2, and delta) and assemble various pentameric receptor subtypes with unknown subunit compositions; however, the rules guiding receptor subunit assembly are unclear. Here, removal of intact alpha6 protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied. Exon 8 of the mouse alpha6 subunit gene was disrupted by homologous recombination. In alpha6 -/- granule cells, the delta subunit was selectively degraded as seen by immunoprecipitation, immunocytochemistry, and immunoblot analysis with delta subunit-specific antibodies. The delta subunit mRNA was present at wild-type levels in the mutant granule cells, indicating a post-translational loss of the delta subunit. These results provide genetic evidence for a specific association between the alpha6 and delta subunits. Because in alpha6 -/- neurons the remaining alpha1, beta2/3, and gamma2 subunits cannot rescue the delta subunit, certain potential subunit combinations may not be found in wild-type cells.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe Glycine Site on the NMDA Receptor: Structure-Activity Relationships and Therapeutic PotentialPaul D. Leeson and Leslie L. IversenCite this: J. Med. Chem. 1994, 37, 24, 4053–4067Publication Date (Print):November 1, 1994Publication History Published online1 May 2002Published inissue 1 November 1994https://pubs.acs.org/doi/10.1021/jm00050a001https://doi.org/10.1021/jm00050a001research-articleACS PublicationsRequest reuse permissionsArticle Views1297Altmetric-Citations277LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

PURPOSE Trastuzumab deruxtecan (T-DXd) 5.4 and 6.4 mg/kg showed robust antitumor activity in multiple cancer indications; however, T-DXd 5.4 mg/kg has not been evaluated in patients with previously treated human epidermal growth factor receptor 2–mutant ( HER2m; defined as single-nucleotide variants and exon 20 insertions) metastatic non–small-cell lung cancer (mNSCLC). METHODS DESTINY-Lung02, a blinded, multicenter, phase II study, investigated T-DXd 5.4 mg/kg once every 3 weeks for the first time in previously treated (platinum-containing therapy) patients with HER2m mNSCLC and further assessed T-DXd 6.4 mg/kg once every 3 weeks in this population. The primary end point was confirmed objective response rate (ORR) per RECIST v1.1 by blinded independent central review. RESULTS One hundred fifty-two patients were randomly assigned 2:1 to T-DXd 5.4 or 6.4 mg/kg once every 3 weeks. As of December 23, 2022, the median duration of follow-up was 11.5 months (range, 1.1-20.6) with 5.4 mg/kg and 11.8 months (range, 0.6-21.0) with 6.4 mg/kg. Confirmed ORR was 49.0% (95% CI, 39.0 to 59.1) and 56.0% (95% CI, 41.3 to 70.0) and median duration of response was 16.8 months (95% CI, 6.4 to not estimable [NE]) and NE (95% CI, 8.3 to NE) with 5.4 and 6.4 mg/kg, respectively. Median treatment duration was 7.7 months (range, 0.7-20.8) with 5.4 mg/kg and 8.3 months (range, 0.7-20.3) with 6.4 mg/kg. Grade ≥ 3 drug-related treatment-emergent adverse events occurred in 39 of 101 (38.6%) and 29 of 50 (58.0%) patients with 5.4 and 6.4 mg/kg, respectively. 13 of 101 (12.9%) and 14 of 50 (28.0%) patients had adjudicated drug-related interstitial lung disease (2.0% grade ≥ 3 in each arm) with 5.4 and 6.4 mg/kg, respectively. CONCLUSION T-DXd demonstrated clinically meaningful responses at both doses. Safety profile was acceptable and generally manageable, favoring T-DXd 5.4 mg/kg.

Cerebellar granule cells express six GABA A receptor subunits abundantly (α 1 , α 6 , β 2 , β 3 , γ 2 , and δ) and assemble various pentameric receptor subtypes with unknown subunit compositions; however, the rules guiding receptor subunit assembly are unclear. Here, removal of intact α 6 protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied. Exon 8 of the mouse α 6 subunit gene was disrupted by homologous recombination. In α 6 −/− granule cells, the δ subunit was selectively degraded as seen by immunoprecipitation, immunocytochemistry, and immunoblot analysis with δ subunit-specific antibodies. The δ subunit mRNA was present at wild-type levels in the mutant granule cells, indicating a post-translational loss of the δ subunit. These results provide genetic evidence for a specific association between the α 6 and δ subunits. Because in α 6 −/− neurons the remaining α 1 , β 2/3 , and γ 2 subunits cannot rescue the δ subunit, certain potential subunit combinations may not be found in wild-type cells.

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged α1-, β2-, or γ2-subunits of the hetero-oligomeric γ-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the α1(c-Myc)β2γ2 and the α1β2(c-Myc)γ2 receptors was twice that obtained with α1β2γ2(c-Myc), suggesting that there are 2× α-, 2× β-, and 1× γ-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining α1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2, but no significant energy transfer was observed with α1β2γ2(c-Myc) receptors, indicating the absence of a second γ-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, α1β2γ2, is composed of two copies each of the α- and β-subunits and one copy of the γ-subunit (i.e.(α1)2(β2)2(γ2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins. We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged α1-, β2-, or γ2-subunits of the hetero-oligomeric γ-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the α1(c-Myc)β2γ2 and the α1β2(c-Myc)γ2 receptors was twice that obtained with α1β2γ2(c-Myc), suggesting that there are 2× α-, 2× β-, and 1× γ-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining α1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2, but no significant energy transfer was observed with α1β2γ2(c-Myc) receptors, indicating the absence of a second γ-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, α1β2γ2, is composed of two copies each of the α- and β-subunits and one copy of the γ-subunit (i.e.(α1)2(β2)2(γ2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins. γ-aminobutyric acid, type A γ-aminobutyric acid monoclonal antibody europium cryptate minimum essential medium fetal calf serum Understanding the assembly and molecular interactions of proteins is a rapidly growing field in biology. Many techniques have been used to probe the interaction of proteins including, for example, immunoprecipitation, yeast two-hybrid analysis, circular dichroism, and more recently fluorescence energy transfer between two proteins in close proximity (1Mathis G. Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar, 2Thomas D.D. Carlsen W.F. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5746-5750Crossref PubMed Scopus (158) Google Scholar, 3Damjanovich S. Bene L. Abdelkrim A. Goldman C.K. Sharrow S. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13134-13139Crossref PubMed Scopus (120) Google Scholar). We have used the technique of fluorescence resonance energy transfer to investigate the stoichiometric assembly of a GABAA1receptor, a member of the major inhibitory ligand-gated ion channel family in the brain. It is proposed that the GABAA receptors are hetero-oligomeric pentamers composed of α, β, and a third subunit type, γ, δ, or ε. This is based on an analogy with other ligand-gated ion channels such as the nicotinic acetylcholine receptor (4Raftery M.A. Hunkapiller M.W. Strader C.D. Hood L.E. Science. 1980; 208: 1454-1457Crossref PubMed Scopus (360) Google Scholar, 5Cooper C.E. Couturier S. Ballivet M. Nature. 1991; 350: 235-238Crossref PubMed Scopus (402) Google Scholar), their size as measured by gel filtration and sucrose density centrifugation (6Mamalaki C. Barnard E.A. Stephenson F.A. J. Neurochem. 1989; 52: 124-134Crossref PubMed Scopus (31) Google Scholar, 7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar), and their image under electron microscopy (8Nayeem N. Green T.P. Martin I.L. Barnard E.A. J. Neurochem. 1994; 62: 815-818Crossref PubMed Scopus (378) Google Scholar). To date, 15 GABAA subunits have been cloned (α1–6, β1–4, γ1–3, δ, and ε), and expression studies reveal that for the most characterized subtypes at least one α, one β, and one γ-subunit are required to recover a fully functional GABAA receptor (9Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1150) Google Scholar). We have investigated the stoichiometry of the most abundant GABAAreceptor isoform in the brain, α1β2γ2, which is responsible for at least some of the therapeutic effects of benzodiazepines, barbiturates, and steroids (10McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1085) Google Scholar). Receptors modified with a c-Myc epitope on either the α1-, β2-, or γ2- subunit were expressed transiently in HEK293 cells. It has previously been shown that receptors transiently transfected in these cells using the same methods as employed here are appropriately assembled into αβγ-hetero-oligomers and are expressed on the cell surface. Other subunit assemblies do not reach the cell surface and are retained in the endoplasmic reticulum (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). mAbs to the c-Myc epitope were derivatized with the fluorescence donor, europium cryptate (EuK), or the fluorescent energy acceptor, XL-665. Europium, encaged by cryptate, emits a strong long lived fluorescent signal at 620 nm when illuminated with light at 337 nm from a nitrogen laser, which can be time-resolved from short lived background fluorescence. In addition to being used as a single label, analogous to a radiolabel for example, EuK also serves as an energy donor. The fluorescent signal produced by EuK can be transferred to an acceptor molecule if it is in close enough proximity. The recipient molecule for this fluorescent resonance energy transfer is a modified allophycocyanine, XL665, which fluoresces at 665 nm (for review of the homogeneous time-resolved fluorescence technology see Mathis (1Mathis G. Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar)). The transfer of energy from europium cryptate-labeled c-Myc antibodies to XL-665-labeled antibodies is therefore indicative of the two antibodies being in close proximity. Because the energy transfer is 50% at a distance of 9.5 nm (1Mathis G. Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar), this would require that the antibodies are very closely associated, for example in the same macromolecular complex. We used EuK-labeled c-Myc mAbs to quantify the number of subunits present on intact cells expressing α1β2γ2 subunits where each of the subunits was epitope-tagged with c-Myc and compared this with the number of receptors present using conventional radioligand binding. Fluorescence energy transfer was then used to confirm the stoichiometry of the receptor as (α1)2(β2)2(γ2)1. [methyl-3H]Ro15-1788 (87.0 Ci/mmol) was from NEN Life Science Products, Hertfordshire, United Kingdom. Flunitrazepam, GABA, and fetal calf serum were from Sigma. Minimal essential medium (MEM) was from Life Technologies, Inc. The GABAAreceptor α1-subunit was epitope-tagged by site-directed mutagenesis using methods described previously (13Wingrove P.B. Wafford K.A. Bain C. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4569-4573Crossref PubMed Scopus (216) Google Scholar). The epitope sequence (EQKLISEEDL) was introduced between Glu34and Leu35 (these two residues becoming the first and last amino acid of the epitope tag), just C-terminal to the putative signal peptide cleavage site. The β2 and γ2GABAA receptor subunits were epitope-tagged using a modified version of the pcDNA1Amp eukaryotic expression vector (pcDNA1AmpSignalMyc). This vector was constructed from the α1 c-Myc cDNA described above and contains the 5′-untranslated region of bovine GABAAα1-subunit (GenBank accession no. X05717), the signal peptide and six amino acids of the mature α1-subunit, and the c-Myc epitope tag sequence (EQKLISEEDL (14Evan G.I. Lewis G. Ramsay G. Bishop J.L. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar)) followed by a small polylinker into which the mature polypeptide of the subunit of interest can be inserted. The human β2-subunit c-Myc construct contains amino acids Glu38–Asn474 of β2, and the human γ2-subunit c-Myc construct contains amino acids Tyr46–le467. All constructs were confirmed by DNA sequencing. Constructs were prepared using polymerase chain reaction to generate the appropriate β2 and γ2 sequences, which were then subcloned into the pcDNA1AmpSignalMyc vector. DNAs were prepared for transfection by CsCl centrifugation. Transient transfection into HEK293 cells was performed exactly as described previously in detail using a 1:1:1 ratio of α1, β2, and γ2 cDNAs (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 15Wingrove P.B. Thompson S.A. Wafford K.A. Whiting P.J. Mol. Pharmacol. 1997; 52: 874-881Crossref PubMed Scopus (95) Google Scholar). The benzodiazepine site of the GABA receptor was labeled by the antagonist [3H]Ro15-1788, a radioligand frequently used to quantify receptors, because only fully assembled αβγ heterotrimers bind this ligand (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar, 12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16Duggan M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar). Nonspecific binding was determined using 10 μmflunitrazepam. Binding to the GABA binding site was carried out with [3H]muscimol, and 100 μm GABA was used to define nonspecific binding. Radioligand binding assays were carried out in a total volume of 0.5 ml in 10 mmKH2PO4, 100 mm KCl, pH 7.4, for 1 h at 4 °C prior to termination through Whatman GF/C filters followed by 3× 3-ml washes with cold assay buffer and scintillation counting. In binding studies carried out at one concentration of [3H]Ro15-1788, this was 1.8 nm. GABAA receptors solubilized from stably transfected cells were immunoprecipitated using anti-α1-, β-, or γ2-subunit antisera bound to protein A-Sepharose (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar). 50 μl of polyclonal antiserum were incubated with 50 μl of packed protein A beads in a total volume of 1 ml of Tris-buffered saline for 1 h at room temperature. Receptors were solubilized from the cells using a deoxycholate/Triton buffer (1% Triton X-100, 0.5% deoxycholate, 0.1 mm KCl, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 100 mm Tris-HCl, pH 8.2) by mixing cell membranes for 1 h at 4 °C with a detergent buffer at a protein concentration of 1 mg/ml. Aliquots (500 μl) of detergent-solubilized cell membranes were incubated with antibodies immobilized on protein A beads overnight at 4 °C. The receptor immobilized on protein A beads was washed three times with Tris-buffered saline/0.1% Tween 20 by centrifugation and resuspension and was finally resuspended in 0.5 ml of Tris-buffered saline, and 10–50-μl aliquots of packed beads were used for [3H]Ro15-1788 binding. The monoclonal anti-c-Myc mAb, 9-E10 (ATCC no. CRL-1729, Ref. 14Evan G.I. Lewis G. Ramsay G. Bishop J.L. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar) was used for these experiments. An antibody was purified from hybridoma supernatant by Cymbus Biotech, UK, and this was then derivatized with either the fluorophore, europium cryptate (at an average ratio of 9 molecules of europium cryptate per antibody molecule), or with the fluorescence acceptor, XL665 (at a ratio of 1 molecule of XL-665 per antibody molecule), by Cis-Bio International, Marcoule, France. Labeling of c-Myc mAb with EuK at this ratio gave 418 counts of fluorescence/s/fmol of antibody. Intact cells, transiently transfected with GABAA receptors with c-Myc-tagged subunits, were harvested by scraping, washed once by centrifugation (20 s at 1,000 rpm in a bench top Eppendorf Microcentrifuge), and resuspended in MEM + 5% FCS. [3H]Ro15-1788 binding (1.8 nm) was carried out to determine receptor density on intact cells. Cells containing the equivalent of 100 fmol of receptor were incubated with various concentrations of europium cryptate-derivatized mAb (0.3–30 nm) in MEM + 5% FCS at room temperature, in a volume of 1 ml for 1 h on a rotamix wheel. After 3× 1-ml washes in MEM + 5% FCS, the cells were resuspended in 190 μl of MEM + 5% FCS, and the fluorescence signal derived from europium cryptate-labeled c-Myc mAbs was quantified in a 96-well low fluorescence microplate on the Packard discovery fluorescence plate reader, following the addition of 10 μl of 1 m KF (final concentration, 50 mm KF). In preliminary experiments the inclusion of 5% FCS was found to improve the of the cells, and KF was to europium cryptate and of mAbs were in excess nm) to each and the plate was a transfer was the receptor been incubated with for 1 and the signal was for to h of the α1-, β2-, and γ2-subunits was to a c-Myc epitope tag at the and cells expressing c-Myc-tagged α-, β-, or γ-subunits or receptors were by GABAA receptors were measured using two [3H]muscimol, a radioligand for the GABA binding and [3H]Ro15-1788, a radioligand for the benzodiazepine binding which only to fully assembled receptors that an α-, β-, and γ-subunit (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar, D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1150) Google Scholar). In preliminary experiments it was to confirm that a homogeneous of fully assembled receptors was being expressed in the cells. This was in two the number of binding for and [3H]Ro15-1788 was the of two binding and one [3H]Ro15-1788 binding site in a GABA receptor in receptors immunoprecipitated from (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar, K. Ragan C.I. Whiting P.J. McKernan R.M. Mol. Pharmacol. 1994; Google Scholar). if the of receptors is assembled in cell the ratio of the for binding be It is that receptors be expressed that are composed of α- and β-subunits but these would not bind [3H]Ro15-1788, and the ratio would therefore be of and [3H]Ro15-1788 binding was carried out in cells expressing the α1-, β2-, and binding of was of protein with a of binding of [3H]Ro15-1788 was of protein with a of nm The ratio of the was which is in with the expression of αβγ receptors and no significant expression of receptors that only the of antibodies the α1-, β2-, and γ2-subunit to the [3H]Ro15-1788 binding from a solubilized cell was The of receptors be immunoprecipitated with antibodies the α1-, β-, or γ2-subunits β, and of [3H]Ro15-1788 binding This that the receptors that bind [3H]Ro15-1788 at least one α-, one β-, and one The number of c-Myc-tagged subunits was quantified by with anti-c-Myc mAb derivatized with europium cryptate, as in 1 the subunit proposed by N. K. J. Neurosci. 1997; PubMed Google Scholar) as a The of a c-Myc tag on the of the GABAAreceptor subunits not the expression of the The number of binding present on cells was by the addition of the epitope and of [3H]Ro15-1788 binding that there was no major in the of receptors for ligand is as α1β2γ2 α1(c-Myc)β2γ2 α1β2γ2(c-Myc) The density of receptors was by the expression of subunits with a c-Myc epitope being of This is in with studies where GABAA receptors were tagged with c-Myc and or of the GABAA receptor (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). experiments were carried out to determine the for the binding of EuK-labeled c-Myc shown in the fluorescence signal by EuK c-Myc at 620 nm was with receptor the binding of EuK-labeled c-Myc to the receptor was to at least 100 fmol of fluorescence from the plate and other for the fluorescence in the absence of and this was as shown in A. Binding of EuK c-Myc to cells expressing receptors was significant were to to this binding by following with mAb and of cells with to binding on the cells. of these nonspecific binding to cells by more and were not each a type receptors expressed c-Myc epitope and nonspecific binding to these cells was from the total fluorescence which was analogous to the employed for nonspecific binding in a radioligand binding binding experiments were carried out using cells expressing α1β2γ2 and cells expressing receptors where either the α-, β-, or γ-subunit was epitope-tagged with Nonspecific binding of EuK c-Myc to cells expressing an receptor was from total binding at each concentration of antibody and was 50% at nm antibody. In binding was and a and EuK-labeled c-Myc bound to more in the α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2 compared with α1β2γ2(c-Myc) as in To the was on six using nm and the fluorescence counts are shown in 4 A. The ratio of fluorescence to α1β2(c-Myc)γ2, and α1β2γ2(c-Myc) was compared as shown in 4 B. This that there are twice as EuK c-Myc on receptors where the α- or is tagged compared with receptors where the γ-subunit is tagged of signal observed at 620 and 665 nm from cells expressing α1β2(c-Myc)γ2, and shown are the of six experiments. The fluorescence counts observed at 620 nm for α1(c-Myc)β2γ2 were and for α1β2(c-Myc)γ2 were were that observed with α1β2γ2(c-Myc), The and the ratio for binding to α1β2γ2(c-Myc) were for each and are shown in B. The of binding for α1(c-Myc) and were not from in the fluorescence energy transfer where was measured at 665 there was no significant between the fluorescence counts produced by and α1β2(c-Myc)γ2 but were more which was not more A of the for [3H]Ro15-1788 and fluorescence can be used to the number of binding for the binding site. α1(c-Myc)β2γ2 there are fluorescence of binding using nm antibody and 1.8 + this is equivalent to fluorescence of binding that there are 418 fluorescence counts of of antibody there are to be antibody binding binding site. The and of in this of of antibody would be more to to an of the number of antibody binding therefore the observation that there is more one antibody binding binding site the most stoichiometry of benzodiazepine site. The of the c-Myc antibodies for the GABAA subunits was nm as shown in A concentration of nm was for the fluorescence transfer studies because this would half-maximally the with nm c-Myc mAb, cells were washed and incubated with excess c-Myc mAb, and fluorescence at 665 nm was measured in to laser shown in 4 a signal is indicating energy transfer when either the or the β2-subunit is epitope-tagged and but not when the γ2-subunit is Because energy transfer can only when there are two c-Myc in close two subunits in the same receptor this that the stoichiometry of the GABAA receptor under is (α1)2(β2)2(γ2)1. the of signal with α1β2γ2(c-Myc) be as that receptors are not expressed as on the cell surface. this were the it be to energy transfer between γ2-subunits on To date, the stoichiometry of the GABAA receptor has been and Many have observed that a receptor can two of M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar, H. Seeburg P.H. J. 1991; Scopus Google Scholar, J. 1991; PubMed Scopus Google Scholar, S. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.M. Quirk K. Ragan C.I. Whiting 1991; Full Text PDF PubMed Scopus Google Scholar). has been obtained on two of γ-subunit can in a single receptor with two studies that two γ-subunits can be present A. A.L. J. Neurochem. 1994; PubMed Scopus Google Scholar, K. Ragan C.I. Whiting P.J. McKernan R.M. Mol. Pharmacol. 1994; Google Scholar). have been studies on the of two β-subunits because the of the β-subunits has the of which between them. have been studies that the stoichiometry of the The first have an M. U. L. H. J.A. 5: PubMed Scopus Google Scholar) the 2× α, 1× β, and 2× based on measuring the by of amino acids on either of the S. J. Neurosci. 1996; PubMed Google Scholar) used a method of a in to the of the receptor to GABA in to the number of subunits In to M. U. L. H. J.A. 5: PubMed Scopus Google Scholar), conclude that the stoichiometry is 2× α, 2× β, and 1× γ, as do J.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) from studies of subunit The most used antibody of subunits to determine the ratio of subunits present from and conclude that the is also 2× α, 2× β, and 1× N. K. J. Neurosci. 1997; PubMed Google Scholar). The here has used fluorescently derivatized mAbs to quantify subunits to the benzodiazepine binding site on the receptor and fluorescent resonance energy transfer to confirm that the stoichiometry of the receptor is This method has other previously described It intact therefore only receptors that are expressed on the surface are therefore to be are (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, T.P. J. Neurosci. PubMed Google Scholar). It of receptor by only epitope which not the expression or of the receptor (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). small of transiently transfected are this has general applicability to other multisubunit cell surface proteins and can be used to investigate the stoichiometry of subunits in composed of subunits and in other ligand-gated ion We and Mathis of Cis-Bio for for to the assay and for the transfected cells.

The specific mechanisms underlying general anesthesia are primarily unknown. The intravenous general anesthetic etomidate acts by potentiating GABA A receptors, with selectivity for β2 and β3 subunit-containing receptors determined by a single asparagine residue. We generated a genetically modified mouse containing an etomidate-insensitive β2 subunit (β2 N265S) to determine the role of β2 and β3 subunits in etomidate-induced anesthesia. Loss of pedal withdrawal reflex and burst suppression in the electroencephalogram were still observed in the mutant mouse, indicating that loss of consciousness can be mediated purely through β3-containing receptors. The sedation produced by subanesthetic doses of etomidate and during recovery from anesthesia was present only in wild-type mice, indicating that the β2 subunit mediates the sedative properties of anesthetics. These findings show that anesthesia and sedation are mediated by distinct GABA A receptor subtypes.

Among hypnotic agents that enhance GABAA receptor function, etomidate is unusual because it is selective for beta2/beta3 compared with beta1 subunit-containing GABAA receptors. Mice incorporating an etomidate-insensitive beta2 subunit (beta(2N265S)) revealed that beta2 subunit-containing receptors mediate the enhancement of slow-wave activity (SWA) by etomidate, are required for the sedative, and contribute to the hypnotic actions of this anesthetic. Although the anatomical location of the beta2-containing receptors that mediate these actions is unknown, the thalamus is implicated. We have characterized GABAA receptor-mediated neurotransmission in thalamic nucleus reticularis (nRT) and ventrobasalis complex (VB) neurons of wild-type, beta2(-/-), and beta(2N265S) mice. VB but not nRT neurons exhibit a large GABA-mediated tonic conductance that contributes approximately 80% of the total GABAA receptor-mediated transmission. Consequently, although etomidate enhances inhibition in both neuronal types, the effect of this anesthetic on the tonic conductance of VB neurons is dominant. The GABA-enhancing actions of etomidate in VB but not nRT neurons are greatly suppressed by the beta(2N265S) mutation. The hypnotic THIP (Gaboxadol) induces SWA and at low, clinically relevant concentrations (30 nM to 3 microM) increases the tonic conductance of VB neurons, with no effect on VB or nRT miniature IPSCs (mIPSCs) or on the holding current of nRT neurons. Zolpidem, which has no effect on SWA, prolongs VB mIPSCs but is ineffective on the phasic and tonic conductance of nRT and VB neurons, respectively. Collectively, these findings suggest that enhancement of extrasynaptic inhibition in the thalamus may contribute to the distinct sleep EEG profiles of etomidate and THIP compared with zolpidem.

A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABAA receptors that contain the alpha5 subunit (alpha5GABAARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by alpha5GABAARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not alpha5 null mutant (alpha5-/-) mice. In addition, etomidate impaired memory performance of WT but not alpha5-/- mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The alpha5-/- mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and alpha5-/- mice. Deletion of the alpha5 subunit of the GABAARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABAAR subtype pharmacology.