Differential assembly diversifies GABAA receptor structures and signalling.

Type A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits1,2 and can be modulated by essential medicines including general anaesthetics and benzodiazepines3. Human GABAAR subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABAARs assembled from α4, ß3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors4-7. This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABAAR arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data8,9, we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABAARs.

Competitive Antagonism of Etomidate Action by Diazepam: In Vitro GABAA Receptor and In Vivo Zebrafish Studies.

BACKGROUND: Recent cryo-electron microscopic imaging studies have shown that in addition to binding to the classical extracellular benzodiazepine binding site of the α1ß3γ2L γ-aminobutyric acid type A (GABAA) receptor, diazepam also binds to etomidate binding sites located in the transmembrane receptor domain. Because such binding is characterized by low modulatory efficacy, the authors hypothesized that diazepam would act in vitro and in vivo as a competitive etomidate antagonist. METHODS: The concentration-dependent actions of diazepam on 20 µM etomidate-activated and 6 µM GABA-activated currents were defined (in the absence and presence of flumazenil) in oocyte-expressed α1ß3γ2L GABAA receptors using voltage clamp electrophysiology. The ability of diazepam to inhibit receptor labeling of purified α1ß3γ2L GABAA receptors by [H]azietomidate was assessed in photoaffinity labeling protection studies. The impact of diazepam (in the absence and presence of flumazenil) on the anesthetic potencies of etomidate and ketamine was compared in a zebrafish model. RESULTS: At nanomolar concentrations, diazepam comparably potentiated etomidate-activated and GABA-activated GABAA receptor peak current amplitudes in a flumazenil-reversible manner. The half-maximal potentiating concentrations were 39 nM (95% CI, 27 to 55 nM) and 26 nM (95% CI, 16 to 41 nM), respectively. However, at micromolar concentrations, diazepam reduced etomidate-activated, but not GABA-activated, GABAA receptor peak current amplitudes in a concentration-dependent manner with a half-maximal inhibitory concentration of 9.6 µM (95% CI, 7.6 to 12 µM). Diazepam (12.5 to 50 µM) also right-shifted the etomidate-concentration response curve for direct activation without reducing the maximal response and inhibited receptor photoaffinity labeling by [H]azietomidate. When administered with flumazenil, 50 µM diazepam shifted the etomidate (but not the ketamine) concentration-response curve for anesthesia rightward, increasing the etomidate EC50 by 18-fold. CONCLUSIONS: At micromolar concentrations and in the presence of flumazenil to inhibit allosteric modulation via the classical benzodiazepine binding site of the GABAA receptor, diazepam acts as an in vitro and in vivo competitive etomidate antagonist.

Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors.

Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1ß3 and α1ß3γ2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the ß subunit M4 (ß3Pro-415, ß3Leu-417, and ß3Thr-418) and M3 (ß3Arg-309) helices located at the base of a pocket in the ß+-α- subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3ß-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAAR ß+-α- subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.

A potent photoreactive general anesthetic with novel binding site selectivity for GABAA receptors.

The pentameric γ-aminobutyric acid type A receptors (GABAARs) are the major inhibitory ligand-gated ion channels in the central nervous system. They mediate diverse physiological functions, mutations in them are associated with mental disorders and they are the target of many drugs such as general anesthetics, anxiolytics and anti-convulsants. The five subunits of synaptic GABAARs are arranged around a central pore in the order ß-α-ß-α-γ. In the outer third of the transmembrane domain (TMD) drugs may bind to five homologous intersubunit binding sites. Etomidate binds between the pair of ß - α subunit interfaces (designated as ß+/α-) and R-mTFD-MPAB binds to an α+/ß- and an γ+/ß- subunit interface (a ß- selective ligand). Ligands that bind selectively to other homologous sites have not been characterized. We have synthesized a novel photolabel, (2,6-diisopropyl-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)methanol or pTFD-di-iPr-BnOH). It is a potent general anesthetic that positively modulates agonist and benzodiazepine binding. It enhances GABA-induced currents, shifting the GABA concentration-response curve to lower concentrations. Photolabeling-protection studies show that it has negligible affinity for the etomidate sites and high affinity for only one of the two R-mTFD-MPAB sites. Exploratory site-directed mutagenesis studies confirm the latter conclusions and hint that pTFD-di-iPr-BnOH may bind between the α+/ß- and α+/γ- subunits in the TMD, making it an α+ ligand. The latter α+/γ- site has not previously been implicated in ligand binding. Thus, pTFD-di-iPr-BnOH is a promising new photolabel that may open up a new pharmacology for synaptic GABAARs.

Binding site location on GABAA receptors determines whether mixtures of intravenous general anaesthetics interact synergistically or additively in vivo.

BACKGROUND AND PURPOSE: General anaesthetics can act on synaptic GABAA receptors by binding to one of three classes of general anaesthetic sites. Canonical drugs that bind selectively to only one class of site are etomidate, alphaxalone, and the mephobarbital derivative, R-mTFD-MPAB. We tested the hypothesis that the general anaesthetic potencies of mixtures of such site-selective agents binding to the same or to different sites would combine additively or synergistically respectively. EXPERIMENTAL APPROACH: The potency of general anaesthetics individually or in combinations to cause loss of righting reflexes in tadpoles was determined, and the results were analysed using isobolographic methods. KEY RESULTS: The potencies of combinations of two or three site-selective anaesthetics that all acted on a single class of site were strictly additive, regardless of which single site was involved. Combinations of two or three site-selective anaesthetics that all bound selectively to different sites always interacted synergistically. The strength of the synergy increased with the number of separate sites involved such that the percentage of each agent's EC50 required to cause anaesthesia was just 35% and 14% for two or three sites respectively. Propofol, which binds non-selectively to the etomidate and R-mTFD-MPAB sites, interacted synergistically with each of these agents. CONCLUSIONS AND IMPLICATIONS: The established pharmacology of the three anaesthetic binding sites on synaptic GABAA receptors was sufficient to predict whether a mixture of anaesthetics interacted additively or synergistically to cause loss of righting reflexes in vivo. The principles established here have implications for clinical practice.

Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the α1ß3γ2 GABAAR Transmembrane Domain.

GABAA receptors (GABAARs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([3H]azietomidate) and mephobarbital [[3H]1-methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid ([3H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the α1ß3γ2 GABAAR transmembrane domain at ß +-α - (ß + sites) and α +-ß -/γ +-ß - (ß - sites) subunit interfaces. We now use competition photolabeling with [3H]azietomidate and [3H]R-mTFD-MPAB to identify para-substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to ß +, while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to ß - sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the α +-ß - and γ +-ß - sites. However, we discovered four compounds that bind with different affinities to the two ß - interface sites. Two of these bind with higher affinity to one of the ß - sites than to the ß + sites. We deduce that 4-benzoyl-propofol binds with >100-fold higher affinity to the γ +-ß - site than to the α +-ß - or ß +-α - sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the α +-ß - site than to the ß + and γ +-ß - sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABAAR transmembrane domain, a property that may facilitate the development of subtype selective GABAAR PAMs.

Author Correction: GABAA receptor signalling mechanisms revealed by structural pharmacology.

In Fig. 5b, d, the arrows showing transmembrane domain rotations were inadvertently pointing clockwise instead of anticlockwise. Similarly, 'anticlockwise' should have been 'clockwise' in the sentence 'This conformational change of the ECD triggers a clockwise rotation of the TMD.' In Extended Data Table 1, the units of the column 'Model resolution' should have been Å instead of Å2. These errors have been corrected online.

GABAA receptor signalling mechanisms revealed by structural pharmacology.

Type-A γ-aminobutyric (GABAA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABAA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1ß3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABAA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABAA receptor modulators.

Cryo-EM structure of the human α1ß3γ2 GABAA receptor in a lipid bilayer.

Type A γ-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system1,2. Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia3,4. Among the numerous assemblies that are theoretically possible, the most prevalent in the brain are the α1ß2/3γ2 GABAA receptors5. The ß3 subunit has an important role in maintaining inhibitory tone, and the expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in ß1-ß3 triple knockout neurons6. So far, efforts to generate accurate structural models for heteromeric GABAA receptors have been hampered by the use of engineered receptors and the presence of detergents7-9. Notably, some recent cryo-electron microscopy reconstructions have reported 'collapsed' conformations8,9; however, these disagree with the structure of the prototypical pentameric ligand-gated ion channel the Torpedo nicotinic acetylcholine receptor10,11, the large body of structural work on homologous homopentameric receptor variants12 and the logic of an ion-channel architecture. Here we present a high-resolution cryo-electron microscopy structure of the full-length human α1ß3γ2L-a major synaptic GABAA receptor isoform-that is functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator 'megabody' and is in a desensitized conformation. Each GABAA receptor pentamer contains two phosphatidylinositol-4,5-bisphosphate molecules, the head groups of which occupy positively charged pockets in the intracellular juxtamembrane regions of α1 subunits. Beyond this level, the intracellular M3-M4 loops are largely disordered, possibly because interacting post-synaptic proteins are not present. This structure illustrates the molecular principles of heteromeric GABAA receptor organization and provides a reference framework for future mechanistic investigations of GABAergic signalling and pharmacology.

Self-Driving Cars and Engineering Ethics: The Need for a System Level Analysis.

The literature on self-driving cars and ethics continues to grow. Yet much of it focuses on ethical complexities emerging from an individual vehicle. That is an important but insufficient step towards determining how the technology will impact human lives and society more generally. What must complement ongoing discussions is a broader, system level of analysis that engages with the interactions and effects that these cars will have on one another and on the socio-technical systems in which they are embedded. To bring the conversation of self-driving cars to the system level, we make use of two traffic scenarios which highlight some of the complexities that designers, policymakers, and others should consider related to the technology. We then describe three approaches that could be used to address such complexities and their associated shortcomings. We conclude by bringing attention to the "Moral Responsibility for Computing Artifacts: The Rules", a framework that can provide insight into how to approach ethical issues related to self-driving cars.

Inhibitable photolabeling by neurosteroid diazirine analog in the ß3-Subunit of human hetereopentameric type A GABA receptors.

Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxiolytic effects due to their positive modulatory interactions with the GABAA receptors in the brain. Binding sites for these neurosteroids have been recently identified at subunit interfaces in the transmembrane domain (TMD) of homomeric ß3 GABAA receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABAA receptor α subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABAA receptors. Here, we report on the synthesis and pharmacological characterization of several photoaffinity analogs of pregnanolone and allopregnanolone, of which 21-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoxy]allopregnanolone (21-pTFDBzox-AP) was the most potent ligand. It is a partial positive modulator of the human α1ß3 and α1ß3γ2L GABAA receptors at sub-micromolar concentrations. [3H]21-pTFDBzox-AP photoincorporated in a pharmacologically specific manner into the α and ß subunits of those receptors, with the ß3 subunit photolabeled most efficiently. Importantly, photolabeling by [3H]21-pTFDBzox-AP was inhibited by the positive steroid modulators alphaxalone, pregnanolone and allopregnanolone, but not by inhibitory neurosteroid pregnenolone sulfate or by two potent general anesthetics and GABAAR positive allosteric modulators, etomidate and an anesthetic barbiturate. The latter two ligands bind to sites at subunit interfaces in the GABAAR that are different from those interacting with neurosteroids. 21-pTFDBzox-AP's potency and pharmacological specificity of photolabeling indicate its suitability for characterizing neurosteroid binding sites in native GABAA receptors.

Etomidate and Etomidate Analog Binding and Positive Modulation of γ-Aminobutyric Acid Type A Receptors: Evidence for a State-dependent Cutoff Effect.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Naphthalene-etomidate, an etomidate analog containing a bulky phenyl ring substituent group, possesses very low γ-aminobutyric acid type A (GABAA) receptor efficacy and acts as an anesthetic-selective competitive antagonist. Using etomidate analogs containing phenyl ring substituents groups that range in volume, we tested the hypothesis that this unusual pharmacology is caused by steric hindrance that reduces binding to the receptor's open state. METHODS: The positive modulatory potencies and efficacies of etomidate and phenyl ring-substituted etomidate analogs were electrophysiology defined in oocyte-expressed α1ß3γ2L GABAA receptors. Their binding affinities to the GABAA receptor's two classes of transmembrane anesthetic binding sites were assessed from their abilities to inhibit receptor labeling by the site-selective photolabels [H]azi-etomidate and tritiated R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid. RESULTS: The positive modulatory activities of etomidate and phenyl ring-substituted etomidate analogs progressively decreased with substituent group volume, reflecting significant decreases in both potency (P = 0.005) and efficacy (P < 0.0001). Affinity for the GABAA receptor's two ß - α anesthetic binding sites similarly decreased with substituent group volume (P = 0.003), whereas affinity for the receptor's α - ß/γ - ß sites did not (P = 0.804). Introduction of the N265M mutation, which is located at the ß - α binding sites and renders GABAA receptors etomidate-insensitive, completely abolished positive modulation by naphthalene-etomidate. CONCLUSIONS: Steric hindrance selectively reduces phenyl ring-substituted etomidate analog binding affinity to the two ß - α anesthetic binding sites on the GABAA receptor's open state, suggesting that the binding pocket where etomidate's phenyl ring lies becomes smaller as the receptor isomerizes from closed to open.

Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors.

Most general anesthetics enhance GABA type A (GABAA) receptor activity at clinically relevant concentrations. Sites of action of volatile anesthetics on the GABAA receptor remain unknown, whereas sites of action of many intravenous anesthetics have been identified in GABAA receptors by using photolabeling. Here, we used photoactivatable analogs of isoflurane (AziISO) and sevoflurane (AziSEVO) to locate their sites on α1ß3γ2L and α1ß3 GABAA receptors. As with isoflurane and sevoflurane, AziISO and AziSEVO enhanced the currents elicited by GABA. AziISO and AziSEVO each labeled 10 residues in α1ß3 receptors and 9 and 8 residues, respectively, in α1ß3γ2L receptors. Photolabeled residues were concentrated in transmembrane domains and located in either subunit interfaces or in the interface between the extracellular domain and the transmembrane domain. The majority of these transmembrane residues were protected from photolabeling with the addition of excess parent anesthetic, which indicated specificity. Binding sites were primarily located within α+/ß- and ß+/α- subunit interfaces, but residues in the α+/γ- interface were also identified, which provided a basis for differential receptor subtype sensitivity. Isoflurane and sevoflurane did not always share binding sites, which suggests an unexpected degree of selectivity.-Woll, K. A., Zhou, X., Bhanu, N. V., Garcia, B. A., Covarrubias, M., Miller, K. W., Eckenhoff, R. G. Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors.

High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4ß3δ subunits.

The inhibitory γ-aminobutyric acid type A receptors are implicated in numerous physiological processes, including cognition and inhibition of neurotransmission, rendering them important molecular targets for many classes of drugs. Functionally, the entire GABAAR family of receptors can be subdivided into phasic, fast acting synaptic receptors, composed of α-, ß- and γ-subunits, and tonic extrasynaptic receptors, many of which contain the δ-subunit in addition to α- and ß-subunits. Whereas the subunit arrangement of the former group is agreed upon, that of the αßδ GABAARs remains unresolved by electrophysiological and pharmacological research. To resolve such issues will require biophysical techniques that demand quantities of receptor that have been previously unavailable. Therefore, we have engineered a stable cell line with tetracycline inducible expression of human α4-, ß3- and N-terminally Flag-tagged δ-subunits. This cell line achieved a specific activity between 15 and 20 pmol [3H]muscimol sites/mg of membrane protein, making it possible to obtain 1 nmole of purified α4ß3δ GABAAR from sixty 15-cm culture dishes. When induced, these cells exhibited agonist-induced currents with characteristics comparable to those previously reported for this receptor and a pharmacology that included strong modulation by etomidate and the δ-subunit-specific ligand, DS2. Immunoaffinity purification and reconstitution in CHAPS/asolectin micelles resulted in the retention of equilibrium allosteric interactions between the separate agonist, anesthetic and DS2 sites. Moreover, all three subunits retained glycosylation. The establishment of this well-characterized cell line will allow molecular level studies of tonic receptors to be undertaken.

Tethering IL2 to Its Receptor IL2Rß Enhances Antitumor Activity and Expansion of Natural Killer NK92 Cells.

IL2 is an immunostimulatory cytokine for key immune cells including T cells and natural killer (NK) cells. Systemic IL2 supplementation could enhance NK-mediated immunity in a variety of diseases ranging from neoplasms to viral infection. However, its systemic use is restricted by its serious side effects and limited efficacy due to activation of T regulatory cells (Tregs). IL2 signaling is mediated through interactions with a multi-subunit receptor complex containing IL2Rα, IL2Rß, and IL2Rγ. Adult natural killer (NK) cells express only IL2Rß and IL2Rγ subunits and are therefore relatively insensitive to IL2. To overcome these limitations, we created a novel chimeric IL2-IL2Rß fusion protein of IL2 and its receptor IL2Rß joined via a peptide linker (CIRB). NK92 cells expressing CIRB (NK92CIRB) were highly activated and expanded indefinitely without exogenous IL2. When compared with an IL2-secreting NK92 cell line, NK92CIRB were more activated, cytotoxic, and resistant to growth inhibition. Direct contact with cancer cells enhanced the cytotoxic character of NK92CIRB cells, which displayed superior in vivo antitumor effects in mice. Overall, our results showed how tethering IL2 to its receptor IL2Rß eliminates the need for IL2Rα and IL2Rß, offering a new tool to selectively activate and empower immune therapy. Cancer Res; 77(21); 5938-51. ©2017 AACR.

Competitive Antagonism of Anesthetic Action at the γ-Aminobutyric Acid Type A Receptor by a Novel Etomidate Analog with Low Intrinsic Efficacy.

BACKGROUND: The authors characterized the γ-aminobutyric acid type A receptor pharmacology of the novel etomidate analog naphthalene-etomidate, a potential lead compound for the development of anesthetic-selective competitive antagonists. METHODS: The positive modulatory potencies and efficacies of etomidate and naphthalene-etomidate were defined in oocyte-expressed α1ß3γ2L γ-aminobutyric acid type A receptors using voltage clamp electrophysiology. Using the same technique, the ability of naphthalene-etomidate to reduce currents evoked by γ-aminobutyric acid alone or γ-aminobutyric acid potentiated by etomidate, propofol, pentobarbital, and diazepam was quantified. The binding affinity of naphthalene-etomidate to the transmembrane anesthetic binding sites of the γ-aminobutyric acid type A receptor was determined from its ability to inhibit receptor photoaffinity labeling by the site-selective photolabels [H]azi-etomidate and R-[H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid. RESULTS: In contrast to etomidate, naphthalene-etomidate only weakly potentiated γ-aminobutyric acid-evoked currents and induced little direct activation even at a near-saturating aqueous concentration. It inhibited labeling of γ-aminobutyric acid type A receptors by [H]azi-etomidate and R-[H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid with similar half-maximal inhibitory concentrations of 48 µM (95% CI, 28 to 81 µM) and 33 µM (95% CI, 20 to 54 µM). It also reduced the positive modulatory actions of anesthetics (propofol > etomidate ~ pentobarbital) but not those of γ-aminobutyric acid or diazepam. At 300 µM, naphthalene-etomidate increased the half-maximal potentiating propofol concentration from 6.0 µM (95% CI, 4.4 to 8.0 µM) to 36 µM (95% CI, 17 to 78 µM) without affecting the maximal response obtained at high propofol concentrations. CONCLUSIONS: Naphthalene-etomidate is a very low-efficacy etomidate analog that exhibits the pharmacology of an anesthetic competitive antagonist at the γ-aminobutyric acid type A receptor.

Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.

Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but the locations of their GABAAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAAR, 11ß-(4-azido-2,3,5,6-tetrafluorobenzoyloxy)allopregnanolone, (F4N3Bzoxy-AP) and 11-aziallopregnanolone (11-AziAP). Both photoprobes acted with equal or higher potency than alphaxalone as general anesthetics and potentiators of GABAAR responses, left-shifting the GABA concentration - response curve for human α1ß3γ2 GABAARs expressed in Xenopus oocytes, and enhancing [3H]muscimol binding to α1ß3γ2 GABAARs expressed in HEK293 cells. With EC50 of 110 nM, 11-AziAP is one the most potent general anesthetics reported. [3H]F4N3Bzoxy-AP and [3H]11-AziAP, at anesthetic concentrations, photoincorporated into α- and ß-subunits of purified α1ß3γ2 GABAARs, but labeling at the subunit level was not inhibited by alphaxalone (30 µM). The enhancement of photolabeling by 3H-azietomidate and 3H-mTFD-MPAB in the presence of either of the two steroid photoprobes indicates the neurosteroid binding site is different from, but allosterically related to, the etomidate and barbiturate sites. Our observations are consistent with two hypotheses. First, F4N3Bzoxy-AP and 11-aziAP bind to a high affinity site in such a pose that the 11-photoactivatable moiety, that is rigidly attached to the steroid backbone, points away from the protein. Second, F4N3Bzoxy-AP, 11-aziAP and other steroid anesthetics, which are present at very high concentration at the lipid-protein interface due to their high lipophilicity, act via low affinity sites, as proposed by Akk et al. (Psychoneuroendocrinology2009, 34S1, S59-S66).

This "Ethical Trap" Is for Roboticists, Not Robots: On the Issue of Artificial Agent Ethical Decision-Making.

In this paper we address the question of when a researcher is justified in describing his or her artificial agent as demonstrating ethical decision-making. The paper is motivated by the amount of research being done that attempts to imbue artificial agents with expertise in ethical decision-making. It seems clear that computing systems make decisions, in that they make choices between different options; and there is scholarship in philosophy that addresses the distinction between ethical decision-making and general decision-making. Essentially, the qualitative difference between ethical decisions and general decisions is that ethical decisions must be part of the process of developing ethical expertise within an agent. We use this distinction in examining publicity surrounding a particular experiment in which a simulated robot attempted to safeguard simulated humans from falling into a hole. We conclude that any suggestions that this simulated robot was making ethical decisions were misleading.

General Anesthetic Binding Sites in Human α4ß3δ γ-Aminobutyric Acid Type A Receptors (GABAARs).

Extrasynaptic γ-aminobutyric acid type A receptors (GABAARs),which contribute generalized inhibitory tone to the mammalian brain, are major targets for general anesthetics. To identify anesthetic binding sites in an extrasynaptic GABAAR, we photolabeled human α4ß3δ GABAARs purified in detergent with [3H]azietomidate and a barbiturate, [3H]R-mTFD-MPAB, photoreactive anesthetics that bind with high selectivity to distinct but homologous intersubunit binding sites in the transmembrane domain of synaptic α1ß3γ2 GABAARs. Based upon 3H incorporation into receptor subunits resolved by SDS-PAGE, there was etomidate-inhibitable labeling by [3H]azietomidate in the α4 and ß3 subunits and barbiturate-inhibitable labeling by [3H]R-mTFD-MPAB in the ß3 subunit. These sites did not bind the anesthetic steroid alphaxalone, which enhanced photolabeling, or DS-2, a δ subunit-selective positive allosteric modulator, which neither enhanced nor inhibited photolabeling. The amino acids labeled by [3H]azietomidate or [3H]R-mTFD-MPAB were identified by N-terminal sequencing of fragments isolated by HPLC fractionation of enzymatically digested subunits. No evidence was found for a δ subunit contribution to an anesthetic binding site. [3H]azietomidate photolabeling of ß3Met-286 in ßM3 and α4Met-269 in αM1 that was inhibited by etomidate but not by R-mTFD-MPAB established that etomidate binds to a site at the ß3+-α4- interface equivalent to its site in α1ß3γ2 GABAARs. [3H]Azietomidate and [3H]R-mTFD-MPAB photolabeling of ß3Met-227 in ßM1 established that these anesthetics also bind to a homologous site, most likely at the ß3+-ß3- interface, which suggests a subunit arrangement of ß3α4ß3δß3.