Differential assembly diversifies GABA receptor structures and signalling.

Type A γ-aminobutyric acid receptors (GABARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits and can be modulated by essential medicines including general anaesthetics and benzodiazepines. Human GABAR 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.

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 α1β3γ2L 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 α1β3γ2L GABAA receptors using voltage clamp electrophysiology.

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

Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABA receptors (GABARs) with 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 GABAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABARs by photoaffinity labeling with [H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([H]21-TFDBzox-AP), a potent GABAR PAM.

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

The pentameric γ-aminobutyric acid type A receptors (GABARs) 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 GABARs are arranged around a central pore in the order β-α-β-α-γ.

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

Background And Purpose: General anaesthetics can act on synaptic GABA 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.

Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the 132 GABAR Transmembrane Domain.

GABA receptors (GABARs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs).

Author Correction: GABA 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.

GABA receptor signalling mechanisms revealed by structural pharmacology.

Type-A γ-aminobutyric (GABA) 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 GABA receptors remains largely unknown.

Cryo-EM structure of the human α1β3γ2 GABA receptor in a lipid bilayer.

Type A γ-aminobutyric acid (GABA) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system. Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia. Among the numerous assemblies that are theoretically possible, the most prevalent in the brain are the α1β2/3γ2 GABA receptors.

Inhibitable photolabeling by neurosteroid diazirine analog in the β3-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 GABA receptors in the brain. Binding sites for these neurosteroids have been recently identified at subunit interfaces in the transmembrane domain (TMD) of homomeric β3 GABA receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABA receptor α subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABA 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 α1β3γ2L GABAA receptors.

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

Most general anesthetics enhance GABA type A (GABA) receptor activity at clinically relevant concentrations. Sites of action of volatile anesthetics on the GABA receptor remain unknown, whereas sites of action of many intravenous anesthetics have been identified in GABA receptors by using photolabeling. Here, we used photoactivatable analogs of isoflurane (AziISO) and sevoflurane (AziSEVO) to locate their sites on αβγ and αβ GABA receptors.

High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ 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-, β- and γ-subunits, and tonic extrasynaptic receptors, many of which contain the δ-subunit in addition to α- and β-subunits. Whereas the subunit arrangement of the former group is agreed upon, that of the αβδ GABAARs remains unresolved by electrophysiological and pharmacological research.

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.

Tethering IL2 to Its Receptor IL2Rβ 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).

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 α1β3γ2L 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.

Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.

Neuroactive steroids are potent positive allosteric modulators of GABA receptors (GABAR), but the locations of their GABAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAR, 11β-(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 GABAR responses, left-shifting the GABA concentration - response curve for human α1β3γ2 GABARs expressed in Xenopus oocytes, and enhancing [H]muscimol binding to α1β3γ2 GABARs expressed in HEK293 cells.

General Anesthetic Binding Sites in Human α4β3δ γ-Aminobutyric Acid Type A Receptors (GABAARs).

Extrasynaptic γ-aminobutyric acid type A receptors (GABARs),which contribute generalized inhibitory tone to the mammalian brain, are major targets for general anesthetics. To identify anesthetic binding sites in an extrasynaptic GABAR, we photolabeled human α4β3δ GABARs purified in detergent with [H]azietomidate and a barbiturate, [H]R-mTFD-MPAB, photoreactive anesthetics that bind with high selectivity to distinct but homologous intersubunit binding sites in the transmembrane domain of synaptic α1β3γ2 GABARs. Based upon H incorporation into receptor subunits resolved by SDS-PAGE, there was etomidate-inhibitable labeling by [H]azietomidate in the α4 and β3 subunits and barbiturate-inhibitable labeling by [H]R-mTFD-MPAB in the β3 subunit.

Contrasting Effects of the γ-Aminobutyric Acid Type A Receptor β3 Subunit N265M Mutation on Loss of Righting Reflexes Induced by Etomidate and the Novel Anesthetic Barbiturate R-mTFD-MPAB.

Background: Previous studies have shown that etomidate modulates γ-aminobutyric acid type A receptors by binding at the β-α subunit interface within the transmembrane domain of receptors that incorporate β2 or β3 subunits. Introducing an asparagine-to-methionine (N265M) mutation at position 265 of the β3 subunit, which sits within the etomidate-binding site, attenuates the hypnotic effect of etomidate in vivo. It was reported recently that the photoactivatable barbiturate R-mTFD-MPAB also acts on γ-aminobutyric acid type A receptors primarily by binding to a homologous site at the γ-β interface.

Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes.

IV general anesthetics, including propofol, etomidate, alphaxalone, and barbiturates, produce important actions by enhancing γ-aminobutyric acid type A (GABAA) receptor activation. In this article, we review scientific studies that have located and mapped IV anesthetic sites using photoaffinity labeling and substituted cysteine modification protection. These anesthetics bind in transmembrane pockets between subunits of typical synaptic GABAA receptors, and drugs that display stereoselectivity also show remarkably selective interactions with distinct interfacial sites.

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.

Contrasting actions of a convulsant barbiturate and its anticonvulsant enantiomer on the α1 β3 γ2L GABAA receptor account for their in vivo effects.

Key Points: Most barbiturates are anaesthetics but unexpectedly a few are convulsants whose mechanism of action is poorly understood. We synthesized and characterized a novel pair of chiral barbiturates that are capable of photolabelling their binding sites on GABAA receptors. In mice the S-enantiomer is a convulsant, but the R-enantiomer is an anticonvulsant.

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β- Interface.

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C.

Dodecyl maltopyranoside enabled purification of active human GABA type A receptors for deep and direct proteomic sequencing.

The challenge in high-quality membrane proteomics is all about sample preparation prior to HPLC, and the cell-to-protein step poses a long-standing bottleneck. Traditional protein extraction methods apply ionic or poly-disperse detergents, harsh denaturation, and repeated protein/peptide precipitation/resolubilization afterward, but suffer low yield, low reproducibility, and low sequence coverage. Contrary to attempts to subdue, we resolved this challenge by providing proteins nature-and-activity-promoting conditions throughout preparation.