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).

A photoreactive analog of allopregnanolone enables identification of steroid-binding sites in a nicotinic acetylcholine receptor.

Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABA receptors (GABAR) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABAR-positive allosteric modulators (PAMs), whereas alphaxalone and most neuroactive steroids are nAChR inhibitors. In this report, we used 11β-(-azidotetrafluorobenzoyloxy)allopregnanolone (FNBzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABAR PAM, to characterize steroid-binding sites in the αβγδ nAChR in its native membrane environment.

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.

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 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.

Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABAAR modulation. [(3)H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[(3)H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABAAR transmembrane domain.

Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.

At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR.

Anion binding modes in meso-substituted hexapyrrolic calix[4]pyrrole isomers.

We report on the synthesis of a new receptor for anions, meso-substituted hexapyrrolic calix[4]pyrrole 1. The calix[4]pyrrole's core features two additional pyrrole side-arms suspended above or below the calix[4]pyrrole core. This hexapyrrolic calix[4]pyrrole 1 is formed as cis- and trans-configurational isomers, the structures of which have been determined by single crystal X-ray diffraction.

Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor.

GABA type A receptors (GABAAR), the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABAARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β(+)-α(-) subunit interfaces, which also contain the GABA-binding sites in the extracellular domain.

Allyl m-trifluoromethyldiazirine mephobarbital: an unusually potent enantioselective and photoreactive barbiturate general anesthetic.

We synthesized 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (14), a trifluoromethyldiazirine-containing derivative of general anesthetic mephobarbital, separated the racemic mixture into enantiomers by chiral chromatography, and determined the configuration of the (+)-enantiomer as S by X-ray crystallography. Additionally, we obtained the (3)H-labeled ligand with high specific radioactivity. R-(-)-14 is an order of magnitude more potent than the most potent clinically used barbiturate, thiopental, and its general anesthetic EC(50) approaches those for propofol and etomidate, whereas S-(+)-14 is 10-fold less potent.

p-(4-Azipentyl)propofol: a potent photoreactive general anesthetic derivative of propofol.

We synthesized 2,6-diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]phenol (p-(4-azipentyl)propofol), or p-4-AziC5-Pro, a novel photoactivable derivative of the general anesthetic propofol. p-4-AziC5-Pro has an anesthetic potency similar to that of propofol. Like propofol, the compound potentiates inhibitory GABA(A) receptor current responses and allosterically modulates binding to both agonist and benzodiazepine sites, assayed on heterologously expressed GABA(A) receptors.