Faculty Opinions recommendation of Numerous classes of general anesthetics inhibit etomidate binding to gamma-aminobutyric acid type A (GABAA) receptors.

Background Classic benzodiazepine agonists induce their clinical effects by binding to a site on gamma-aminobutyric acid type A (GABAA) receptors and enhancing receptor activity. There are conflicting data regarding whether the benzodiazepine site is allosterically coupled to gamma-aminobutyric acid binding versus the channel open-close (gating) equilibrium. The authors tested the hypothesis that benzodiazepine site ligands modulate alpha1beta2gamma2L GABAA receptor gating both in the absence of orthosteric agonists and when the orthosteric sites are occupied. Methods GABAA receptors were recombinantly expressed in Xenopus oocytes and studied using two-microelectrode voltage clamp electrophysiology. To test gating effects in the absence of orthosteric agonist, the authors used spontaneously active GABAA receptors containing a leucine-to-threonine mutation at residue 264 on the alpha1 subunit. To examine effects on gating when orthosteric sites were fully occupied, they activated wild-type receptors with high concentrations of a partial agonist, piperidine-4-sulfonic acid. Results In the absence of orthosteric agonists, the channel activity of alpha1L264Tbeta2gamma2L receptors was increased by diazepam and midazolam and reduced by the inverse benzodiazepine agonist FG7142. Flumazenil displayed very weak agonism and blocked midazolam from further activating mutant channels. In wild-type receptors activated with saturating concentrations of piperidine-4-sulfonic acid, midazolam increased maximal efficacy. Conclusions Independent of orthosteric site occupancy, classic benzodiazepines modulate the gating equilibrium in alpha1beta2gamma2L GABAA receptors and are therefore allosteric coagonists. A Monod-Wyman-Changeux coagonist gating model quantitatively predicts these effects, suggesting that benzodiazepines minimally alter orthosteric ligand binding.

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A Conserved Tyrosine in the β2Subunit M4 Segment Is a Determinant of γ-Aminobutyric Acid Type A Receptor Sensitivity to Propofol

Anesthesiology ◽

10.1097/01.anes.0000278875.36639.2c ◽

2007 ◽

Vol 107 (3) ◽

pp. 412-418 ◽

Cited By ~ 39

Author(s):

James E. Richardson ◽

Paul S. Garcia ◽

Kate K. O'Toole ◽

Jason M. C. Derry ◽

Shannon V. Bell ◽

...

Keyword(s):

Binding Site ◽

Type A ◽

Receptor Activation ◽

Aminobutyric Acid ◽

Gamma Aminobutyric Acid ◽

General Anesthetics ◽

Receptor Function ◽

293 Cells ◽

Acid Type ◽

Normal Sensitivity

Background The gamma-aminobutyric acid type A receptor (GABAA-R) beta subunits are critical targets for the actions for several intravenous general anesthetics, but the precise nature of the anesthetic binding sites are unknown. In addition, little is known about the role the fourth transmembrane (M4) segment of the receptor plays in receptor function. The aim of this study was to better define the propofol binding site on the GABAA-R by conducting a tryptophan scan in the M4 segment of the beta2 subunit. Methods Seven tryptophan mutations were introduced into the C-terminal end of the M4 segment of the GABAA-R beta2 subunit. GABAA-R subunit complementary DNAs were transfected into human embryonic kidney 293 cells grown on glass coverslips. After transfection (36-72 h), coverslips were transferred to a perfusion chamber to assay receptor function. Cells were whole cell patch clamped and exposed to GABA, propofol, etomidate, and pregnenolone. Chemicals were delivered to the cells using two 10-channel infusion pumps and a rapid solution exchanger. Results All tryptophan mutations were well tolerated, and with one exception, all resulted in minimal changes in receptor activation by GABA. One mutation, beta2(Y444W), selectively suppressed the ability of propofol to enhance receptor function while retaining normal sensitivity to etomidate and pregnenolone. Conclusions This is the first report of a mutation that selectively reduces propofol sensitivity without altering the action of etomidate. The reduction in propofol sensitivity is consistent with the loss of a hydrogen bond within the propofol binding site. These results also suggest a possible orientation of the propofol molecule within its binding site.

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Tryptophan and Cysteine Mutations in M1 Helices of α1β3γ2L γ-Aminobutyric Acid Type A Receptors Indicate Distinct Intersubunit Sites for Four Intravenous Anesthetics and One Orphan Site

Anesthesiology ◽

10.1097/aln.0000000000001390 ◽

2016 ◽

Vol 125 (6) ◽

pp. 1144-1158 ◽

Cited By ~ 22

Author(s):

Anahita Nourmahnad ◽

Alex T. Stern ◽

Mayo Hotta ◽

Deirdre S. Stewart ◽

Alexis M. Ziemba ◽

...

Keyword(s):

Gabaa Receptor ◽

Gabaa Receptors ◽

Barbituric Acid ◽

Channel Gating ◽

Type A ◽

Aminobutyric Acid ◽

General Anesthetics ◽

Cysteine Modification ◽

Intravenous Anesthetics ◽

Acid Type

Abstract Background γ-Aminobutyric acid type A (GABAA) receptors mediate important effects of intravenous general anesthetics. Photolabel derivatives of etomidate, propofol, barbiturates, and a neurosteroid get incorporated in GABAA receptor transmembrane helices M1 and M3 adjacent to intersubunit pockets. However, photolabels have not been consistently targeted at heteromeric αβγ receptors and do not form adducts with all contact residues. Complementary approaches may further define anesthetic sites in typical GABAA receptors. Methods Two mutation-based strategies, substituted tryptophan sensitivity and substituted cysteine modification–protection, combined with voltage-clamp electrophysiology in Xenopus oocytes, were used to evaluate interactions between four intravenous anesthetics and six amino acids in M1 helices of α1, β3, and γ2L GABAA receptor subunits: two photolabeled residues, α1M236 and β3M227, and their homologs. Results Tryptophan substitutions at α1M236 and positional homologs β3L231 and γ2L246 all caused spontaneous channel gating and reduced γ-aminobutyric acid EC50. Substituted cysteine modification experiments indicated etomidate protection at α1L232C and α1M236C, R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid protection at β3M227C and β3L231C, and propofol protection at α1M236C and β3M227C. No alphaxalone protection was evident at the residues the authors explored, and none of the tested anesthetics protected γ2I242C or γ2L246C. Conclusions All five intersubunit transmembrane pockets of GABAA receptors display similar allosteric linkage to ion channel gating. Substituted cysteine modification and protection results were fully concordant with anesthetic photolabeling at α1M236 and β3M227 and revealed overlapping noncongruent sites for etomidate and propofol in β+–α– interfaces and R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid and propofol in α+–β– and γ+–β– interfaces. The authors’ results identify the α+–γ– transmembrane interface as a potentially unique orphan modulator site.

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Opposing Actions of Etomidate on Cortical Theta Oscillations Are Mediated by Different γ-Aminobutyric Acid Type A Receptor Subtypes

Anesthesiology ◽

10.1097/00000542-200502000-00017 ◽

2005 ◽

Vol 102 (2) ◽

pp. 346-352 ◽

Cited By ~ 13

Author(s):

Berthold Drexler ◽

Claire L. Roether ◽

Rachel Jurd ◽

Uwe Rudolph ◽

Bernd Antkowiak

Keyword(s):

Gabaa Receptors ◽

Type A ◽

Mutant Mice ◽

Theta Oscillations ◽

Aminobutyric Acid ◽

Receptor Subtypes ◽

Gamma Aminobutyric Acid ◽

Wild Type ◽

Action Potential Firing ◽

Acid Type

Background Cortical networks generate diverse patterns of rhythmic activity. Theta oscillations (4-12 Hz) are commonly observed during spatial learning and working memory tasks. The authors ask how etomidate, acting predominantly via gamma-aminobutyric acid type A (GABAA) receptors containing beta2 or beta3 subunits, affects theta activity in vitro. Methods To characterize the effects of etomidate, the authors recorded action potential firing together with local field potentials in slice cultures prepared from the neocortex of the beta3(N265M) knock-in mutant and wild type mice. Actions of etomidate were studied at 0.2 microm, which is approximately 15% of the concentration causing immobility ( approximately 1.5 microm). Results In preparations derived from wild type and beta3(N265M) mutant mice, episodes of ongoing activity spontaneously occurred at a frequency of approximately 0.1 Hz and persisted for several seconds. Towards the end of these periods, synchronized oscillations in the theta band developed. These oscillations were significantly depressed in slices from beta3(N265M) mutant mice (P < 0.05). In this preparation etomidate acts almost exclusively via beta2 subunit containing GABAA receptors. In contrast, no depression was observed in slices from wild type mice, where etomidate potentiates both beta2- and beta3-containing GABAA receptors. Conclusions At concentrations assumed to cause sedation and amnesia, etomidate depresses theta oscillations via beta2-containing GABAA receptors but enhances these oscillations by acting on beta3 subunit containing receptors. This indicates that the overall effect of the anesthetic reflects a balance between enhancement and inhibition produced by different GABAA receptor subtypes.

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