scholarly journals New 3-, 8-disubstituted pyrazolo[5,1-c][1,2,4]benzotriazines useful for studying the interaction with the HBp-3 area (hydrogen bond point area) in the benzodiazepine site on the γ-aminobutyric acid type A (GABAA) receptor

2011 ◽  
Vol 19 (10) ◽  
pp. 3074-3085 ◽  
Author(s):  
Gabriella Guerrini ◽  
Giovanna Ciciani ◽  
Fabrizio Bruni ◽  
Silvia Selleri ◽  
Fabrizio Melani ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Gabaa Receptor ◽  
Type A ◽  
Aminobutyric Acid ◽  
Acid Type ◽  
Benzodiazepine Site

Classic Benzodiazepines Modulate the Open–Close Equilibrium in α1β2γ2Lγ-Aminobutyric Acid Type A Receptors

2005 ◽  
Vol 102 (4) ◽  
pp. 783-792 ◽  
Author(s):  
Dirk Rüsch ◽  
Stuart A. Forman
Keyword(s):  
Gabaa Receptors ◽  
Sulfonic Acid ◽  
Site Occupancy ◽  
Type A ◽  
Aminobutyric Acid ◽  
Clinical Effects ◽  
Gamma Aminobutyric Acid ◽  
Wild Type ◽  
Acid Type ◽  
Benzodiazepine Site

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.

Multiple assembly signals in γ-aminobutyric acid (type A) receptor subunits combine to drive receptor construction and composition

2003 ◽  
Vol 31 (4) ◽  
pp. 875-879 ◽  
Author(s):  
K. Bollan ◽  
L.A. Robertson ◽  
H. Tang ◽  
C.N. Connolly
Keyword(s):  
Gabaa Receptor ◽  
Gabaa Receptors ◽  
Type A ◽  
Aminobutyric Acid ◽  
Single Amino Acid ◽  
Functional Surface ◽  
Surface Receptors ◽  
Acid Type ◽  
Absolute Requirement ◽  
Receptor Assembly

Mammalian γ-aminobutyric acid type A (GABAA) receptors are constructed from a large repertoire of subunits (α1–α6, β1–β3, γ1–γ3, δ, ∊, θ and π) into a pentameric ion channel. GABAA receptor assembly occurs within the endoplasmic reticulum (ER) and involves interactions with chaperone molecules. Only specific subunit combinations can produce functional surface receptors (with a fixed stoichiometry); other subunit combinations are retained within the ER and degraded. Thus, receptor assembly occurs by defined pathways to limit the diversity of GABAA receptors. The key to understanding how receptor diversity is achieved and controlled is the identification of assembly signals capable of distinguishing between other subunit partners. Analysis of an assembly box in α1 (residues 57–68) has revealed an absolute requirement for this region in the assembly of αβ receptors. Furthermore, a selective requirement for a single amino acid (R66) is observed for the assembly of α1β2, but not α1β1 or α1β3, receptors. In addition, we have characterized an assembly signal in the β3 subunit that is capable of driving the assembly of β3, γ2β3 and α1β3 receptors. Interestingly, this signal does not appear to utilize the α1 assembly box, suggesting the presence of alternative assembly signals within the α1 subunit. Although this β3 signal is sufficient to permit the formation of βγ receptors it is not necessary, suggesting that alternative assembly signals also exist within the β3 subunit. These findings support the belief that GABAA receptor assembly occurs via multiple defined pathways that may be determined by subunit availability.

scholarly journals Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αβγ and αβδ Expression Using Flow Cytometry and Electrophysiology

2016 ◽  
Vol 291 (39) ◽  
pp. 20440-20461 ◽  
Author(s):  
Emmanuel J. Botzolakis ◽  
Katharine N. Gurba ◽  
Andre H. Lagrange ◽  
Hua-Jun Feng ◽  
Aleksandar K. Stanic ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Gabaa Receptor ◽  
Type A ◽  
Aminobutyric Acid ◽  
Acid Type

scholarly journals γ-Aminobutyric Acid Type A Receptor Modulation by Etomidate Analogs

2016 ◽  
Vol 124 (3) ◽  
pp. 651-663 ◽  
Author(s):  
Ervin Pejo ◽  
Peter Santer ◽  
Lei Wang ◽  
Philip Dershwitz ◽  
S. Shaukat Husain ◽  
...  
Keyword(s):  
Gabaa Receptor ◽  
Response Curve ◽  
Type A ◽  
Field Analysis ◽  
Aminobutyric Acid ◽  
Molecular Field ◽  
Comparative Molecular Field Analysis ◽  
Structural Elements ◽  
Receptor Modulation ◽  
Acid Type

Abstract Background Etomidate is a highly potent anesthetic agent that is believed to produce hypnosis by enhancing γ-aminobutyric acid type A (GABAA) receptor function. The authors characterized the GABAA receptor and hypnotic potencies of etomidate analogs. The authors then used computational techniques to build statistical and graphical models that relate the potencies of these etomidate analogs to their structures to identify the specific molecular determinants of potency. Methods GABAA receptor potencies were defined with voltage clamp electrophysiology using α1β3γ2 receptors harboring a channel mutation (α1[L264T]) that enhances anesthetic sensitivity (n = 36 to 60 measurements per concentration–response curve). The hypnotic potencies of etomidate analogs were defined using a loss of righting reflexes assay in Sprague Dawley rats (n = 9 to 21 measurements per dose–response curve). Three-dimensional quantitative structure–activity relationships were determined in silico using comparative molecular field analysis. Results The GABAA receptor and hypnotic potencies of etomidate and the etomidate analogs ranged by 91- and 53-fold, respectively. These potency measurements were significantly correlated (r2 = 0.72), but neither measurement correlated with drug hydrophobicity (r2 = 0.019 and 0.005, respectively). Statistically significant and predictive comparative molecular field analysis models were generated, and a pharmacophore model was built that revealed both the structural elements in etomidate analogs associated with high potency and the interactions that these elements make with the etomidate-binding site. Conclusions There are multiple specific structural elements in etomidate and etomidate analogs that mediate GABAA receptor modulation. Modifying any one element can alter receptor potency by an order of magnitude or more.

scholarly journals 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

2016 ◽  
Vol 125 (6) ◽  
pp. 1144-1158 ◽  
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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