title: Quercetin antagonism of GABAAρ₁ receptors is prevented by ascorbic acid through a redox-independent mechanism.
abstract: Quercetin is a natural flavonoid widely distributed in plants that acts as a neuroprotective agent and modulates the activity of different synaptic receptors and ion channels, including the ionotropic GABA receptors. GABA(Aρ₁) receptors were shown to be antagonized by quercetin, but the mechanisms underlying these antagonistic actions are still unknown. We have analyzed here if the antagonistic action produced by quercetin on GABA(Aρ₁) receptors was related to its redox activity or due to alternative mechanism/s. Homomeric GABA(Aρ₁) receptors were expressed in frog oocytes and GABA-evoked responses electrophysiologically recorded. Quercetin effects on GABA(Aρ₁) receptors were examined in the absence or presence of ascorbic acid. Chemical protection of cysteines by selective sulfhydryl reagents and site directed mutagenesis experiments were also used to determine ρ₁ subunit residues involved in quercetin actions. Quercetin antagonized GABA(Aρ₁) receptor responses in a dose-dependent, fast and reversible manner. Quercetin inhibition was prevented in the presence of ascorbic acid, but not by thiol reagents that modify the extracellular Cys-loop of these receptors. H141, an aminoacidic residue located near to the ρ₁ subunit GABA binding site, was involved in the allosteric modulation of GABA(Aρ₁) receptors by several agents including ascorbic acid. Quercetin similarly antagonized GABA-evoked responses mediated by mutant (H141D)GABA(Aρ₁) and wild-type receptors, but prevention exerted by ascorbic acid on quercetin effects was impaired in mutant receptors. Taken together the present results suggest that quercetin antagonistic actions on GABA(Aρ₁) receptors are mediated through a redox-independent allosteric mechanism.
authors.affiliation: Laboratorio de Neurobiología Celular y Molecular, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas, Vuelta de Obligado 2490, C1428ADN Ciudad Autónoma de Buenos Aires, Argentina. calero@gmail.com
authors.forename: Cecilia I
authors.initials: CI
authors.lastname: Calero
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Beltrán González
authors.forename: Javier
authors.initials: J
authors.lastname: Gasulla
authors.forename: Silvia
authors.initials: S
authors.lastname: Alvarez
authors.forename: Pablo
authors.initials: P
authors.lastname: Evelson
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Calvo
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D058787:GABA-A Receptor Antagonists / Q000494:pharmacology*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D011794:Quercetin / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D058787:GABA-A Receptor Antagonists; D011963:Receptors, GABA-A; D006639:Histidine; D011794:Quercetin; D001205:Ascorbic Acid
keywords: Allosteric modulator; Ascorbic acid; Flavonoid; GABA; GABA receptor; N-ethylmaleimide; NEM; Quercetin; γ-aminobutyric acid