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.
Eur J Pharmacol. 2013 Aug 15;714(1-3):274-80. doi: 10.1016/j.ejphar.2013.07.044. Epub 2013 Jul 31.
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.
槲皮素是一种广泛存在于植物中的天然类黄酮，具有神经保护作用，可调节不同突触受体和离子通道的活性，包括离子型 GABA 受体。已表明槲皮素可拮抗 GABA(Aρ₁)受体，但这些拮抗作用的机制尚不清楚。我们在此分析了槲皮素对 GABA(Aρ₁)受体的拮抗作用是否与其氧化还原活性有关，或者是否由于替代机制/途径。在青蛙卵母细胞中表达同型 GABA(Aρ₁)受体，并用电生理学方法记录 GABA 诱导的反应。在不存在或存在抗坏血酸的情况下，检查了槲皮素对 GABA(Aρ₁)受体的作用。还使用半胱氨酸选择性巯基试剂的化学保护和定点突变实验来确定参与槲皮素作用的 ρ₁亚基残基。槲皮素以剂量依赖性、快速和可逆的方式拮抗 GABA(Aρ₁)受体反应。抗坏血酸的存在可防止槲皮素抑制，但不能防止修饰这些受体细胞外环半胱氨酸的巯基试剂。位于 ρ₁亚基 GABA 结合位点附近的氨基酸残基 H141 参与了多种试剂（包括抗坏血酸）对 GABA(Aρ₁)受体的变构调节。槲皮素类似地拮抗突变型（H141D）GABA(Aρ₁)和野生型受体介导的 GABA 诱导的反应，但抗坏血酸对槲皮素作用的预防作用在突变型受体中受损。综上所述，目前的结果表明，槲皮素对 GABA(Aρ₁)受体的拮抗作用是通过一种不依赖氧化还原的变构机制介导的。
