Brain and Mind Centre, Sydney Pharmacy School, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia.
Br J Pharmacol. 2021 Apr;178(7):1556-1573. doi: 10.1111/bph.15380. Epub 2021 Feb 27.
GABA receptors containing δ-subunits are notorious for being difficult to study in vitro due to heterogeneity of expressed receptor populations and low GABA-evoked current amplitudes. Thus, there are some published misconceptions and contradictory conclusions made regarding the pharmacology and stoichiometry of δ-containing receptors. The aim of this study was to obtain robust homogenous expression of α1βδ receptors for in-depth investigation.
Novel δ-containing pentameric concatenated constructs were designed. The resulting α1β2δ and α1β3δ GABA receptor concatemers were investigated by two-electrode voltage-clamp electrophysiology using Xenopus laevis oocytes.
First, while homogenous α1βδ GABA receptor pools could not be obtained by manipulating the ratio of injected cRNAs of free α1, β2/3, and δ subunits, concatenated pentameric α1β2δ and α1β3δ constructs resulted in robust expression levels of concatemers. Second, by using optimised constructs that give unidirectional assembly of concatemers, we found that the δ subunit cannot directly participate in GABA binding and receptor activation. Hence, functional δ-containing receptors are likely to all have a conventional 2α:2β:1δ stoichiometry arranged as βαβαδ when viewed counterclockwise from the extracellular side. Third, α1β2/3δ receptors were found to express efficiently in X. laevis oocytes but have a low estimated open probability of ~0.5% upon GABA activation. Because of this, these receptors are uniquely susceptible to positive allosteric modulation by, for example, neurosteroids.
Our data answer important outstanding questions regarding the pharmacology and stoichiometry of α1δ-containing GABA receptors and pave the way for future analysis and drug discovery efforts.
由于表达的受体群体异质性和 GABA 诱发电流幅度低，含 δ 亚单位的 GABA 受体很难在体外进行研究，这是众所周知的。因此，关于含 δ 受体的药理学和计量比，存在一些已发表的误解和相互矛盾的结论。本研究的目的是获得稳健的同质 α1βδ 受体表达，以进行深入研究。
设计了新型含 δ 的五聚体串联构建体。使用 Xenopus laevis 卵母细胞中的双电极电压钳电生理学技术研究由此产生的 α1β2δ 和 α1β3δ GABA 受体串联体。
首先，虽然通过操纵游离 α1、β2/3 和 δ 亚基的注射 cRNA 比例，不能获得同质的 α1βδ GABA 受体池，但串联的五聚体 α1β2δ 和 α1β3δ 构建体导致串联体的表达水平稳健。其次，通过使用优化的构建体，这些构建体使串联体单向组装，我们发现 δ 亚基不能直接参与 GABA 结合和受体激活。因此，功能性含 δ 受体可能都具有传统的 2α：2β：1δ 计量比，当从细胞外侧逆时针观察时，排列为 βαβαδ。第三，发现 α1β2/3δ 受体在 X. laevis 卵母细胞中有效表达，但在 GABA 激活时估计开放概率低至约 0.5%。由于这个原因，这些受体容易受到例如神经甾体的正变构调节。
我们的数据回答了关于含 α1δ 的 GABA 受体药理学和计量比的重要悬而未决的问题，并为未来的分析和药物发现工作铺平了道路。
