Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, Florida, 33612, USA.
Sci Rep. 2017 Aug 10;7(1):7770. doi: 10.1038/s41598-017-08031-9.
Anaesthetic molecules act on synaptic transmission via the allosteric modulation of ligand-gated chloride channels, such as hetero-oligomeric αβγ GABA receptors. To elucidate the overall activation paradigm via allosteric versus orthosteric sites, we used highly homologous, but homo-oligomeric, ρ receptors that are contrastingly insensitive to anaesthetics and respond partially to several full GABA αβγ receptor agonists. Here, we coexpressed varying ratios of RNAs encoding the wild-type and the mutated ρ subunits, which are anaesthetic-sensitive and respond with full efficacy to partial GABA agonists, to generate distinct ensembles of receptors containing five, four, three, two, one, or zero mutated subunits. Using these experiments, we then demonstrate that, in the pentamer, three anaesthetic-sensitive ρ subunits are needed to impart full efficacy to the partial GABA agonists. By contrast, five anaesthetic-sensitive subunits are required for direct activation by anaesthetics alone, and only one anaesthetic-sensitive subunit is sufficient to confer the anaesthetic-dependent potentiation to the GABA current. In conclusion, our data indicate that GABA and anaesthetics holistically activate the GABA ρ receptor through distinct subunit level rearrangements and suggest that in contrast to the global impact of GABA via orthosteric sites, the force of anaesthetics through allosteric sites may not propagate to the neighbouring subunits and, thus, may have only a local and limited effect on the ρ GABA receptor model system.
麻醉分子通过配体门控氯离子通道的变构调节作用于突触传递，例如异源寡聚αβγ GABA 受体。为了阐明通过变构和正位部位的总体激活范式，我们使用高度同源但同聚的 ρ 受体，这些受体对麻醉剂的反应相对不敏感，并且对几种全 GABA αβγ 受体激动剂部分反应。在这里，我们共表达了编码野生型和突变 ρ 亚基的 RNA 的不同比例，这些亚基对麻醉剂敏感并且对部分 GABA 激动剂具有完全功效反应，以产生包含五个、四个、三个、两个、一个或零个突变亚基的不同受体集合。使用这些实验，我们然后证明，在五聚体中，需要三个对麻醉剂敏感的 ρ 亚基才能赋予部分 GABA 激动剂完全功效。相比之下，仅通过麻醉剂直接激活需要五个对麻醉剂敏感的亚基，而仅一个对麻醉剂敏感的亚基足以赋予 GABA 电流依赖于麻醉剂的增强作用。总之，我们的数据表明，GABA 和麻醉剂通过不同的亚基水平重排整体激活 GABA ρ 受体，并表明与通过正位部位的 GABA 的整体影响相比，通过变构部位的麻醉剂的力可能不会传递到相邻的亚基，因此，对 ρ GABA 受体模型系统的影响可能是局部和有限的。
