Hannan Saad, Mortensen Martin, Smart Trevor G
Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Neuropharmacology. 2015 Jun;93:28-40. doi: 10.1016/j.neuropharm.2015.01.001. Epub 2015 Jan 26.
The snake neurotoxin α-bungarotoxin (α-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, α-Bgtx is also used as an imaging tool for fluorophore-labelling studies, and given the structural conservation within the pentameric ligand-gated ion channel family, we assessed whether α-Bgtx could bind to recombinant and native γ-aminobutyric type-A receptors (GABAARs). Applying fluorophore-linked α-Bgtx to recombinant αxβ1/2γ2 GABAARs expressed in HEK-293 cells enabled clear cell-surface labelling of α2β1/2γ2 contrasting with the weaker staining of α1/4β1/2γ2, and no labelling for α3/5/6β1/2γ2. The labelling of α2β2γ2 was abolished by bicuculline, a competitive antagonist at GABAARs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABAARs. Labelling by α-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor β-α subunit interface forms part of the α-Bgtx binding site. Using whole-cell recording, high concentrations of α-Bgtx (20 μM) inhibited GABA-activated currents at all αxβ2γ2 receptors examined, but at lower concentrations (5 μM), α-Bgtx was selective for α2β2γ2. Using α-Bgtx, at low concentrations, permitted the selective inhibition of α2 subunit-containing GABAARs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, α-Bgtx can act as an inhibitor at recombinant and native GABAARs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus.
蛇神经毒素α-银环蛇毒素（α-Bgtx）是烟碱型乙酰胆碱受体（nAChRs）的竞争性拮抗剂，被广泛用于研究其功能和细胞表面表达。越来越多的研究中，α-Bgtx也被用作荧光团标记研究的成像工具，鉴于五聚体配体门控离子通道家族内部的结构保守性，我们评估了α-Bgtx是否能与重组和天然的γ-氨基丁酸A型受体（GABAARs）结合。将荧光团连接的α-Bgtx应用于在HEK-293细胞中表达的重组αxβ1/2γ2 GABAARs，能够清晰地对α2β1/2γ2进行细胞表面标记，与之形成对比的是α1/4β1/2γ2的染色较弱，而α3/5/6β1/2γ2则没有标记。GABAARs的竞争性拮抗剂荷包牡丹碱和在nAChRs和GABAARs中以类似方式起作用的筒箭毒碱（d-Tc）均可消除α2β2γ2的标记。GABA也可减少α-Bgtx的标记，这表明受体β-α亚基界面处的GABA结合位点构成了α-Bgtx结合位点的一部分。使用全细胞记录法，高浓度的α-Bgtx（20 μM）可抑制所有检测的αxβ2γ2受体上GABA激活的电流，但在较低浓度（5 μM）时，α-Bgtx对α-2β2γ2具有选择性。低浓度使用α-Bgtx可选择性抑制海马齿状回颗粒细胞中含α2亚基的GABAARs，降低突触电流幅度而不影响GABA介导的强直电流。总之，α-Bgtx可作为重组和天然GABAARs的抑制剂，并可作为一种选择性工具来抑制海马体中的相位电流而非强直电流。
