Department of Physiology, University College Cork, Ireland.
Neurochem Int. 2012 May;60(6):543-54. doi: 10.1016/j.neuint.2012.02.014. Epub 2012 Feb 18.
Although transient receptor potential (TRP) channel biology research has expanded rapidly in recent years, the field is hampered by the widely held, but relatively poorly investigated, belief that most of the pharmacological tools used to investigate TRP channel function may not be particularly selective for their intended targets. The objective of this study was therefore to determine if this was indeed the case by systematically evaluating the effects of three routinely used putative TRP channel antagonists, SKF 96365, flufenamic acid (FF) and 2-aminoethoxydiphenyl borate (2-APB) against one of the most widely expressed CNS receptor subtypes CNS, the human α1β2γ2 GABA(A) receptor. Using whole cell patch-clamp recording to record responses to rapidly applied GABA in the absence and presence of the three putative antagonists in turn we found that SKF 96365 (1-100 μM) and FF (1-100 μM) significantly inhibited GABA responses of recombinant human α1β2γ2 GABA(A) receptor stably expressed in HEK293 cells with IC(50) values of 13.4 ± 5.1 and 1.9 ± 1.4 μM, respectively, suppressing the maximal response to GABA at all concentrations used in a manner consistent with a non-competitive mode of action. SKF 96365 and FF also both significantly reduced desensitisation and prolonged the deactivation kinetics of the receptors to GABA (1mM; P<0.05). 2-APB (10-1000 μM) also inhibited responses to GABA at all concentrations used with an IC(50) value of 16.7 ± 5.4 μM (n=3-5) but had no significant effect on the activation, desensitisation or deactivation kinetics of the GABA responses. Taken together this investigation revealed that these widely utilised TRP channel antagonists display significant 'off-target' effects at concentrations that are routinely used for the study of TRP channel function in numerous biological systems and as such, data which is obtained utilising these compounds should be interpreted with caution.
尽管瞬时受体电位 (TRP) 通道生物学研究近年来迅速发展，但该领域受到广泛存在的阻碍，即相对缺乏研究的信念，即用于研究 TRP 通道功能的大多数药理学工具可能对其预期靶点不是特别有选择性。因此，本研究的目的是通过系统评估三种常用的假定 TRP 通道拮抗剂 SKF 96365、氟芬酸 (FF) 和 2-氨基乙氧基二苯硼酸盐 (2-APB) 对一种最广泛表达的中枢神经系统受体亚型 CNS（人 α1β2γ2 GABA(A) 受体）的影响，来确定这是否确实如此。我们使用全细胞膜片钳记录在不存在和存在三种假定拮抗剂的情况下依次记录快速施加 GABA 时的反应，发现 SKF 96365（1-100 μM）和 FF（1-100 μM）显著抑制重组人 α1β2γ2 GABA(A) 受体在 HEK293 细胞中的 GABA 反应，IC50 值分别为 13.4 ± 5.1 和 1.9 ± 1.4 μM，以非竞争性作用模式抑制所有使用浓度下 GABA 的最大反应。SKF 96365 和 FF 还显著减少了受体对 GABA（1mM；P<0.05）的脱敏作用并延长了失活动力学。2-APB（10-1000 μM）也抑制了所有使用浓度下的 GABA 反应，IC50 值为 16.7 ± 5.4 μM（n=3-5），但对 GABA 反应的激活、脱敏或失活动力学没有显著影响。总之，这项研究表明，这些广泛使用的 TRP 通道拮抗剂在浓度下显示出明显的“脱靶”效应，这些浓度在许多生物系统中用于研究 TRP 通道功能，并且因此，利用这些化合物获得的数据应谨慎解释。
