Shu Hong-Jin, Lu Xinguo, Bracamontes John, Steinbach Joe Henry, Zorumski Charles F, Mennerick Steven
Department of Psychiatry, Washington University in St. Louis, School of Medicine, St. Louis, MO, United States.
Department of Anesthesiology, Washington University in St. Louis, School of Medicine, St. Louis, MO, United States.
Front Synaptic Neurosci. 2021 Nov 18;13:763411. doi: 10.3389/fnsyn.2021.763411. eCollection 2021.
GABA receptors (GABARs) play a crucial role in inhibition in the central nervous system. GABARs containing the δ subunit mediate tonic inhibition, have distinctive pharmacological properties and are associated with disorders of the nervous system. To explore this receptor sub-class, we recently developed mice with δ-containing receptors rendered resistant to the common non-competitive antagonist picrotoxin (PTX). Resistance was achieved with a knock-in point mutation (T269Y; T6'Y) in the mouse genome. Here we characterize pharmacological and biophysical features of GABARs containing the mutated subunit to contextualize results from the KI mice. Recombinant receptors containing δ T6'Y plus WT α4 and WT β2 subunits exhibited 3-fold lower EC values for GABA but not THIP. GABA EC values in native receptors containing the mutated subunit were in the low micromolar range, in contrast with some published results that have suggested nM sensitivity of recombinant receptors. Rectification properties of δ-containing GABARs were similar to γ2-containing receptors. Receptors containing δ T6'Y had marginally weaker sensitivity to positive allosteric modulators, likely a secondary consequence of differing GABA sensitivity. Overexpression of δT6'Y in neurons resulted in robust PTX-insensitive IPSCs, suggesting that δ-containing receptors are readily recruited by synaptically released GABA. Overall, our results give context to the use of δ receptors with the T6'Y mutation to explore the roles of δ-containing receptors in inhibition.
γ-氨基丁酸受体（GABARs）在中枢神经系统的抑制过程中发挥着关键作用。含有δ亚基的GABARs介导持续性抑制，具有独特的药理学特性，并与神经系统疾病相关。为了探究这一受体亚类，我们最近培育出了对常见非竞争性拮抗剂印防己毒素（PTX）具有抗性的含δ受体小鼠。通过在小鼠基因组中进行敲入点突变（T269Y；T6'Y）实现了抗性。在此，我们对含有突变亚基的GABARs的药理学和生物物理学特征进行了表征，以便将敲入（KI）小鼠的实验结果置于背景中。含有δ T6'Y加上野生型α4和野生型β2亚基的重组受体对γ-氨基丁酸（GABA）的半数有效浓度（EC）值降低了3倍，但对4,5,6,7-四氢异噁唑并[5,4-c]吡啶-3-醇（THIP）没有影响。含有突变亚基的天然受体中的GABA EC值处于低微摩尔范围内，这与一些已发表的结果不同，那些结果表明重组受体的敏感度为纳摩尔级。含δ的GABARs的整流特性与含γ2的受体相似。含有δ T6'Y 的受体对正性变构调节剂的敏感性略弱，这可能是GABA敏感性不同的次要结果。在神经元中过表达δT6'Y会导致产生强大的对PTX不敏感的抑制性突触后电流（IPSCs），这表明含δ的受体很容易被突触释放的GABA所募集。总体而言，我们的结果为使用具有T6'Y突变的δ受体来探究含δ受体在抑制过程中的作用提供了背景信息。
