Jatczak-Śliwa Magdalena, Terejko Katarzyna, Brodzki Marek, Michałowski Michał A, Czyzewska Marta M, Nowicka Joanna M, Andrzejczak Anna, Srinivasan Rakenduvadhana, Mozrzymas Jerzy W
Laboratory of Neuroscience, Department of Biophysics, Wrocław Medical University, Wrocław, Poland.
Department of Molecular Physiology and Neurobiology, University of Wrocław, Wrocław, Poland.
Front Cell Neurosci. 2018 Aug 28;12:237. doi: 10.3389/fncel.2018.00237. eCollection 2018.
GABA receptors (GABARs) play a crucial inhibitory role in the CNS. Benzodiazepines (BDZs) are positive modulators of specific subtypes of GABARs, but the underlying mechanism remains obscure. Early studies demonstrated the major impact of BDZs on binding and more recent investigations indicated gating, but it is unclear which transitions are affected. Moreover, the upregulation of GABAR spontaneous activity by BDZs indicates their impact on receptor gating but the underlying mechanisms remain unknown. Herein, we investigated the effect of a BDZ (flurazepam) on the spontaneous and GABA-induced activity for wild-type (WT, αβγ) and mutated (at the orthosteric binding site αF64) GABARs. Surprisingly, in spite of the localization at the binding site, these mutations increased the spontaneous activity. Flurazepam (FLU) upregulated this activity for mutants and WT receptors to a similar extent by affecting opening/closing transitions. Spontaneous activity affected GABA-evoked currents and is manifested as an overshoot after agonist removal that depended on the modulation by BDZs. We explain the mechanism of this phenomenon as a cross-desensitization of ligand-activated and spontaneously active receptors. Moreover, due to spontaneous activity, FLU-pretreatment and co-application (agonist + FLU) protocols yielded distinct results. We provide also the first evidence that GABAR may enter the desensitized state in the absence of GABA in a FLU-dependent manner. Based on our data and model simulations, we propose that FLU affects agonist-induced gating by modifying primarily preactivation and desensitization. We conclude that the mechanisms of modulation of spontaneous and ligand-activated GABAR activity concerns gating but distinct transitions are affected in spontaneous and agonist-evoked activity.
γ-氨基丁酸受体（GABARs）在中枢神经系统中发挥着至关重要的抑制作用。苯二氮䓬类药物（BDZs）是GABARs特定亚型的正性调节剂，但其潜在机制仍不清楚。早期研究证明了BDZs对结合的主要影响，而最近的研究表明其对门控有影响，但尚不清楚哪些转变受到了影响。此外，BDZs对GABAR自发活性的上调表明它们对受体门控有影响，但其潜在机制仍然未知。在此，我们研究了一种BDZ（氟西泮）对野生型（WT，αβγ）和突变型（在正构结合位点αF64处）GABARs的自发活性和γ-氨基丁酸（GABA）诱导活性的影响。令人惊讶的是，尽管这些突变位于结合位点，但它们增加了自发活性。氟西泮（FLU）通过影响开放/关闭转变，将突变体和野生型受体的这种活性上调到相似的程度。自发活性影响GABA诱发的电流，并表现为激动剂去除后的过冲，这取决于BDZs的调节。我们将这种现象的机制解释为配体激活受体和自发激活受体的交叉脱敏。此外，由于自发活性，FLU预处理和共应用（激动剂+FLU）方案产生了不同的结果。我们还提供了首个证据，表明GABAR可能以FLU依赖的方式在没有GABA的情况下进入脱敏状态。基于我们的数据和模型模拟，我们提出FLU主要通过改变预激活和脱敏来影响激动剂诱导的门控。我们得出结论，自发激活和配体激活的GABAR活性的调节机制涉及门控，但自发活性和激动剂诱发活性中受影响的转变不同。
