Modulation of neuronal and recombinant GABAA receptors by redox reagents
Modulation of neuronal and recombinant GABAA receptors by redox reagents
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DOI：
10.1111/j.1469-7793.1999.0035z.x
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发表时间：
1999-05-15
影响因子：
5.5
通讯作者：
Smart, TG
中科院分区：
文献类型：
--
作者：
Amato, A;Connolly, CN;Smart, TG
1.采用重组人胚肾细胞表达的小鼠受体亚基和大鼠神经元GABA（A）受体，结合全细胞和单通道膜片钳技术，研究了GABA（A）受体中假定的二硫桥的功能作用及其对氧化和还原的敏感性.还原剂二硫苏糖醇（DTT）可逆地增强α 1 β 1或α 1 β 2受体的GABA激活反应（I（GABA）），而氧化剂5，5 '-二硫代-双-（2-硝基苯甲酸）（DTNB）引起抑制。I（GABA）的氧化还原调节是独立的GABA浓度，膜电位和受体激动剂，并不影响GABA EC（50）或希尔系数。内源性抗氧化剂还原型谷胱甘肽（GSH）也增强α 1 β 2受体中的I（GABA），而氧化形式的DTT和谷胱甘肽（GSSG）均引起较小的抑制作用.由α 1 β 1 γ 2S或α 1 β 2 γ 2S组成的重组受体对DTT和DTNB的敏感性显著降低。4.对于神经元GABA（A）受体，I（GABA）是增强的氟安定和相对不受氧化还原试剂。然而，在培养的交感神经元，烟碱乙酰胆碱激活的反应被DTT抑制，而在小脑颗粒神经元，NMDA激活的电流增强DTT和抑制：DTNB。单个GABA激活的离子通道电流显示α 1 β 1结构的电导为16 pS。DTT不影响电导或由停留时间直方图确定的单个开放时间常数，但通过影响通道开放概率而增加平均开放时间，而不增加细胞表面受体的数量. DTT和DTNB的作用的动力学模型表明，该受体存在于氧化和还原形式之间的平衡。DTT增加进入还原受体形式和脱敏状态的速率。DTNB逆转了这些动力学效应。我们的成果。表明由α和β亚基形成的GABA（A）受体对氧化还原剂的调节敏感。在受体中包含γ 2亚基，或从一些神经元GABA受体记录，导致对DTT和DTNB的敏感性降低。鉴于中枢神经系统某些区域存在α β亚单位复合物，以及内源性氧化还原化合物的产生和释放，天然GABA（A）受体可能受到氧化还原机制的调节。
1. The functional role played by the postulated disulphide bridge in gamma-aminobutyric acid type A (GABA(A)) receptors and its susceptibility to oxidation and reduction were studied using recombinant (murine receptor subunits expressed in human embryonic kidney cells) and rat neuronal GABA(A) receptors in conjunction with whole-cell and single channel patch-clamp techniques.2. The reducing agent dithiothreitol (DTT) reversibly potentiated GABA-activated responses (I(GABA)) of alpha 1 beta 1 or alpha 1 beta 2 receptors while the oxidizing reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) caused inhibition. Redox modulation of I(GABA) was independent of GABA concentration, membrane potential and the receptor agonist and did not affect the GABA EC(50) or Hill coefficient. The endogenous antioxidant reduced glutathione (GSH) also potentiated I(GABA) in alpha 1 beta 2 receptors, while both the oxidized form of DTT and glutathione (GSSG) caused small inhibitory effects.3. Recombinant receptors composed of alpha 1 beta 1 gamma 2S or alpha 1 beta 2 gamma 2S were considerably less sensitive to DTT and DTNB.4. For neuronal GABA(A) receptors, I(GABA) was enhanced flurazepam and relatively unaffected by redox reagents. However, in cultured sympathetic neurones, nicotinic acetylcholine-activated responses were inhibited by DTT whilst in cerebellar granule neurones, NMDA-activated currents were potentiated by DTT and inhibited by: DTNB.5. Single GABA-activated ion channel currents exhibited a conductance of 16 pS for alpha 1 beta 1 constructs. DTT did not affect the conductance or individual open time constants determined from dwell time histograms, but increased the mean open time by affecting the channel open probability without increasing the number of cell surface receptors.6. A kinetic model of the effects of DTT and DTNB suggested that the receptor existed in equilibrium between oxidized and reduced forms. DTT increased the rate of entry into reduced receptor forms and also into desensitized states. DTNB reversed these kinetic effects.7. Our results. indicate that GABA(A) receptors formed by alpha and beta subunits are susceptible to regulation by redox agents. Inclusion of the gamma 2 subunit in the receptor, or recording from some neuronal GABA, receptors, resulted in reduced sensitivity to DTT and DTNB. Given the suggested existence of alpha beta subunit complexes in some areas of the central nervous system together with the generation and release of endogenous redox compounds, native GABA(A) receptors may be subject to regulation by redox mechanisms.