Post-Hypoxic Regulation of GABA-A Receptor Function

描述（由申请人提供）：中风和缺氧经常引起癫痫发作或肌阵挛，神经兴奋性过度紊乱。缺氧诱导的高兴奋性与抑制性GABAA受体（GABAARs）功能障碍有关，但其潜在机制尚不清楚。GABAARs是由γ -氨基丁酸（GABA）激活的氯离子通道，由亚基组成，决定其药理学和动力学性质。我们在体外研究了缺氧对NT2-N神经元细胞和原代皮层神经元GABAAR功能和亚基mRNA表达的影响。短暂缺氧后1小时最大gaba诱发电流增加，但48小时后下降至对照组的60%，与α -1、α -5、β -2和γ -2亚基mrna减少有关。这些变化与bHLH转录因子、缺氧诱导因子-1 α (HIF1a)的诱导和钙通过缺氧诱导去极化打开的电压门控通道进入有关。我们的长期目标是了解缺氧诱导GABAAR神经可塑性的机制，具体目标如下：1。确定缺氧后GABAAR电流早期升高的机制。假设1:GABAAR电流的初始增加与GABAAR通道密度增加或磷酸化有关。我们将使用全细胞、穿孔贴片和单通道记录来测量GABAAR电流、药理学和缺氧后GABAAR电流的单通道特性。2. 确定转录变化是否解释了GABAAR电流的晚期减少。假设2：缺氧后48小时GABAAR电流的减少与GABAAR亚基转录的改变有关，特定亚基的改变解释了GABAAR药理学的改变。我们将使用全细胞记录和RT-PCR来评估GABAAR药理学和亚基表达的变化。3. 确定HIF-1a是否参与GABAAR亚基表达的改变。假设3：缺氧诱导HIF-1a与其他信号机制一起参与GABAAR亚基表达的调节。我们将确定a.缺氧是否会改变HIF-1a表达，b. HIF-1a升高是否会重现缺氧相关的GABAR变化，c.电压门控钙通道是否介导GABAAR调节。这些研究暗示了缺氧后高兴奋性的新机制，并可能导致缺氧后癫痫发作和肌阵挛的新治疗方法。

DESCRIPTION (provided by applicant): Stroke and hypoxia frequently cause seizures or myoclonus, disorders of excessive neuronal excitability. Hypoxia-induced hyperexcitability is linked to dysfunction of inhibitory GABAA receptors (GABAARs), but the underlying mechanisms are poorly understood. GABAARs are chloride channels activated by gamma-aminobutyric acid (GABA), composed of subunits that determine their pharmacology and kinetic properties. We have studied the effects of hypoxia on GABAAR function and subunit mRNA expression in NT2-N neuronal cells and primary cortical neurons in vitro. Maximal GABA-evoked currents increased 1 h after transient hypoxia but then decreased to 60% of control after 48 h, associated with reductions in alpha-1, alpha-5, beta-2 and gamma-2 subunit mRNAs. These changes are associated with induction of the bHLH transcription factor, hypoxia-inducible factor-1alpha (HIF1a) and calcium entry via voltage-gated channels opened by hypoxia-induced depolarizaton. Our long-term goal is to understand the mechanisms of hypoxia-induced GABAAR neuroplasticity, with these specific aims: 1. Determine the mechanisms underlying the early increase in GABAAR current after hypoxia. Hypothesis 1 is that the initial increase in GABAAR current is related to increased GABAAR channel density or phosphorylation. We will use whole-cell, perforated patch and single channel recordings to measure GABAAR currents, pharmacology, and single channel properties of post-hypoxic GABAAR currents. 2. Determine whether transcriptional changes account for the late reduction in GABAAR currents. Hypothesis 2 is that the reduction in GABAAR currents 48 h after hypoxia is related to altered GABAAR subunit transcription, and that specific subunit changes account for altered GABAAR pharmacology. We will use whole-cell recordings and RT-PCR to assess changes in GABAAR pharmacology and subunit expression. 3. Determine whether HIF-1a is involved in altered GABAAR subunit expression. Hypothesis 3 is that hypoxic induction of HIF-1a participates in regulating GABAAR subunit expression in concert with other signaling mechanisms. We will determine a. whether hypoxia alters HIF-1a expression, b. whether elevated HIF-1a reproduces hypoxia-related GABAR changes, and c. whether voltage-gated calcium channels mediate GABAAR regulation. These studies implicate a novel mechanism underlying post- hypoxic hyperexcitability, and could result in new treatments for post-hypoxic seizures and myoclonus.