Post-Hypoxic Regulation of GABA-A Receptor Function

中风和缺氧经常引起癫痫发作或肌阵挛，神经元过度兴奋的疾病。 缺氧诱导的过度兴奋与抑制性GABAA受体（GABAARs）功能障碍有关，但 对潜在的机制知之甚少。GABAAR是由γ-氨基丁酸激活的氯离子通道 氨基丁酸（GABA），由决定其药理学和动力学特性的亚基组成。我们有 研究了缺氧对NT 2-N神经细胞GABAAR功能和亚基mRNA表达的影响 和原代皮层神经元。短暂缺氧1h后GABA诱发的最大电流增加 但48 h后下降至对照组的60%，与a1、a5、02和y2亚基的减少有关 mRNA。这些变化与缺氧诱导的bHLH转录因子的诱导有关， 因子-1a（HIF 1a）和钙离子通过缺氧诱导的去极化开放的电压门控通道进入。 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：1.确定GABAAR电流早期增加的机制， 缺氧假设1是GABAAR电流的初始增加与GABAAR增加有关 通道密度或磷酸化。我们将使用全细胞，穿孔贴片和单通道记录 测量GABAAR电流、药理学和缺氧后GABAAR的单通道特性 水流2.确定转录变化是否是GABAAR晚期减少的原因 水流假设2：缺氧48小时后GABAAR电流的减少与缺氧后GABAAR电流的改变有关。 GABAAR亚基转录，特定的亚基变化解释了GABAAR药理学的改变。 我们将使用全细胞记录和RT-PCR来评估GABAAR药理学和亚单位的变化， 表情3.确定HIF-1a是否参与GABAAR亚基表达的改变。 假设3：低氧诱导HIF-1 α参与调节GABAAR亚单位的表达。 与其他信号机制相协调。我们将确定A。缺氧是否改变HIF-1 a表达，B. 升高的HIF-1a是否再现缺氧相关的GABAR变化，以及c.无论是电压门控 钙通道介导GABAAR调节。这些研究暗示了一种新的机制， 缺氧过度兴奋，并可能导致新的治疗缺氧后癫痫发作和肌阵挛。

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 y-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 a1, a5, 02 and y2 subunit mRNAs. These changes are associated with induction of the bHLH transcription factor, hypoxia-inducible factor-1a(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.