Post-Hypoxic Regulation of GABA-A Receptor Function

中风和缺氧经常导致癫痫发作或肌阵挛，即过度的神经元兴奋性障碍。 低氧诱导的过度兴奋性与抑制性GABAA受体(GABAARs)功能障碍有关，但 人们对潜在的机制知之甚少。GABA受体是由γ-氨基丁酸激活的氯离子通道 酸(GABA)，由决定其药理和动力学性质的亚基组成。我们有 研究低氧对NT2-N神经细胞GABAAR功能及亚单位mRNA表达的影响 和体外培养的原代皮质神经元。短暂性缺氧1h后最大GABA诱发电流增加 但在48小时后下降到对照组的60%，并伴随着a1、a5、02和y2亚基的减少 MRNAs。这些变化与低氧诱导的bhlh转录因子的诱导有关。 因子-1a(HIF1a)和钙离子通过低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低氧诱导的GABAAR神经可塑性的机制， 具体目标：1.确定伽巴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。我们将使用全单元、穿孔贴片和单通道录音 测量缺氧后GABAAR电流、药理学和单通道特性 洋流。2.确定转录变化是否是GABAAR后期减少的原因 洋流。假设2低氧后48小时GABA电流的减少与改变有关 GABAAR亚基转录，并且特定亚基的变化解释了GABAAR药理的改变。 我们将使用全细胞记录和RT-PCR来评估GABAAR药理和亚基的变化 表情。3.确定HIF-1a是否参与改变的GABAAR亚基表达。 假设3低氧诱导HIF-1a参与调节GABAAR亚单位的表达。 与其他信令机制协调一致。我们将确定a.缺氧是否改变HIF-1a的表达，b. 升高的HIF-1a是否复制缺氧相关的GABAR变化，以及C.是否有电压门控 钙通道介导GABAAR的调节。这些研究揭示了一种新的后遗症发生机制。 低氧高兴奋性，并可能导致新的治疗低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.