Post-Hypoxic Regulation of GABA-A Receptor Function

描述(申请人提供)：中风和缺氧经常导致癫痫发作或肌阵挛，过度的神经元兴奋性障碍。低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，由决定其药理和动力学特性的亚基组成。我们研究了低氧对体外培养的NT2-N神经元和原代皮质神经元GABAAR功能和亚单位mRNA表达的影响。最大GABA诱发电流在短暂低氧后1h升高，48h后降至对照组的60%，并伴有α-1、α-5、β-2和γ-2亚单位mRNAs的减少。这些变化与bHLH转录因子、低氧诱导因子-1a(HIF1a)的诱导和低氧诱导去极化开放的电压门控通道钙内流有关。我们的长期目标是了解低氧诱导GABAAR神经可塑性的机制，具体目的有：1.确定低氧后早期GABAAR电流增加的机制。假设1是GABAAR电流的初始增加与GABAAR通道密度的增加或磷酸化有关。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.确定转录变化是否是GABAAR电流后期减少的原因。假说2认为，缺氧48小时后GABAAR电流的减少与GABAAR亚基转录改变有关，而特定亚基的改变解释了GABAAR药理改变。我们将使用全细胞记录和RT-PCR来评估GABAAR药理和亚单位表达的变化。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，并与其他信号机制协调。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.电压门控钙通道是否介导GABAAR调节。这些研究暗示了缺氧后过度兴奋的新机制，并可能导致新的治疗缺氧后癫痫发作和肌阵挛的方法。

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.