Post-Hypoxic Regulation of GABA-A Receptor Function

GABA-A 受体功能的缺氧后调节

• 批准号: 7157555
• 负责人: Lazar John GREENFIELD
• 金额: $ 25.92万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7157555
• 关键词: Accounting; Affect; BHLH Protein; Behavior; Calcium; Calcium Channel; Cell Nucleus; Cells; Cerebral Hypoxia; Chloride Channels; Chloride Ion; Chlorides; Complementary DNA; Condition; Deferoxamine; Disease; Disruption; Dominant-Negative Mutation; Electric Capacitance; Exposure to; Functional disorder; Genes; Genetic Transcription; Goals; Hypoxia; Hypoxia Inducible Factor; Immunoblotting; In Vitro; Iodoacetamide; Kinetics; Link; Measures; Mediating; Messenger RNA; Modeling; Myoclonus; Neuronal Plasticity; Neurons; Oxygen; Pharmacology; Phosphorylation; Physiological; Property; Proteins; Rattus; Regulation; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Role; Seizures; Signal Pathway; Signal Transduction; Stroke; Time; Transactivation; Transfection; Whole-Cell Recordings; clinically significant; cytotoxicity; density; gamma-Aminobutyric Acid; in vivo; inhibitor/antagonist; mRNA Expression; mutant; neuronal excitability; novel; prevent; receptor; receptor function; voltage; voltage gated channel

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.

描述（由申请人提供）：中风和缺氧经常导致癫痫发作或肌阵挛，以及神经元过度兴奋的疾病。缺氧引起的过度兴奋与抑制性 GABAA 受体 (GABAAR) 功能障碍有关，但其潜在机制尚不清楚。 GABAAR 是由 γ-氨基丁酸 (GABA) 激活的氯离子通道，由决定其药理学和动力学特性的亚基组成。我们在体外研究了缺氧对 NT2-N 神经元细胞和原代皮质神经元 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 小时后下降至对照的 60%，这与 alpha-1、alpha-5、beta-2 和 gamma-2 亚基 mRNA 的减少有关。这些变化与 bHLH 转录因子、缺氧诱导因子 1α (HIF1a) 的诱导以及缺氧诱导的去极化打开的电压门控通道的钙进入有关。我们的长期目标是了解缺氧诱导的 GABAAR 神经可塑性的机制，具体目标如下： 1. 确定缺氧后 GABAAR 电流早期增加的机制。假设1是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. 确定转录变化是否是 GABAAR 电流后期减少的原因。假设2是缺氧48小时后GABAAR电流的减少与GABAAR亚基转录的改变有关，并且特定亚基的变化解释了GABAAR药理学的改变。我们将使用全细胞记录和 RT-PCR 来评估 GABAAR 药理学和亚基表达的变化。 3. 确定 HIF-1a 是否参与改变的 GABAAR 亚基表达。假设3是HIF-1a的缺氧诱导与其他信号传导机制共同参与调节GABAAR亚基表达。我们将确定a.缺氧是否会改变 HIF-1a 表达，b．升高的 HIF-1a 是否会重现缺氧相关的 GABAR 变化，以及 c．电压门控钙通道是否介导 GABAAR 调节。这些研究揭示了缺氧后过度兴奋的新机制，并可能为缺氧后癫痫发作和肌阵挛带来新的治疗方法。