Regulation of GABAA Receptor Cell Surface Expression

GABAA 受体细胞表面表达的调节

• 批准号: 7688408
• 负责人: NANCY J LEIDENHEIMER
• 金额: $ 43.49万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7688408
• 关键词: 4-Aminobutyrate aminotransferase; Affect; Aftercare; Allylglycine; Antibodies; Anxiety; Barbiturates; Benzodiazepines; Binding; Biochemical; Biogenesis; Biotinylation; Brain; Calnexin; Cell Surface Receptors; Cell surface; Cells; Cerebrum; Chemicals; Chloride Channels; Confocal Microscopy; Dendrites; Disease; Drug Delivery Systems; Endoplasmic Reticulum; Epilepsy; Equilibrium; Flow Cytometry; Fluorescence; GABA Receptor; Golgi Apparatus; Harvest; Immunoprecipitation; Incubated; Infection; Label; Ligands; Mediating; Membrane; Mental disorders; Methionine; Molecular Chaperones; Neuraxis; Neurons; Neurotransmitter Receptor; Neurotransmitters; Physiologic pulse; Physiological; Process; Production; Proteins; Recombinants; Regulation; Research; Schizophrenia; Site; Sleep Disorders; Sodium Dodecyl Sulfate-PAGE; Streptavidin; Structure; Surface; System; Techniques; Therapeutic; Therapeutic Agents; Time; Toxic effect; Vigabatrin; Viral; Virus; Western Blotting; barbituric acid salt; gamma-Aminobutyric Acid; inhibitor/antagonist; nervous system disorder; neurotransmission; particle; postsynaptic; prevent; receptor; research study; small hairpin RNA; tool; trafficking; transduction efficiency; uptake

The GABAA receptor is a ligand-gated chloride channel that, upon binding the neurotransmitter GABA, mediates neuronal inhibition throughout the brain. In addition to binding GABA, the receptor is allosterically modulated by a variety of therapeutic agents, such as benzodiazepines (BZ) and barbiturates, that bind to distinct sites on the receptor. Many GABAA receptor subunits (α1-6, β1-3, γ1-3, δ, ε, θ and π) exist and are preferentially assembled into pentameric structures within the endoplasmic reticulum (ER). Once assembled, the receptors are exported to the cell surface to participate in GABAergic neurotransmission. Surprisingly, little is known regarding receptor biogenesis despite the obvious importance of this process for maintaining appropriate levels of cell surface receptors. Using a recombinant expression system, we have recently discovered that the neurotransmitter GABA can act as a ligand chaperone in the ER to promote receptor biogenesis, thus increasing surface expression of GABAA receptors. The proposed studies will further investigate this finding. These experiments will examine 1) if GABA acts as a physiological ligand chaperone of native GABAA receptors in primary neuronal cultures; 2) whether the GABA chaperone effect displays receptor subtype selectivity; 3) whether AAV-GAD67 transduction can promote GABA chaperoning in neuronal cultures and 4) the mechanism by which the ligand chaperoning occurs. Experiments will be conducted on recombinant GABAA receptors expressed in HEK 293 cells as well as native receptors in primary neuronal cultures. A multifaceted approach involving fluorescence confocal microscopy, flow cytometry and biochemical techniques will be used.

GABAA受体是一种配体门控氯离子通道，在结合神经递质GABA后，介导整个大脑的神经元抑制。除了结合GABA外，受体还通过多种治疗剂（如苯二氮卓类（BZ）和巴比妥类）进行变构调节，这些治疗剂结合受体上的不同位点。存在许多GABAA受体亚基（α1-6、β1-3、γ1-3、δ、ε、θ和π），并且在内质网（ER）内优先组装成五聚体结构。一旦组装，受体被输出到细胞表面以参与GABA能神经传递。令人惊讶的是，很少有人知道关于受体的生物发生，尽管这一过程中维持适当水平的细胞表面受体的明显重要性。使用重组表达系统，我们最近发现，神经递质GABA可以作为ER中的配体伴侣，以促进受体生物合成，从而增加GABAA受体的表面表达。拟议的研究将进一步调查这一发现。这些实验将检查1）GABA是否在原代神经元培养物中充当天然GABAa受体的生理配体伴侣; 2）GABA伴侣效应是否显示受体亚型选择性; 3）AAV-GAD 67转导是否可以促进神经元培养物中的GABA伴侣作用和4）配体伴侣作用发生的机制。将对HEK 293细胞中表达的重组GABAA受体以及原代神经元培养物中的天然受体进行实验。一个多方面的方法，涉及荧光共聚焦显微镜，流式细胞术和生化技术将被使用。