Regulation of GABAA Receptor Cell Surface Expression

GABAA受体是一种配体门控的氯离子通道，与神经递质GABA结合后，在整个大脑中介导神经元抑制。除了结合GABA外，该受体还受到多种治疗药物的变构调节，如苯二氮卓类药物（BZ）和巴比妥酸盐，它们与受体上的不同位点结合。许多GABAA受体亚基（α1-6, β1-3, γ1-3, δ, ε， θ和π）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参与gaba能神经传递。令人惊讶的是，尽管这一过程对于维持细胞表面受体的适当水平具有明显的重要性，但对受体的生物发生知之甚少。利用重组表达系统，我们最近发现神经递质GABA可以作为内质网中的配体伴侣，促进受体的生物发生，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。这些实验将检验1)GABA是否在原代神经元培养中作为天然GABAA受体的生理配体伴侣；2) GABA伴侣效应是否表现出受体亚型选择性；3) AAV-GAD67的转导是否能促进GABA在神经元培养中的伴侣行为，4)这种配体伴侣行为发生的机制。实验将在HEK 293细胞中表达重组GABAA受体，以及在原代神经元培养中表达天然受体。将采用多方面的方法，包括荧光共聚焦显微镜、流式细胞术和生化技术。

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.