Regulation of GABAA Receptor Cell Surface Expression

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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.