Abstract
GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors.
Highlights
The neurotransmitter γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system
Neurons Expressing Surface GABAA Receptors Contain the Neurotransmitter GABA and its Degradative Enzyme GABA Transaminase For GABA to act as a ligand chaperone, GABA must be present in neurons that express surface GABAA receptors
In the present study we extend these findings using primary neuronal cultures to show that endogenous GABA acts as a cognate ligand chaperone for neuronal GABAA receptors
Summary
The neurotransmitter γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. 30% of synapses in the brain contain GABAA receptors (Nutt, 2006), a subtype of GABA receptor that mediates fast inhibitory neurotransmission. Upon binding GABA, an integral chloride channel within the receptor is gated, allowing chloride influx leading to membrane hyperpolarization and neuronal inhibition. GABAA receptors belong to the Cys-loop ligand-gated ion channel superfamily, whose other members include the nicotinic acetylcholine, glycine and serotonin type 3 receptors (Olsen and Sieghart, 2008). Cys-loop ligand-gated ion channels are pentameric in structure, with each subunit possessing a large extracellular N-terminus, four membrane-spanning domains (M1–M4), a large cytoplasmic loop between M3 and M4, and a short extracellular C-terminus. 19 GABAA receptor subunits have been identified only a limited number of receptor subtypes exist based on developmental/tissue expression and assembly residues that specify oligomerization interfaces (Olsen and Sieghart, 2009). Of particular relevance to the present study, the receptor topology places the GABA binding pocket within the lumen of the RER during receptor biogenesis
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