The neurotransmitter -aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. Approximately 30% of synapses in the brain contain GABAA receptors (Nutt, 2006), a subtype of GABA receptor that mediates fast inhibitory neurotransmission. The GABAA receptor is associated with a variety of neurological (epilepsy, insomnia) and psychiatric (anxiety, schizophrenia) disorders (Mohler, 2006;Charych et al., 2009) and is the target of several classes of therapeutic agents including benzodiazepines, barbiturates and anesthetics (Whiting, 2003). Mutations in GABAA receptor subunits are associated with several genetically- based epilepsies (Macdonald et al., 2010). Some of these mutants are retained in the endoplasmic reticulum (ER) due to inefficient folding and processing, resulting in deficits in cell surface expression. Such """"""""conformational"""""""" diseases are not unique and the cell surface expression of some disease-associated, ER-retained mutants can be rescued by ligand or pharmacological chaperones. A variety of disease mutants show promise for rescue by pharmacological chaperones, including those associated with cystic fibrosis, retinitis pigmentosa, long QT syndrome and Fabry's disease (Bernier et al., 2004). Clinical trials with pharmacological chaperones for the treatment of Fabry's disease are now underway (Fan and Ishii, 2010). In this proposal, we hypothesize that ligand chaperoning may be used to rescue the surface expression of ER-retained, epilepsy-associated GABAA receptor mutants. In support of this notion, we have recently shown that GABA can act as a ligand chaperone in the early secretory pathway to increase the cell surface expression of recombinant, wild type GABAA receptors (Eshaq et al., 2010). Our above hypothesis will be tested by two specific aims.
In Specific Aim 1 we will determine if the cell surface expression of epilepsy-associated GABAA receptor mutants expressed in HEK 293 cells can be rescued by GABA treatment.
In Specific aim 2 we will determine whether the cell surface expression of epilepsy-associated GABAA receptor mutants expressed in primary cerebral cortical cultures can be rescued by elevating intracellular GABA levels with the GABA transaminase inhibitor vigabatrin. For these experiments, receptor cell surface expression will be measured using immunofluorescence confocal microscopy and flow cytometry.
The GABAA receptor is a neurotransmitter receptor that can carry various genetic mutations that either cause or are associated with epilepsy. These mutations impair the ability of the cell to properly synthesize the receptor. The proposed research investigates a possible mechanism for aiding the correct synthesis of these mutant receptors. Thus, these studies have relevance to the treatment of epilepsy.
|Leidenheimer, Nancy J; Ryder, Katelyn G (2014) Pharmacological chaperoning: a primer on mechanism and pharmacology. Pharmacol Res 83:10-9|