The GABAA receptor is a ligand-gated chloride channel that mediates the fast inhibitory effects of the neurotransmitter GABA (g-aminobutyric acid). Over seventeen different receptor subunits exist and multiple receptor subtypes are present in vivo. The GABAA receptor is regulated by protein phosphorylation through multiple kinases. This regulation is complex, involving a variety of subunits and multiple second messenger system pathways. We have recently shown that activation of calcium phospholipid-dependent protein kinase (PKC) decreases GABAA receptor function via internalization of GABAA receptors. The regulation of GABAA receptor cell surface expression by PKC remains to be characterized. It has recently been established that GABAA receptors undergo clathrin-dependent constitutive endocytosis. We hypothesize that the receptor also undergoes PKC-regulated clathrin-dependent endocytosis. Furthermore, we hypothesize that the dileucine and tyrosine AP2 adaptin recognition motifs on the receptor play selective roles in constitutive vs. PKC-regulated endocytosis and also are involved in determining the post-endocytic sorting of the receptor to recycling vs. lysosomal pathways. Lastly, we hypothesize that activation of the PKC-linked metabotropic glutamate receptors mGluR 115 may represent a mechanism by which PKC-dependent GABAA receptor endocytosis occurs in neurons.
The specific aims of this proposal are: 1)To determine if the AP2 adaptin recognition motifs present on the GABAA receptor play a role in clathrin-dependent constitutive GABAA receptor endocytosis; 2) To determine whether PKC activation promotes clathrin-dependent GABAA receptor endocytosis; 3) To investigate mGluR1/5 receptor regulation of GABAA receptor cell surface expression in neurons. The proposed experiments will be performed in HEK 293 cells expressing recombinant receptors and in primary neuronal cultures. Cell surface receptors will be visualized by confocal microscopy using indirect immunofluorescence and fluorescent protein tags. Complementary biochemical experiments using chymotrypsin digest/Immunoblotting blotting techniques will also be used to measure receptor cell surface expression. Experiments using whole-cell patch-clamp recordings will provide a functional correlate for the confocal microscopy and biochemical studies. The proposed studies will aid in the understanding of GABAA receptor function and neuronal plasticity. The involvement of GABAA receptors in anxiety, sleep and seizure disorders underscores the importance of understanding the cellular and molecular mechanisms of this receptor.
