Protein Kinase C Phosphorylation Regulates Membrane Insertion of GABAA Receptor Subtypes That Mediate Tonic Inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, gamma-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from alpha 4 beta 2/3 with or without the delta subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the alpha 4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the beta 3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the delta subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of alpha 4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the alpha 4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of alpha 4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the alpha 4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.