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

Abstract

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, ␥-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 ␣4␤2/3 with or without the ␦ 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 ␣4 subunit is phosphorylated at Ser 443 by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the ␤3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the ␦ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of ␣4 subunit-containing GABA A Rs is dependent on Ser 443 . Mechanistically, phosphorylation of Ser 443 acts to increase the stability of the ␣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 ␣4 subunit-containing GABA A Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the ␣4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA A R subtypes that mediate tonic inhibition. ␥-Aminobutyric acid type A receptors (GABA A Rs)2 constitute the major inhibitory ligand-gated receptors in the adult central nervous system and are responsible for both phasic and tonic forms of inhibition (1). These receptors are pentameric, anion-selective ion channels that can be assembled from eight subunit classes: ␣(1-6), ␤(1-3), ␥(1-3), ␦, ⑀, , , and (1-3)(2-3). This large number of receptor subunits provides the basis for a significant degree of heterogeneity of GABA A R structure and function. However, previous studies suggest that in the brain, the majority of phasic inhibition is dependent upon a few GABA A Rs subunits, namely the ␣, ␤, and ␥2 subunits located within synaptic sites (2, 3). In the adult brain, these receptors are specific targets for brief exposures to high concentrations of GABA, resulting in short lived, but significant, hyperpolarization. In contrast, tonic inhibition is characterized by a sustained reduction in the cell's input resistance, effectively reducing the probability of action potential generation (1, 4). Tonic inhibition is the result of persistent activation by GABA A Rs consisting primarily of ␣, ␤, and ␦ subunits located within peri-or extrasynaptic sites (1). With respect to specific brain regions, extrasynaptic GABA A Rs that mediate tonic inhibition in the thalamus and dentate gyrus of the hippocampus are composed of the ␣4 and ␤2/3 subunits with or without the ␦ subunit (5-9). Verificati...

Persistent CaMKII Activation Mediates Learning-Induced Long-Lasting Enhancement of Synaptic Inhibition

“…Indeed, a central role for synaptic inhibition in learning has been shown in several brain areas (Donato et al, 2013;Bañuelos et al, 2014;Courtin et al, 2014;Kfir et al, 2014;Sparta et al, 2014). The mechanisms underlying learning-induced and activity-induced plasticity of synaptic inhibition have been the subject of an increasing number of studies (Rivera et al, 2004;Fiumelli et al, 2005;Saar et al, 2012;McKay et al, 2013;Camiré and Topolnik, 2014;Kfir et al, 2014).…”

“…CaMKII phosphorylation of the GABA A receptor was shown to regulate the expression of the cell surface receptors and their function (Houston et al, 2009;Saliba et al, 2012). PKCdependent phosphorylation was shown to enhance receptor surface stability (Abramian et al, 2010) and reduce its current (Krishek et al, 1994;Brandon et al, 2000). PKA can enhance or reduce inhibitory synaptic transmission by differential phosphorylation of two sites on GABA A ␤3 subunits (McDonald et al, 1998).…”

3β-Methyl-Neurosteroid Analogs Are Preferential Positive Allosteric Modulators and Direct Activators of Extrasynapticδ-Subunitγ-Aminobutyric Acid Type A Receptors in the Hippocampus Dentate Gyrus Subfield

“…PKC activator potentiates THDOC-enhanced GABA-gated currents in recombinant receptors (Leidenheimer and Chapell, 1997), while inhibition of PKC reduces the THDOC and AP-prolonged decay time of mIPSCs in murine neurons (Fancsik et al, 2000;Harney et al, 2003). THDOC also promotes the phosphorylation of the S443 within the 4-subunit and the upregulation of 4-containing GABA-A receptors in the cell membrane, leading to increased tonic inhibition (Abramian et al, 2010;Abramian et al, 2014). The -subunit is predominately assembled with the 4-and 6-subunit (Jones et al, 1997;Peng et al, 2002;Spigelman et al, 2003).…”

“…Protein kinase activity influences the surface expression of GABA-A receptors and the neurosteroid sensitivity. Several GABA-A receptor subunits are substrates of PKC including the 4-and -subunits (Abramian et al, 2010;Adams et al, 2015). PKC activator potentiates THDOC-enhanced GABA-gated currents in recombinant receptors (Leidenheimer and Chapell, 1997), while inhibition of PKC reduces the THDOC and AP-prolonged decay time of mIPSCs in murine neurons (Fancsik et al, 2000;Harney et al, 2003).…”

“…Two additional serine residues of the -subunits (Ser-408 and Ser-383) are phosphorylated by PKA and CaMKII, respectively (McDonald et al, 1998;Saliba et al, 2012). Additionally, the serine residues (Ser-443) within the intracellular domain of the 4-subunit, and the Ser-408 and Ser-409 in 3-subunits are phosphorylated by PKC (Leidenheimer and Chapell, 1997;Fancsik et al, 2000;Harney et al, 2003;Abramian et al, 2010;Abramian et al, 2014;Adams et al, 2015). However, the extent of functional impact of PKC activity on the neurosteroid potentiation of extrasynaptic GABA-A receptor-mediated tonic inhibition remains unclear.…”