Protein Kinase Cϵ Regulates γ-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of γ2 Subunits

Abstract

Ethanol enhances ␥-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA A receptors, especially at low concentrations achieved during social drinking. We postulated that the ⑀ isoform of protein kinase C (PKC⑀) regulates the ethanol sensitivity of GABA A receptors, as mice lacking PKC⑀ show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKC⑀ mutant to selectively inhibit the catalytic activity of PKC⑀. We used this mutant and PKC⑀ ؊/؊ mice to determine that PKC⑀ phosphorylates ␥2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at ␣1␤2␥2 receptors, which is the most abundant GABA A receptor subtype in the brain. Our findings indicate that PKC⑀ phosphorylation of ␥2 regulates the response of GABA A receptors to specific allosteric modulators, and, in particular, PKC⑀ inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol. ␥-Aminobutyrate type A (GABA A )3 receptors mediate the majority of rapid inhibitory neurotransmission in the brain and are an important target for ethanol, the most widely abused drug (1). Ethanol modulation of GABA A receptors was first identified in synaptosomal preparations where intoxicating concentrations (10 -30 mM) enhanced receptor function as measured by 36 Cl uptake assays (2, 3). However, after 30 years of investigation, it is apparent that ethanol enhancement of synaptic GABA A receptors is variable and in some preparations cannot be detected even at anesthetic concentrations (1, 4).GABA A receptors are pentameric protein complexes of subunits from eight classes (␣1-6, ␤1-3, ␥1-3, ␦, ⑀, , , and 1-3) (5). Most receptors are composed of two ␣ subunits and two ␤ subunits that co-assemble with one ␥2 subunit, which anchors these receptors at synapses where they mediate phasic inhibition (6). A minority contain a ␦ subunit instead of ␥2; these receptors are extrasynaptic and mediate tonic inhibition in neurons (7). Because earlier ethanol studies that focused on GABA currents carried by ␥2-containing receptors produced variable results, recent attention has turned to ethanol effects at receptors containing ␦ subunits. Reports from three laboratories found these receptors enhanced by low (Յ30 mM) intoxicating concentrations of ethanol (8 -10). However, two recent studies were unable to demonstrate low dose ethanol sensitivity of ␦-containing GABA A receptors (11, 12), indicating that, like synaptic GABA A receptors, ethanol modulation of extrasynaptic receptors is also variable.The reasons for this high degree of variability are unknown. One possibility is that intracellular signaling pathways may regulate ethanol sensitivity of GABA A receptors, and the activity of such pathways was not controlled for in these studies. This hypothesis is consistent with our findings in mice lacking protein kinase C⑀ (PKC⑀), which show an increased behavioral response to ethanol (13). Ethanol modulation of GABA A receptors is a...

PKCε phosphorylation of the sodium channel NaV1.8 increases channel function and produces mechanical hyperalgesia in mice

“…As shown in Figure 4A, PKCε phosphorylated the intracellular Na v 1.8/L3 loop at a rate similar to phosphorylation of the major intracellular loop of the GABA A γ2S subunit, which contains a PKCε phosphorylation site at S327 (9). Similar to GABA A γ2S, the Na v 1.8/L3 loop was phosphorylated to a maximal stoichiometry of 0.95 ± 0.08 (n = 3), suggesting that it is a true PKCε substrate.…”

“…(A) The top 2 panels show an autoradiogram of phosphorylated intracellular L3 loop (p-L3) and a scanned image of a Coomassie blue-stained gel before autoradiography (L3). The bottom 2 panels are an autoradiogram and gel of PKCε phosphorylation of the large intracellular loop of the GABAA γ2 subunit (p-γ2s), a known PKCε substrate (9). (B) An autoradiogram and Western blot with anti-6xHis antibody, showing that PKCε phosphorylation of the NaV1.8 L3 loop was substantially reduced by alanine substitution at S1452 but not at T1437.…”

“…In this study, we used a chemical genetics approach (9) to specifically detect direct protein substrates of PKCε in dorsal root ganglion (DRG) cells and found that the tetrodotoxin-resistant (TTX-R) sodium channel Na V 1.8 is a PKCε substrate. Na V 1.8 channels are selectively expressed in peripheral sensory neurons of neonatal and adult DRG and trigeminal ganglia (10)(11)(12).…”

Neurogenetic studies of alcohol addiction

“…The authors concluded that PKC epsilon is necessary for ethanol's modulatory effects on GABA A receptors (Besheer et al 2006). Because no drug was available that selectively inhibited PKC epsilon activity over other PKC isoforms, the authors used a chemical strategy to generate an ATP analogue-sensitive PKC epsilon mutant to selectively inhibit catalytic activity of PKC epsilon (Qi et al 2007). Using this mutant, they showed that PKC epsilon affects GABA A receptors by phosphorylating the g2 subunit, thereby affecting receptors with the a1b2g2 subunit composition, an abundant variant in brain.…”

Synaptic Effects Induced by Alcohol

“…Messing and co-workers have shown that activity of the epsilon subunit of protein kinase C (PKC) is necessary for EtOH potentiation of γ2-subunitcontaining GABA A receptors expressed heterologously in a mammalian cell line (Qi et al 2007). This PKC action appears to involve phosphorylation of a specific serine residue on the γ2 subunit.…”