Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABA _A receptors

Abstract

Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABA A Rs). Although neurosteroids are accepted allosteric modulators of GABA A Rs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABA A Rs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABA A R subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABA A R subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABA A Rs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.PKC | tonic current | receptor insertion | current rundown N eurosteroids are synthesized de novo in the brain from cholesterol, or steroid hormone precursors. Raising neurosteroid levels in the CNS causes anxiolysis, sedation/hypnosis, anticonvulsant action, and anesthesia and reduces depressivelike behaviors (1-3). Accordingly, dysregulation of neurosteroid signaling is associated with premenstrual dysphoric disorder, panic disorder, depression, schizophrenia, and bipolar disorder. Neurosteroids exert the majority of their actions via potentiating the activity of γ-aminobutyric acid receptors (GABA A Rs), which mediate the majority of fast synaptic inhibition in the adult brain. Accordingly, at low nanomolar concentrations they potentiate GABA-dependent currents, whereas at micromolar concentrations they directly activate GABA A Rs (4-8).GABA A Rs are Cl − -preferring pentameric ligand-gated ion channels that assemble from eight families of subunits: α(1-6), β(1-3), γ(1-3), δ, e, ө, π, and ρ(1-3) (9, 10). Receptor subtypes composed of α1-3βγ subunits largely mediate synaptic or phasic inhibition, whereas those constructed from α4-6β1-3, with or without γ/δ subunits, are principal determinants of tonic inhibition (11-13). Neurosteroids have been shown to bind GABA A Rs at an allosteric site distinct from that of GABA, benzodiazepines, or barbiturates (9, 14). Hosie et al. identified residues located within the transmembrane domain of GABA A R α and β subunits that are critical for the direct activation (α1-6; Threonine 236, β1-3; Tyrosine 284) and allosteric potentiation (α1-6 Asparagine 407, and α1-6 Glutamine 246) of neurosteroids (15-17). Accordingly, mutation of glutamine 241 (Q241) within the α1-6 subunits prevents allosteric potentiation of GABA A R composed of αβγ and αβδ subunits by neurosteroids (15,16).In addition to modulating channel gating, neurosteroids exert...

Diabetes induces GABA receptor plasticity in murine vagal motor neurons

“…It is possible that GABA release or reuptake are altered, but no change was observed in phasic current frequency or resting tonic current, suggesting that ambient GABA concentrations are not different. Rather, functional regulation of ␦-subunits often results from posttranslational modification (Abramian et al 2014;Goodkin et al 2008). A serine residue in the ␣ 4 -subunit serves as a phosphorylation site on the ␦-containing receptor (Abramian et al 2010;Joshi et al 2013), which mediates trafficking of the receptor in the membrane.…”

“…Immunohistochemically labeled ␦-subunits associated with the membrane and increased functional expression appear as clustered or enlarged puncta on or close to the surface of the neuron (Abramian et al 2014;Mangan et al 2005). Regardless of treatment, ␦-containing receptor labeling in the DMV was less robust than in the cerebellum, which has high ␦-subunit expression.…”

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).…”

“…Although GX does not have a metabotropic effect on GABA-A receptor phosphorylation and trafficking, inhibition of PKC still dampens the potentiation of tonic inhibition by neurosteroids. This is likely because sustained application of the PKC inhibitor causes decreased phosphorylation and subsequent internalization of receptors, and therefore, reduced tonic current potentiation (Comenencia-Ortiz et al, 2014).…”

“…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.…”