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...

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Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

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