Role of serotonergic signaling in GABAA receptor phosphorylation and functional expression.
Doctoral thesis, UCL (University College London).
γ-aminobutyric acid type-A (GABAA) receptors are heteropentameric ligand-gated chloride channels that mediate the majority of fast synaptic inhibition in the brain. Emerging evidence indicates that their functional expression is subject to dynamic modulation by phosphorylation. However, the cell signaling molecules responsible for regulating GABAA receptor phosphorylation and thus the efficacy of neuronal inhibition remain to be identified. The β subunits are of particular interest in this context as their intracellular domains contain conserved serine residues (S409 in β1, S410 in β2 and S408/9 in β3), known substrates for a number of protein kinases, including PKA and PKC. In vitro binding experiments showed that phosphorylation and/or mutation of these residues confers a reduction in binding of GABAA receptor β subunits to the μ2 adaptin of the clathrin adaptor protein (AP)-2 complex - a critical regulator of GABAA receptor endocytosis and surface number. Consistent with this, coimmunoprecipitation of AP2-μ2 adaptin with endogenous GABAA receptor β3 subunits was significantly reduced in cultured neurons treated with a potent inhibitor of S408/9 dephosphorylation that was accompanied by an increase in the stability of GABAA receptor β3 subunits at the cell surface. Interestingly, recent studies have implicated PKA and PKC in the mediation of serotonergic modulation of GABAA receptor activity in the prefrontal cortex, suggesting that phosphorylation of GABAA receptor β subunits may underlie this regulation. To address this, a phospho-specific antibody directed against β3 at S408/9 was developed. Immunoblotting with anti-pS408/9-β3 demonstrated a PKC-dependent increase in the phosphorylation state of GABAA receptor β3 subunits following enhanced 5-hydroxytryptamine type-2 (5-HT2) receptor activation ex vivo. Moreover, in vivo biochemical and immunohistochemical studies revealed region-specific increases in GABAA receptor β3 subunit phosphorylation in mice dosed with the selective serotonin reuptake inhibitor (SSRI) fluoxetine (Prozac™), a commonly prescribed antidepressant. Together, the results presented herein suggest that the phospho-dependent increase in GABAA receptor functional expression may underlie the therapeutic action of SSRIs.
|Title:||Role of serotonergic signaling in GABAA receptor phosphorylation and functional expression|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Neuroscience, Physiology and Pharmacology|
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