Adams, J.M.; (2010) Co-regulation of GABAA receptors by neurosteroids and protein kinases. Doctoral thesis , UCL (University College London).

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Abstract

γ-aminobutyric acid type-A (GABAA) receptors mediate inhibitory synaptic transmission in the CNS where neurosteroids and protein kinases are their most potent endogenous modulators. Acting individually, these can either enhance or depress receptor function depending on the type of neurosteroid or kinase present, and the subunit combination of the receptor. However, in vivo, these agents probably act in concert to modulate GABAA receptors and precisely ‘fine-tune’ inhibitory neurotransmission; although how this is achieved remains unclear. Therefore, the relationship between these two modulators, at α1β3γ2L and α4β3δ GABAA receptors, expressed in HEK293 cells, was investigated using whole-cell patch clamp electrophysiology. At α1β3γ2L receptors, the potentiation of GABA responses by tetrahydrodeoxycorticosterone (THDOC) was reduced by PKC inhibition and enhanced by PKC activation, implying a role for this kinase in regulating neurosteroid potentiation. By comparison, neurosteroid potentiation was reduced at α1β3S408A,S409Aγ2L receptors, and was unaltered by PKC inhibitors or activators indicating that phosphorylation of the β3 subunit, by PKC, is important for regulating neurosteroid activity. To determine whether ‘extrasynaptic-type’ GABAA receptors are modulated similarly, experiments were also undertaken with α4β3δ and α4β3S408A,S409Aδ receptors. Neurosteroid potentiation was significantly reduced at both receptor subtypes after treatment with the protein kinase inhibitor, staurosporine. Staurosporine was notably less effective at α4β3S408A,S409Aδ receptors, suggesting that, although β3 subunit phosphorylation may play a role in the regulation of neurosteroid potentiation at α4β3δ receptors, it does not fully account for this modulation. Biochemical experiments on α4 subunits identified a new Ser/Thr phosphorylation site (S443). THDOC-mediated potentiation at α4S443Aβ3S408A,S409Aδ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of both the α4 and β3 subunits is required for the regulation of neurosteroid activity at α4β3δ receptors. Furthermore, Western blot analysis for wild-type α1β3γ2L receptors, revealed that THDOC increased phosphorylation of β3S408,S409 implying a ‘reverse’ pathway exists for neurosteroids to modulate the phosphorylation state of the GABAA receptor. Overall, these findings provide an important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which fine-tuning of GABAergic inhibitory transmission may be achieved by two endogenous neuromodulators.

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