title: Modulation of neurosteroid potentiation by protein kinases at synaptic- and extrasynaptic-type GABAA receptors.
abstract: GABAA receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology. For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3(S408A,S409A)γ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3(S408A,S409A)δ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4(S443A)β3(S408A,S409A)δ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3(S408,S409) implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors. Overall, these findings provide important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously tuned by two endogenous neuromodulators.
authors.affiliation: Department of Neuroscience, Physiology & Pharmacology, UCL, Gower Street, London WC1E 6BT, UK.
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Adams
authors.affiliation: Department of Neuroscience, Physiology & Pharmacology, UCL, Gower Street, London WC1E 6BT, UK.
authors.forename: Philip
authors.initials: P
authors.lastname: Thomas
authors.affiliation: Department of Neuroscience, Physiology & Pharmacology, UCL, Gower Street, London WC1E 6BT, UK. Electronic address: t.smart@ucl.ac.uk.
authors.forename: Trevor G
authors.initials: TG
authors.lastname: Smart
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003900:Desoxycorticosterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013569:Synapses / Q000187:drug effects* / Q000502:physiology; D014162:Transfection
chemical_list: D018377:Neurotransmitter Agents; D047428:Protein Kinase Inhibitors; D011963:Receptors, GABA-A; D003900:Desoxycorticosterone; C009413:tetrahydrodeoxycorticosterone; D011494:Protein Kinases; D019311:Staurosporine
keywords: Extrasynaptic GABA(A) receptors; Neurosteroid; Phosphorylation; Protein kinase; Synaptic GABA(A) receptors