Abstract: Gamma-aminobutyric acid type A receptors (GABAARs) are the principal mediators of inhibitory transmission in the mammalian central nervous system. GABAARs can be localized at postsynaptic inhibitory specializations or at extrasynaptic sites. While synaptic GABAARs are activated transiently following the release of GABA from presynaptic vesicles, extrasynaptic GABAARs are activated contin... read moreuously by resting GABA concentrations and thus mediate tonic inhibition. In the hippocampus and thalamus, extrasynaptic GABAARs are predominantly composed of alpha4 beta2/3 and delta subunits. The tonic inhibitory currents mediated by extrasynaptic GABAARs control neuronal excitability and the strength of synaptic transmission. However, the mechanisms by which neurons control the functional properties of extrasynaptic GABAARs had not yet been explored. Phosphorylation of residues within synaptic GABAAR subunits is critical for the assembly, trafficking and cell surface stability of synaptic GABAAR subtypes. Here we have identified serines 443 in the alpha4 and serines 408/409 in the beta3 subunit as the principal sites for PKC phosphorylation in extrasynaptic GABAAR subtypes. Steroid metabolites of progesterone and deoxycortisone (known as neurosteroids) have been shown to be potent positive allosteric modulators of extrasynaptic GABAARs. However, the exact mechanisms by which neurosteroids alter extrasynaptic GABAARs function are not well understood. Previous experiments have suggested that Protein Kinase C (PKC) activity is required for neurosteroid-mediated modulation of GABAARs. We show that the deoxycortisone metabolite, Tetrahydrodeoxycorticosterone (THDOC) induces the phosphorylation of the alpha4 GABAAR subunit on serine 443 (S443) dependent upon the ability of this agent to activate PKC. We analyzed the functional significance of THDOC induced phosphorylation of the alpha4 subunit using patch-clamp recording and TIRF microscopy. Collectively these approaches revealed that THDOC increases the cell surface stability of alpha4-containing GABAARs by promoting their insertion into the plasma membrane, a phenomenon critically dependent on S443 in the alpha4 subunit and on S408/409 within the beta3 subunit when co-expressed. Significantly, the ability of THDOC to increase the expression levels of GABAARs was independent of the delta subunit and glutamine 246 (Q246) in the alpha4 subunit, a site critical for allosteric neurosteroid potentiation. Collectively we have identified a novel mechanism by which neurosteroids can induce long-term changes in the expression levels of GABAARs via PKC dependent phosphorylation of the alpha4 subunit.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Neuroscience.
Advisor: Stephen Moss.
Committee: Jamie Maguire, Maribel Rios, Kathleen Dunlap, and Shelley Russek.