Abstract
Neuroactive steroids (NASs) are synthesized within the brain and exert profound effects on behavior. These effects are primarily believed to arise from the activities of NASs as positive allosteric modulators (PAMs) of the GABA-type A receptor (GABAAR). NASs also activate a family of G protein-coupled receptors known as membrane progesterone receptors (mPRs). Here, using surface-biotinylation assays and electrophysiology techniques, we examined mPRs' role in mediating the effects of NAS on the efficacy of GABAergic inhibition. Selective mPR activation enhanced phosphorylation of Ser-408 and Ser-409 (Ser-408/9) within the GABAAR β3 subunit, which depended on the activity of cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC). mPR activation did not directly modify GABAAR activity and had no acute effects on phasic or tonic inhibition. Instead, mPR activation induced a sustained elevation in tonic current, which was blocked by PKA and PKC inhibition. Substitution of Ser-408/9 to alanine residues also prevented the effects of mPR activation on tonic current. Furthermore, this substitution abolished the effects of sustained NAS exposure on tonic inhibition. Interestingly, the allosteric effects of NAS on GABAergic inhibition were independent of Ser-408/9 in the β3 subunit. Additionally, although allosteric effects of NAS on GABAergic inhibition were sensitive to a recently developed "NAS antagonist," the sustained effects of NAS on tonic inhibition were not. We conclude that metabotropic effects of NAS on GABAergic inhibition are mediated by mPR-dependent modulation of GABAAR phosphorylation. We propose that this mechanism may contribute to the varying behavioral effects of NAS.
Highlights
Neuroactive steroids (NASs) are synthesized within the brain and exert profound effects on behavior
To examine the role that membrane progesterone receptors (mPRs) may play in mediating the effects of neurosteroids on GABAergic inhibition, we exposed hippocampal slices to ORG OD 02-0 (ORG), which selectively activates mPRs but not nuclear progesterone receptors [14]
We examined the effects of ORG on GABA-type A receptor (GABAAR) phosphorylation in the presence of 10 M GF 109203X (GFX), a selective inhibitor of protein kinase C (PKC), and 1 M KT5720 (KT), a selective inhibitor of protein kinase A (PKA), for 10 min
Summary
MPR agonists potentiate GABAAR phosphorylation and plasma membrane accumulation Previous studies have shown that endogenous and selected synthetic neurosteroids, including ALLO, tetrahydrodeoxycorticosterone (THDOC), and SAGE-516, exert sustained effects on GABAergic inhibition. The ratio of pS408/9/3 immunoreactivity was determined and compared between treatments as detailed previously (10, 18 –20) Under these conditions, ORG significantly increased Ser-408/9 phosphorylation to 138.9 Ϯ 20.0% of control (Fig. 1A; p Ͻ 0.05; n ϭ 6 mice). Our surface fractions were free of the cytosolic protein actin, demonstrating the robustness of our surface protein biotinylation procedure These experiments suggest that ORG and P4 mimic the effects of NAS on 3 subunit phosphorylation and induce a selective increase in the plasma membrane accumulation of GABAARs
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