Neuroactive steroids (NASs) such as allopregnanolone (ALLO) play a central role in regulating behavior via their potent anxiolytic, anticonvulsant, sedative, and hypnotic actions. Accordingly, modifications in the levels of NASs contribute to anxiety, autism spectrum disorders, depression, epilepsy, and premenstrual syndrome. Classically, NASs are thought to act by rapidly boosting neuronal inhibition by positive allosteric modulation of the activity of ?-aminobutyric acid type A receptors (GABAARs). In addition to their allosteric actions, we have recently shown that NASs act via a protein kinase C-dependent mechanism to enhance the phosphorylation of residues including Serine?s 408 and 409 in the ?3 subunit (S408/9), a process that increases GABAAR number on the plasma membrane leading to a sustained increase in the efficacy of GABAergic inhibition. Although we have shown that NASs do not directly activate PKC, the mechanism by which NASs lead to changes in phosphorylation of GABAAR subunits are unknown. It is emerging that in addition to their positive allosteric modulation of GABAARs, NASs can directly activate membrane progesterone receptors (mPRs); G-protein coupled receptors that regulate PKC signaling. However, no information is available on the role that mPRs play in regulating GABAAR activity. Likewise, the behavioral significance of the sustained mPR-mediated metabotropic actions of NASs remains unexplored. To address these issues we have created mice in which S408/9 in the ?3 subunit have been mutated to alanines, mutations that are predicted to reduce the metabotropic actions of NASs on GABAAR function. Preliminary studies using these tools have allowed us to formulate a central hypothesis that will be tested here; NASs activate mPRs to enhance the phosphorylation of GABAARs on residues including S408/9 in the ?3 subunit, a mechanism that underlies their anticonvulsant efficacy. In contrast, their anxiolytic efficacy is mediated via allosteric potentiation of GABAAR activity, a process dependent upon Q241 in the ?2 subunit. Our experiments will focus on the following aims.
Aim 1. To test the hypothesis that the ability of NAS to induce sustained effects on GABAergic inhibition is dependent upon S408/9A in the ?3 subunit.
Aim 2. To test the hypothesis that the anxiolytic, and anticonvulsant efficacy of NASs is dependent upon S408/9 in the ?3 subunit.
Aim 3. To test the hypothesis that NASs mediate their metabotropic effects on GABAARs via the activation of mPRs. Collectively, our proposal will identify the molecular mechanisms by which NAS exert their therapeutic actions. This information may aid the development of new therapeutic strategies to alleviate the burdens of anxiety, autism spectrum disorders, depression, epilepsy, and premenstrual syndrome.
Neuroactive steroids (NASs) play a central role in regulating behavior via their potent anxiolytic, antidepressant, and anticonvulsant actions. We have recently shown that NASs also act via a protein kinase C-dependent mechanism to enhance the phosphorylation of residues in the ?3 subunit (S408/9) of GABAARs, a process that increases the membrane insertion of GABAARs and a sustained increase in the efficacy of GABAergic inhibition. We will demonstrate that NASs can achieve this sustained effect by directly activating membrane progesterone receptors (mPRs); G-protein coupled receptors that regulate PKC signaling. Therefore, this proposal may aid the development of more efficacious therapies to alleviate the burdens of anxiety, depression, and epilepsy through a mechanism that boosts trafficking of GABAARs to limit neuronal excitability.
