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Modulation of the human ?1 GABAA receptor by inhibitory steroids.

ABSTRACT: Modulators of the ?1 GABAA receptor may be useful in the treatment of visual, sleep, and cognitive disorders. Neuroactive steroids and analogues have been shown to modulate ?1 receptor function, but the molecular mechanisms are poorly understood.We employed electrophysiology and voltage-clamp fluorometry to compare the actions of several neuroactive steroids and analogues on the human ?1 GABAA receptor.Results confirmed that P294S and T298F mutations affect modulation by steroids. The P294S mutation abolished inhibition by (3?,5?)-3-hydroxypregnan-20-one (3?5?P) while the T298F mutation eliminated inhibition by 17?-estradiol. Voltage-clamp fluorometry demonstrated that steroids differing in the presence of a charged group on C3 or nature of substituent on C17 uniquely modified fluorescence changes elicited by GABA in the extracellular domain. The I307Q mutation reversed the inhibitory effect of 3?5?P but was without effect on modulation by (3?,5?)-3-hydroxypregnan-20-one sulfate or 17?-estradiol. The effect of 3?5?P on the fluorescence change generated at Y241C was dependent on whether the steroid acted as an inhibitor or a potentiator. Further, the effect was limited to uncharged 5?-reduced steroids containing an acetyl group on C17.The data demonstrate that steroids and analogues differ with respect to conformational changes elicited by these drugs as well as sensitivity to the effects of mutations. Steroids and analogues could be provisionally divided into three major groups based on their actions on the ?1 GABAA receptor: 5?-reduced uncharged steroids, sulfated and carboxylated steroids, and 17?-estradiol. Further division among 5?-reduced uncharged steroids was based on substituent at position C17.

SUBMITTER: Eaton MM

PROVIDER: S-EPMC4048647 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Modulation of the human ρ1 GABAA receptor by inhibitory steroids.

Eaton Megan M MM Lim You Bin YB Covey Douglas F DF Akk Gustav G

Psychopharmacology 20131207 17

<h4>Rationale</h4>Modulators of the ρ1 GABAA receptor may be useful in the treatment of visual, sleep, and cognitive disorders. Neuroactive steroids and analogues have been shown to modulate ρ1 receptor function, but the molecular mechanisms are poorly understood.<h4>Objectives</h4>We employed electrophysiology and voltage-clamp fluorometry to compare the actions of several neuroactive steroids and analogues on the human ρ1 GABAA receptor.<h4>Results</h4>Results confirmed that P294S and T298F mu ...[more]

PMID: 24317445

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Project description:BACKGROUND: The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity. METHODOLOGY/PRINCIPAL FINDINGS: Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GR-DNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation. CONCLUSIONS/SIGNIFICANCE: The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior.

Dataset Information

Modulation of the human ?1 GABAA receptor by inhibitory steroids.

Publications

Modulation of the human ρ1 GABAA receptor by inhibitory steroids.

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