Multiple functional neurosteroid binding sites on GABAA receptors.
Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the...
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2019-03-01
|
| Series: | PLoS Biology |
| Online Access: | https://doi.org/10.1371/journal.pbio.3000157 |
| _version_ | 1819110318247247872 |
|---|---|
| author | Zi-Wei Chen John R Bracamontes Melissa M Budelier Allison L Germann Daniel J Shin Krishnan Kathiresan Ming-Xing Qian Brad Manion Wayland W L Cheng David E Reichert Gustav Akk Douglas F Covey Alex S Evers |
| author_facet | Zi-Wei Chen John R Bracamontes Melissa M Budelier Allison L Germann Daniel J Shin Krishnan Kathiresan Ming-Xing Qian Brad Manion Wayland W L Cheng David E Reichert Gustav Akk Douglas F Covey Alex S Evers |
| author_sort | Zi-Wei Chen |
| collection | DOAJ |
| description | Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action. |
| first_indexed | 2024-12-22T03:39:49Z |
| format | Article |
| id | doaj.art-a27cab63789b480aa2cf6c0a29f66e70 |
| institution | Directory Open Access Journal |
| issn | 1544-9173 1545-7885 |
| language | English |
| last_indexed | 2024-12-22T03:39:49Z |
| publishDate | 2019-03-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Biology |
| spelling | doaj.art-a27cab63789b480aa2cf6c0a29f66e702022-12-21T18:40:17ZengPublic Library of Science (PLoS)PLoS Biology1544-91731545-78852019-03-01173e300015710.1371/journal.pbio.3000157Multiple functional neurosteroid binding sites on GABAA receptors.Zi-Wei ChenJohn R BracamontesMelissa M BudelierAllison L GermannDaniel J ShinKrishnan KathiresanMing-Xing QianBrad ManionWayland W L ChengDavid E ReichertGustav AkkDouglas F CoveyAlex S EversNeurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action.https://doi.org/10.1371/journal.pbio.3000157 |
| spellingShingle | Zi-Wei Chen John R Bracamontes Melissa M Budelier Allison L Germann Daniel J Shin Krishnan Kathiresan Ming-Xing Qian Brad Manion Wayland W L Cheng David E Reichert Gustav Akk Douglas F Covey Alex S Evers Multiple functional neurosteroid binding sites on GABAA receptors. PLoS Biology |
| title | Multiple functional neurosteroid binding sites on GABAA receptors. |
| title_full | Multiple functional neurosteroid binding sites on GABAA receptors. |
| title_fullStr | Multiple functional neurosteroid binding sites on GABAA receptors. |
| title_full_unstemmed | Multiple functional neurosteroid binding sites on GABAA receptors. |
| title_short | Multiple functional neurosteroid binding sites on GABAA receptors. |
| title_sort | multiple functional neurosteroid binding sites on gabaa receptors |
| url | https://doi.org/10.1371/journal.pbio.3000157 |
| work_keys_str_mv | AT ziweichen multiplefunctionalneurosteroidbindingsitesongabaareceptors AT johnrbracamontes multiplefunctionalneurosteroidbindingsitesongabaareceptors AT melissambudelier multiplefunctionalneurosteroidbindingsitesongabaareceptors AT allisonlgermann multiplefunctionalneurosteroidbindingsitesongabaareceptors AT danieljshin multiplefunctionalneurosteroidbindingsitesongabaareceptors AT krishnankathiresan multiplefunctionalneurosteroidbindingsitesongabaareceptors AT mingxingqian multiplefunctionalneurosteroidbindingsitesongabaareceptors AT bradmanion multiplefunctionalneurosteroidbindingsitesongabaareceptors AT waylandwlcheng multiplefunctionalneurosteroidbindingsitesongabaareceptors AT davidereichert multiplefunctionalneurosteroidbindingsitesongabaareceptors AT gustavakk multiplefunctionalneurosteroidbindingsitesongabaareceptors AT douglasfcovey multiplefunctionalneurosteroidbindingsitesongabaareceptors AT alexsevers multiplefunctionalneurosteroidbindingsitesongabaareceptors |