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A photoreactive analog of allopregnanolone enables identification of steroid-binding sites in a nicotinic acetylcholine receptor
journal contributionposted on 2022-06-08, 16:01 authored by Zhiyi Yu, David C Chiara, Pavel Y Savechenkov, Karol BruzikKarol Bruzik, Jonathan B Cohen
Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABAA receptors (GABAAR) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABAAR-positive allosteric modulators (PAMs), whereas alphaxalone and most neuroactive steroids are nAChR inhibitors. In this report, we used 11β-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F4N3Bzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABAAR PAM, to characterize steroid-binding sites in the Torpedo α2βγδ nAChR in its native membrane environment. We found that F4N3Bzoxy-AP (IC50 = 31 μm) is 7-fold more potent than alphaxalone in inhibiting binding of the channel blocker [3H]tenocyclidine to nAChRs in the desensitized state. At 300 μm, neither steroid inhibited binding of [3H]tetracaine, a closed-state selective channel blocker, or of [3H]acetylcholine. Photolabeling identified three distinct [3H]F4N3Bzoxy-AP-binding sites in the nAChR transmembrane domain: 1) in the ion channel, identified by photolabeling in the M2 helices of βVal-261 and δVal-269 (position M2-13'); 2) at the interface between the αM1 and αM4 helices, identified by photolabeling in αM1 (αCys-222/αLeu-223); and 3) at the lipid-protein interface involving γTrp-453 (M4), a residue photolabeled by small lipophilic probes and promegestone, a steroid nAChR antagonist. Photolabeling in the ion channel and αM1 was higher in the nAChR-desensitized state than in the resting state and inhibitable by promegestone. These results directly indicate a steroid-binding site in the nAChR ion channel and identify additional steroid-binding sites also occupied by other lipophilic nAChR antagonists.
General Anesthetic Sites On Ligand-Gates Ion Channels | Funder: Massachusetts General Hospital | Grant ID: P01GM058448
CitationYu, Z., Chiara, D. C., Savechenkov, P. Y., Bruzik, K. S.Cohen, J. B. (2019). A photoreactive analog of allopregnanolone enables identification of steroid-binding sites in a nicotinic acetylcholine receptor. Journal of Biological Chemistry, 294(19), 7892-7903. https://doi.org/10.1074/jbc.ra118.007172
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Neurosciences1.1 Normal biological development and functioningnicotinic acetylcholine receptors (nAChR)GABA receptoranestheticsteroidion channellipid-protein interactionligand-gated ion channelneurosteroid allopregnanoloneneurotransmitter receptorperfluorophenyl azidephotolabelinglipid–protein interactionAnimalsBinding SitesFish ProteinsMolecular Docking SimulationPregnanoloneReceptors, NicotinicSteroidsTetracaineTorpedoBiochemistry & Molecular BiologyChemical SciencesBiological SciencesMedical and Health Sciences