Abstract
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.
Highlights
Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABAA receptors (GABAAR) and nicotinic acetylcholine receptors
Recent crystal structures of chimeric homopentameric receptors containing GABAAR ␣ subunit transmembrane domain (TMD) identify a binding site for steroid PAMs in the TMD at subunit interfaces [11, 12], which is distinct from the intersubunit sites in the extracellular third of the TMD for etomidate, propofol, and barbiturates identified in heteromeric GABAARs by photolabeling and mutational analyses [13, 14]
For muscle-type nicotinic acetylcholine receptors (nAChRs), single-channel analyses of the acute effects of natural and synthetic steroids indicate a reduction of open channel lifetime [15,16,17], but it is not known whether this results from steroid binding within the ion channel or indirectly as a consequence of interactions at the lipid interface
Summary
For muscle-type nAChRs, single-channel analyses of the acute effects of natural and synthetic steroids indicate a reduction of open channel lifetime [15,16,17], but it is not known whether this results from steroid binding within the ion channel or indirectly as a consequence of interactions at the lipid interface. To further characterize steroid-binding sites in the Torpedo nAChR, we use 11-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F4N3Bzoxy-AP) (Fig. 1), a photoreactive allopregnanolone derivative that is a potent general anesthetic and GABAAR-positive allosteric modulator [27]. In contrast to promegestone, [3H]F4N3Bzoxy-AP photolabeled residues in the nAChR ion channel in the desensitized state Effects of F4N3Bzoxy-AP, alphaxalone, or proadifen on the equilibrium binding to Torpedo nAChR-rich membranes of [3H]ACh (A) or the channel blockers [3H]TCP (؉Carb) and [3H]tetracaine (؉␣-bungarotoxin) (B). Conserved positive charges at the N termini of the M2 helices), which provides a first identification of a steroid-binding site in the nAChR ion channel. [3H]F4N3Bzoxy-AP identified sites accessible from the lipid, one within the ␣ subunit, identified by photolabeling of ␣Cys-222/␣Leu-223 at the interface between the M1 and M4 helices, and a site near the cytoplasmic surface of the TMD, identified by photolabeling of ␥Trp-453 in ␥M4, a residue photolabeled by [3H]promegestone
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