Abstract
Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABA(A) receptors (GABA(A)R) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABA(A)R-positive allosteric modulators (PAMs), whereas alphaxalone and most neuroactive steroids are nAChR inhibitors. In this report, we used 11β-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F(4)N(3)Bzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABA(A)R PAM, to characterize steroid-binding sites in the Torpedo α(2)βγδ nAChR in its native membrane environment. We found that F(4)N(3)Bzoxy-AP (IC(50) = 31 μm) is 7-fold more potent than alphaxalone in inhibiting binding of the channel blocker [(3)H]tenocyclidine to nAChRs in the desensitized state. At 300 μm, neither steroid inhibited binding of [(3)H]tetracaine, a closed-state selective channel blocker, or of [(3)H]acetylcholine. Photolabeling identified three distinct [(3)H]F(4)N(3)Bzoxy-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.