A photoreactive analog of allopregnanolone enables identification of steroid-binding sites in a nicotinic acetylcholine receptor

Abstract

Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABA_A receptors (GABA_AR) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABA_AR-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 GABA_AR 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.

Keywords: GABA receptor; anesthetic; ion channel; ligand-gated ion channel; lipid–protein interaction; neurosteroid allopregnanolone; neurotransmitter receptor; nicotinic acetylcholine receptors (nAChR); perfluorophenyl azide; photolabeling; steroid.

Figure 1.

Chemical structures of F₄N₃Bzoxy-AP, alphaxalone, and promegestone.

Figure 2.

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 (+α-bungarotoxin) (B). Binding was determined by centrifugation at 4 °C. Individual experiments were performed in duplicate, and the data were normalized to the specific binding in the absence of competitor. Pooled data (mean ± S.D.) are plotted for each ligand ([³H]ACh, n = 2; [³H]TCP and [³H]tetracaine, n = 3). For [³H]ACh, total and nonspecific bindings (+100 μm Carb) were 2529 ± 63 cpm and 93 ± 17 cpm, respectively. For [³H]TCP, the total and nonspecific (+ 100 μm PCP) bindings were 4798 ± 55 and 1451 ± 24 cpm, respectively. F₄N₃Bzoxy-AP (●) and alphaxalone (○) inhibited [³H]TCP binding with IC₅₀ values of 31 ± 4 and 209 ± 12 μm, respectively. For [³H]tetracaine, the total and nonspecific (+100 μm tetracaine) bindings were 2204 ± 242 and 941 ± 45 cpm, respectively. At 300 μm, F₄N₃Bzoxy-AP (▴) and alphaxalone (Δ) inhibited [³H]tetracaine binding by <5%.

Figure 3.

[³H]F₄N₃Bzoxy-AP photoincorporation into Torpedo nAChR–rich membranes. Membrane suspensions equilibrated with [³H]F₄N₃Bzoxy-AP (3 μm) were irradiated at 365 nm for 30 min or 254 nm for 2 min in the absence or presence of different cholinergic ligands, and triplicate samples were fractionated by SDS-PAGE. After staining for protein, one gel was prepared for fluorography, and subunit gel bands were excised from the second for ³H determination by liquid scintillation counting. A, representative Coomassie Blue–stained gel lane (lane 0) and a fluorogram of [³H]F₄N₃Bzoxy-AP photoincorporation into nAChR membranes (left, 365 nm; right, 254 nm). Lanes 1/6, no drug (control); lanes 2/7, 1 mm Carb; lanes 3/8, 1 mm Carb and 100 μm PCP; lanes 4/9, 1 mm Carb and 300 μm alphaxalone; lanes 5/10, 1 mm Carb and 100 μm R-mTFD-MPAB. B–D, ³H incorporation into nAChR subunit gel bands after irradiation at 365 nm (B) or 254 nm (C) from the same experiment as the fluorogram and from an independent experiment at 254 nm (D). The average cpm ± S.D. are plotted for samples from two gels. Included in C and D are the p values, where statistically significant (p < 0.05, one-way ANOVA and Tukey's multiple comparison test for pairs of labeling conditions (GraphPad Prism 7)). A, left, electrophoretic mobilities are indicated of the nAChR α, β, γ, and δ subunits, rapsyn (Rsn), the Na⁺/K⁺-ATPase α subunit (α_Na/K), and the mitochondrial voltage-dependent anion channel (34 kDa) and ADP/ATP carrier (32 kDa).

Figure 4.

State-dependent [³H]F₄N₃Bzoxy-AP photolabeling of ion channel residues in βM2 (βVal-261) and δM2 (δVal-269). Subunit fragments beginning at the N termini of βM2 and δM2 were isolated for sequence analysis by Tricine SDS-PAGE and rpHPLC from trypsin and EndoLys-C digests of β and δ subunits, respectively, isolated from nAChRs photolabeled at 365 nm in the absence and presence of Carb. A and C, ³H elution profiles (○, control; ●, 1 mm Carb) during rpHPLC purification of β and δ subunit fragments of ∼10 and ∼14 kDa isolated by Tricine SDS-PAGE. B and D, ³H (○, control; ●, Carb) and PTH-derivative (▵, control; ▴, Carb) released during sequence analysis of fragments beginning at βMet-249 (B, ▵,13 pmol; ▴, 19 pmol) and δMet-257 (D, ▵, 23 pmol; ▴, 32 pmol) from rpHPLC fractions 30–32 (A) and 27–29 (C), respectively. The peaks of ³H release in cycle 13 indicated photolabeling of βM2–13′ (βVal-261) and δM2–13′ (δVal-269) at >5-fold higher efficiency in the presence of Carb than in the absence (Table 1).

Figure 5.

Promegestone inhibition of [³H]F₄N₃Bzoxy-AP photolabeling βM2 and δM2 ion channel residues. ³H (●; +Carb; ♦, +Carb + promegestone) and PTH-derivatives (▴, +Carb; ♢, +Carb + promegestone) released during sequence analysis of fragments beginning at βMet-249 (▴, 25 pmol; ♢, 17 pmol) (A) and δMet-257 (310 pmol, both conditions) isolated from nAChR-rich membranes irradiated at 254 nm in the presence of Carb in the absence or presence of 100 μm promegestone (B). The peaks of ³H release in cycle 13 indicated [³H]F₄N₃Bzoxy-AP photolabeling of βM2–13′ (βVal-261) and δM2–13′ (δVal-269) that was reduced in the presence of promegestone. Based upon sequencing data from two independent photolabeling experiments, promegestone reduced photolabeling efficiency (cpm/pmol) of βVal-261 by 38 ± 18% (five paired samples) and of δVal-269 by 53 ± 24% (three paired samples). B, peaks of release in cycles 16 and 20 indicate photolabeling of the channel-lining residues δM2–16′ (δLeu-272) and δM2–20′ (δGln-276) that was reduced in the presence of promegestone.

Figure 6.

State-dependent [³H]F₄N₃Bzoxy-AP photolabeling in αM1. A–C, α subunit fragments beginning at αHis-186, which extends through αM1, and at αMet-243, the N terminus of αM2, were isolated by rpHPLC from an EndoLys-C digest of αV8–20, isolated by V8 protease in gel digestion of α subunits from nAChRs photolabeled at 254 nm in the presence of Carb. A, ³H elution profile for the rpHPLC fractionation of the EndoLys-C digest. B and C, ³H and PTH-derivatives released while sequencing the fragments beginning at αHis-186 (B) and αMet-243 (C) from rpHPLC fractions 26–28 and 30–32, respectively. D–F, ³H (○, control; ●, Carb; ♦, Carb/promegestone) and PTH-derivatives (▵, control; ▴, Carb; ♢, Carb/promegestone) released while sequencing fragments begin at αIle-210, the N terminus of αM1 (D and F), or at αGln-208 (E). The fragment beginning at αIle-210 was isolated by rpHPLC from trypsin digests, with the sequencing filters treated at cycle 2 with o-phthalaldehyde to prevent further sequencing of peptides not containing a proline at that cycle. The fragment beginning at αGln-208 was isolated for sequencing by first isolating by rpHPLC from an EndoLys-C digest at the αHis-186 fragment, which was sequenced for 18 cycles. The sequencing filter was then treated with cyanogen bromide to cleave at the carboxyl side αMet-207. The peaks of ³H release in cycles 13 and 14 (D and F) or 15 and 16 (E) indicate photolabeling of αCys-222 and αLeu-223 that was enhanced in the presence of Carb compared with control, and promegestone reduced the Carb-enhanced labeling.

Figure 7.

[³H]F₄N₃BzoxyAP photolabeling in αM4 and γM4. ³H (○, control; ●, Carb) and PTH-derivatives (▵, control; ▴, Carb) were released during sequence analysis of subunit fragments beginning at αTyr-401 (A and B) and γAla-450 (C), the N termini of αM4 and γM4, which were isolated by SDS-PAGE and rpHPLC from nAChRs photolabeled at 254 nm (A) or 365 nm (B and C). Gel bands of ∼10 kDa (αV8–10, beginning at αGlu-338/αAsn-339) and 14 kDa (γV8–14, containing fragments beginning at γLeu-373/γIle-413) were isolated by in gel digestion of the subunits with V8 protease (42), and then trypsin and EndoLys-C digests of αV8–10 and γV8–14, respectively, were fractionated by rpHPLC. A and B, no release of ³H above background was detected while sequencing fragments beginning at αTyr-401, the N terminus of αM4, isolated from nAChRs photolabeled in the presence of Carb at 254 nm (A) or 365 nm (B). Based upon the levels of background ³H release, labeling of residues in αM4, if it occurred, was at <0.3 cpm/pmol, i.e. at <10% the efficiency of labeling of αCys-222 or βVal-261 at 254 nm (Table 1). C, the peak of ³H release in cycle 4 indicated photolabeling of γTrp-453 at 4- and 20-fold higher efficiency than the photolabeling in the ion channel of the M2–13′ residues βVal-261 and δVal-269, respectively (Table 1).

Figure 8.

F₄N₃Bzoxy-AP–binding sites in the Torpedo nAChR. A, T. californica nAChR homology model was constructed based on the T. marmorata nAChR structure (PDB code 2BG9 (45)), with modifications to correct for the previously identified error in assignment of amino acids in the M2 and M3 helices (see under “Computational docking”). A, side view of the nAChR extracellular and transmembrane domains (α, yellow; β, brown; γ, green; δ, cyan). B, view of the nAChR TMD from the base of the extracellular domain. C, side view from the lipid of the γ-α subunit interface. D, side view of the ion channel with an α subunit omitted for visualization of photolabeled residues. The amino acids photolabeled by [³H]F₄N₃Bzoxy-AP are shown in stick representation in the ion channel (B and D, magenta), in αM1 (B and C, αCys-222/αLeu-223 (red)), and in γM4 (B and C, γTrp-453, black)). Also included in stick representation in C are: (i) the amino acids in αM4 and γM4 (purple) photolabeled by [³H]promegestone, which also photolabels γTrp-453 (26), and by [¹²⁵I]TID, which also labels αThr-422 and αVal-425 (orange) but not γTrp-453 or αHis-408 (42); (ii) αVal-218 (green) in αM1, photolabeled by a convulsant barbiturate [³H]S-mTFD-MPPB (43); (iii) the amino acids (cyan) in αM1 (αLeu-231) and γM3 (γMet-299) photolabeled by the anesthetic barbiturate [³H]R-mTFD-MPAB (36); and (iv) the amino acids (orange) in αM4, αM1 (αPhe-227/αLeu-228), and γM3 (γPhe-292, γLeu-296, and γAsn-300) photolabeled by [¹²⁵I]TID, which also photolabels αCys-222/αLeu-223 (42). C and D, locations of F₄N₃Bzoxy-AP (molecular volume = 448 ų) docked in the binding sites are shown as Connolly surface representations of the volumes defined by the 10 most energetically favorable binding poses (ion channel, volume = 942 ų and αM1/αM4, volume = 596 ų).