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

  • Zhiyi Yu
    Affiliations
    From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
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  • David C. Chiara
    Affiliations
    From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
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  • Pavel Y. Savechenkov
    Affiliations
    Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612
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  • Karol S. Bruzik
    Affiliations
    Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612
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  • Jonathan B. Cohen
    Correspondence
    To whom correspondence should be addressed:Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.:617-432-1728; Fax:617-432-1639
    Affiliations
    From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
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  • Author Footnotes
    2 The abbreviations used are: PAMpositive allosteric modulatorTMDtransmembrane domainF4N3Bzoxy-AP11β-(p-azidotetrafluorobenzoyloxy)allopregnanoloneTID3-(trifluoromethyl)-3-(m-iodophenyl)diazirineR-mTFD-MPABR-1-methyl-5-allyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acidGABAARγ-aminobutyric acid type A receptornAChRnicotinic acetylcholine receptorCarbcarbamylcholinePCPphencyclidineTCPtenocyclidineAChacetylcholineV8 proteaseS. aureus endopeptidase Glu-CEndoLys-CL. enzymogenes endoproteinase Lys-CTPCKl-1-tosylamido-2-phenylethyl chloromethyl ketoneTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinePTHphenylthiohydantoinrpHPLCreversed-phase high-performance liquid chromatographypLGICpentameric ligand-gated ion channel.
Open AccessPublished:March 28, 2019DOI:https://doi.org/10.1074/jbc.RA118.007172
      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.

      Introduction

      Many steroids, including endogenous 3α-hydroxy metabolites of progesterone and deoxycorticosterone and synthetic analogs, act as potent general anesthetics, sedatives, anxiolytics, or anticonvulsants (
      • Reddy D.S.
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      Clinical potential of neurosteroids for CNS disorders.
      ,
      • Blanco M.J.
      • La D.
      • Coughlin Q.
      • Newman C.A.
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      • Salituro F.G.
      Breakthroughs in neuroactive steroid drug discovery.
      ). They are positive allosteric modulators (PAMs)
      The abbreviations used are: PAM
      positive allosteric modulator
      TMD
      transmembrane domain
      F4N3Bzoxy-AP
      11β-(p-azidotetrafluorobenzoyloxy)allopregnanolone
      TID
      3-(trifluoromethyl)-3-(m-iodophenyl)diazirine
      R-mTFD-MPAB
      R-1-methyl-5-allyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid
      GABAAR
      γ-aminobutyric acid type A receptor
      nAChR
      nicotinic acetylcholine receptor
      Carb
      carbamylcholine
      PCP
      phencyclidine
      TCP
      tenocyclidine
      ACh
      acetylcholine
      V8 protease
      S. aureus endopeptidase Glu-C
      EndoLys-C
      L. enzymogenes endoproteinase Lys-C
      TPCK
      l-1-tosylamido-2-phenylethyl chloromethyl ketone
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
      PTH
      phenylthiohydantoin
      rpHPLC
      reversed-phase high-performance liquid chromatography
      pLGIC
      pentameric ligand-gated ion channel.
      of synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABAARs), anion-selective pentameric ligand-gated channels (pLGICs) (
      • Harrison N.L.
      • Majewska M.D.
      • Harrington J.W.
      • Barker J.L.
      Structure–activity relationships for steroid interaction with the γ-aminobutyric acidA receptor complex.
      ,
      • Hawkinson J.E.
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      Correlation of neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding and γ-aminobutyric acida receptor function.
      • Belelli D.
      • Lambert J.J.
      Neurosteroids: endogenous regulators of the GABAA receptor.
      ), and negative allosteric modulators of muscle and neuronal nicotinic acetylcholine receptors (nAChRs), cation-selective pLGICs (
      • Ke L.
      • Lukas R.J.
      Effects of steroid exposure on ligand binding and functional activities of diverse nicotinic acetylcholine receptor subtypes.
      ,
      • Paradiso K.
      • Sabey K.
      • Evers A.S.
      • Zorumski C.F.
      • Covey D.F.
      • Steinbach J.H.
      Steroid inhibition of rat neuronal nicotinic α4β2 receptors expressed in HEK 293 cells.
      • Arias H.R.
      • Bhumireddy P.
      • Bouzat C.
      Molecular mechanisms and binding site locations for noncompetitive antagonists of nicotinic acetylcholine receptors.
      ). The complex effects of steroid PAMs on GABAAR gating and the locations of residues identified by mutational analyses as steroid sensitivity determinants suggest the existence of multiple steroid-binding sites in the transmembrane domain (TMD) of heteromeric GABAARs (
      • Hosie A.M.
      • Wilkins M.E.
      • da Silva H.M.
      • Smart T.G.
      Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites.
      ,
      • Akk G.
      • Covey D.F.
      • Evers A.S.
      • Steinbach J.H.
      • Zorumski C.F.
      • Mennerick S.
      Mechanisms of neurosteroid interactions with GABA(A) receptors.
      ). Recent crystal structures of chimeric homopentameric receptors containing GABAAR α subunit TMDs identify a binding site for steroid PAMs in the TMD at subunit interfaces (
      • Laverty D.
      • Thomas P.
      • Field M.
      • Andersen O.J.
      • Gold M.G.
      • Biggin P.C.
      • Gielen M.
      • Smart T.G.
      Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.
      ,
      • Miller P.S.
      • Scott S.
      • Masiulis S.
      • De Colibus L.
      • Pardon E.
      • Steyaert J.
      • Aricescu A.R.
      Structural basis for GABAA receptor potentiation by neurosteroids.
      ), 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 (
      • Olsen R.W.
      Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.
      ,
      • Forman S.A.
      • Miller K.W.
      Mapping general anesthetic sites in heteromeric γ-aminobutyric acid type a receptors reveals a potential for targeting receptor subtypes.
      ). A distinct intrasubunit site for pregnenolone sulfate, an inhibitory steroid, was also identified in a chimeric homomeric receptor TMD (
      • Laverty D.
      • Thomas P.
      • Field M.
      • Andersen O.J.
      • Gold M.G.
      • Biggin P.C.
      • Gielen M.
      • Smart T.G.
      Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.
      ).
      For muscle-type nAChRs, single-channel analyses of the acute effects of natural and synthetic steroids indicate a reduction of open channel lifetime (
      • Wachtel R.E.
      • Wegrzynowicz E.S.
      Kinetics of nicotinic acetylcholine ion channels in the presence of intravenous anaesthetics and induction agents.
      ,
      • Bouzat C.
      • Barrantes F.J.
      Modulation of muscle nicotinic acetylcholine receptors by the glucocorticoid hydrocortisone–possible allosteric mechanism of channel blockade.
      • Garbus I.
      • Bouzat C.
      • Barrantes F.J.
      Steroids differentially inhibit the nicotinic acetylcholine receptor.
      ), 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. As cholesterol is present in high concentrations in synaptic membranes and is important for facilitating agonist-induced conformational transitions, inhibition by steroids may result from perturbation of nAChR–cholesterol interactions (
      • Barrantes F.J.
      Structural basis for lipid modulation of nicotinic acetylcholine receptor function.
      ,
      • Baenziger J.E.
      • Domville J.A.
      • Therien J.P.
      The role of cholesterol in the activation of nicotinic acetylcholine receptors.
      • Unwin N.
      Segregation of lipids near acetylcholine-receptor channels imaged by cryo-EM.
      ). Mutations of some amino acids in the M4 helices that are exposed at the lipid interface have as large an effect on channel gating as the substitutions at channel-lining residues (
      • Santiago J.
      • Guzmàn G.R.
      • Rojas L.V.
      • Marti R.
      • Asmar-Rovira G.A.
      • Santana L.F.
      • McNamee M.
      • Lasalde-Dominicci J.A.
      Probing the effects of membrane cholesterol in the Torpedo californica acetylcholine receptor and the novel lipid-exposed mutation α C418W in Xenopus oocytes.
      ,
      • Mitra A.
      • Bailey T.D.
      • Auerbach A.L.
      Structural dynamics of the M4 transmembrane segment during acetylcholine receptor gating.
      ). Also, substitutions at a position in αM4 exposed at the lipid interface reduced hydrocortisone potency (
      • Garbus I.
      • Roccamo A.M.
      • Barrantes F.J.
      Identification of threonine 422 in transmembrane domain α M4 of the nicotinic acetylcholine receptor as a possible site of interaction with hydrocortisone.
      ). A mutation within the M2 ion channel domain that increased channel lifetime also reduces hydrocortisone potency, but there was no evidence for hydrocortisone competition with QX-222, an open channel blocker (
      • Bouzat C.
      • Barrantes F.J.
      Modulation of muscle nicotinic acetylcholine receptors by the glucocorticoid hydrocortisone–possible allosteric mechanism of channel blockade.
      ).
      nAChR-rich membranes that can be isolated from the Torpedo electric organ provide a unique preparation to use photoaffinity-labeling techniques to identify steroid-binding sites in a muscle-type nAChR in its native membrane environment. Radiolabeled, photoreactive analogs of hydrophobic general anesthetics, including propofol, mephobarbital, and etomidate, have been shown to bind to sites in the TMD within the ion channel and to inter- and intra-subunit sites (
      • Hamouda A.K.
      • Jayakar S.S.
      • Chiara D.C.
      • Cohen J.B.
      Photoaffinity labeling of nicotinic receptors: diversity of drug binding sites!.
      ,
      • Forman S.A.
      • Chiara D.C.
      • Miller K.W.
      Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors.
      ). Promegestone, a progestin steroid with intrinsic photoreactivity (Fig. 1), is a potent inhibitor of Torpedo nAChRs expressed in Xenopus oocytes and of binding of [3H]phencyclidine (PCP), a channel blocker, to nAChR-rich membranes (
      • Blanton M.P.
      • Xie Y.
      • Dangott L.J.
      • Cohen J.B.
      The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid–protein interface.
      ). Whereas [3H]promegestone photolabeled amino acids at the lipid interface in the M4 helices, no evidence was found of photolabeling ion channel residues. However, promegestone may have bound in the ion channel without efficient photolabeling.
      Figure thumbnail gr1
      Figure 1Chemical structures of F4N3Bzoxy-AP, alphaxalone, and promegestone.
      To further characterize steroid-binding sites in the Torpedo nAChR, we now use 11β-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F4N3Bzoxy-AP) (Fig. 1), a photoreactive allopregnanolone derivative that is a potent general anesthetic and GABAAR-positive allosteric modulator (
      • Savechenkov P.Y.
      • Chiara D.C.
      • Desai R.
      • Stern A.T.
      • Zhou X.
      • Ziemba A.M.
      • Szabo A.L.
      • Zhang Y.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.
      ). In contrast to promegestone, which upon UV irradiation forms a reactive ketyl radical at the 3-position of the steroid A-ring, F4N3Bzoxy-AP reacts by formation of a stabilized nitrene at the 11-position in the steroid C-ring (
      • Keana J.F.
      • Cai S.X.
      New reagents for photoaffinity labeling: synthesis and photolysis of functionalized perfluorophenyl azides.
      ,
      • Cline M.R.
      • Mandel S.M.
      • Platz M.S.
      Identification of the reactive intermediates produced upon photolysis of p-azidoacetophenone and its tetrafluoro analogue in aqueous and organic solvents: implications for photoaffinity labeling.
      ). Because a nAChR agonist containing the same photoreactive group photolabeled aliphatic and aromatic amino acid side chains within the ACh-binding sites in the nAChR extracellular domain (
      • Nirthanan S.
      • Ziebell M.R.
      • Chiara D.C.
      • Hong F.
      • Cohen J.B.
      Photolabeling the Torpedo nicotinic acetylcholine receptor with 4-azido-2,3,5,6-tetrafluorobenzoylcholine, a partial agonist.
      ), F4N3Bzoxy-AP should have the capacity to photoincorporate into many of the amino acid side chains in the nAChR TMD. We found that, similar to promegestone, F4N3Bzoxy-AP inhibited binding of a channel blocker in the nAChR-desensitized state. In contrast to promegestone, [3H]F4N3Bzoxy-AP photolabeled residues in the nAChR ion channel in the desensitized state (positions βM2–13′ and δM2–13′, numbered from the 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 also identified sites accessible from the lipid, one within the α subunit, identified by photolabeling of αCys-222/αLeu-223 at the interface between the M1 and M4 helices, and a site near the cytoplasmic surface of the TMD, identified by photolabeling of γTrp-453 in γM4, a residue photolabeled by [3H]promegestone.

      Results

      Radioligand binding assays

      We compared the effects of F4N3Bzoxy-AP and alphaxalone on the equilibrium binding of [3H]ACh and of channel blockers that bind preferentially in the nAChR desensitized state stabilized by agonist ([3H]tenocyclidine ([3H]TCP), a PCP analog (
      • Arias H.R.
      • Trudell J.R.
      • Bayer E.Z.
      • Hester B.
      • McCardy E.A.
      • Blanton M.P.
      Noncompetitive antagonist binding sites in the Torpedo nicotinic acetylcholine receptor ion channel. Structure–activity relationship studies using adamantane derivatives.
      )) or in the closed channel state stabilized by the peptide neurotoxin α-bungarotoxin ([3H]tetracaine (
      • Moore M.A.
      • McCarthy M.P.
      Snake venom toxins, unlike smaller antagonists, appear to stabilize a resting state conformation of the nicotinic acetylcholine receptor.
      ,
      • Gallagher M.J.
      • Cohen J.B.
      Identification of amino acids of the Torpedo nicotinic acetylcholine receptor contributing to the binding site for the noncompetitive antagonist [3H]tetracaine.
      )) (Fig. 2). F4N3Bzoxy-AP or alphaxalone even at 100 μm altered [3H]ACh-specific binding by <5%, in contrast to proadifen, a prototypic-desensitizing, noncompetitive antagonist (
      • Boyd N.D.
      • Cohen J.B.
      Desensitization of membrane-bound Torpedo acetylcholine receptor by amine noncompetitive antagonists and aliphatic alcohols: studies of [3H]acetylcholine binding and 22Na+ ion fluxes.
      ), that increased binding by ∼30% with an EC50 of ∼1 μm. Neither F4N3Bzoxy-AP nor alphaxalone at concentrations up to 300 μm had any effect on the binding of [3H]tetracaine to the ion channel in the closed state. In contrast, for nAChRs in the desensitized state stabilized by agonist, both F4N3Bzoxy-AP and alphaxalone inhibited [3H]TCP binding, with F4N3Bzoxy-AP (IC50 = 31 ± 4 μm) 7-fold more potent than alphaxalone (IC50 = 209 ± 12 μm).
      Figure thumbnail gr2
      Figure 2Effects 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%.

      [3H]F4N3Bzoxy-AP photolabeling of Torpedo nAChR-rich membranes

      We compared patterns of nAChR subunit photolabeling after irradiation of nAChR-rich membranes at 365 or 254 nm in the absence or presence of an agonist (carbamylcholine (Carb)), PCP, alphaxalone, or the steroid noncompetitive antagonists alphaxalone or promegestone. After photolabeling with [3H]F4N3Bzoxy-AP (3 μm) and fractionation of membrane polypeptides by SDS-PAGE, the 3H incorporation into the nAChR subunits and other membrane polypeptides was assessed by fluorography (Fig. 3A) for qualitative characterization and by liquid scintillation counting of excised subunit gel bands to quantify photoincorporation (Fig. 3, B–D). As seen by fluorography, after irradiation at 365 nm, the nAChR subunits photolabeled most prominently were the α and γ subunits, with the most prominently photolabeled gel bands those of 34 and 32 kDa previously identified as the voltage-dependent anion channel and ADP/ATP carrier from contaminating mitochondrial fragments (
      • Blanton M.P.
      • Lala A.K.
      • Cohen J.B.
      Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling.
      ). Irradiation at 254 nm resulted in prominent, pharmacologically-specific photolabeling in the nAChR α subunit, with Carb enhancing photolabeling compared with control and PCP inhibiting the Carb-enhanced photolabeling. Quantification of photolabeling by liquid scintillation counting (Fig. 3, B and C) established that for irradiation at 254 nm, Carb enhanced α subunit photolabeling by ∼50%, with the Carb-enhanced photolabeling inhibited by PCP but not by 300 μm alphaxalone or R-mTFD-MPAB, a barbiturate allosteric inhibitor that binds to sites in the ion channel and at the γ–α subunit interface (
      • Hamouda A.K.
      • Stewart D.S.
      • Chiara D.C.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Cohen J.B.
      Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.
      ). To test the effect of a second steroid antagonist on [3H]F4N3Bzoxy-AP photolabeling at 254 nm, we used promegestone, which inhibits with an IC50 of 10 μm the ACh responses of Torpedo nAChRs expressed in Xenopus oocytes and the binding of the channel blocker [3H]PCP to nAChR-rich membranes in the presence of Carb (Fig. 3D) (
      • Blanton M.P.
      • Xie Y.
      • Dangott L.J.
      • Cohen J.B.
      The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid–protein interface.
      ). Carb enhanced nAChR α subunit photolabeling by ∼40%, and promegestone at 300 μm reduced photolabeling to the level seen in the control condition or in the presence of Carb and PCP. Similarly for the β and δ subunits, promegestone inhibited the small enhancement of photolabeling (∼20%) seen in the presence of Carb.
      Figure thumbnail gr3
      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 (αNa/K), and the mitochondrial voltage-dependent anion channel (34 kDa) and ADP/ATP carrier (32 kDa).

      [3H]F4N3Bzoxy-AP photolabeling in the nAChR ion channel

      Pharmacologically-specific photolabeling was most prominent in the α subunit after photolabeling at 254 nm, but we first characterized photolabeling within the M2 channel-forming helices after irradiation at 365 nm, a wavelength that minimizes nonspecific UV-induced protein degradation. Photolabeling was characterized in βM2 and δM2, because fragments beginning at their N termini (βMet-249 and δMet-257) can be isolated readily (
      • White B.H.
      • Cohen J.B.
      Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist.
      ,
      • Arevalo E.
      • Chiara D.C.
      • Forman S.A.
      • Cohen J.B.
      • Miller K.W.
      Gating-enhanced accessibility of hydrophobic sites within the transmembrane region of the nicotinic acetylcholine receptor’s δ-subunit–a time-resolved photolabeling study.
      • Jayakar S.S.
      • Dailey W.P.
      • Eckenhoff R.G.
      • Cohen J.B.
      Identification of propofol-binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog.
      ) at ∼10-fold higher mass levels than the αM2 fragment that requires a more complex purification procedure (
      • Pratt M.B.
      • Husain S.S.
      • Miller K.W.
      • Cohen J.B.
      Identification of sites of incorporation in the nicotinic acetylcholine receptor of a photoactivatible general anesthetic.
      ). Trypsin digests of β subunits isolated from nAChRs photolabeled with [3H]F4N3Bzoxy-AP in the absence or presence of Carb were fractionated by Tricine gel SDS-PAGE and then rpHPLC (Fig. 4A). When the fragment beginning at βMet-249 was sequenced from the major peak of 3H (Fig. 4B), the peak of 3H release in cycle 13 identified photolabeling of βVal-261 (a channel-lining residue, position M2–13′) in the presence of Carb, whereas labeling in the absence of agonist, if it occurred, was at <10% that level. Similarly, after fractionation of an EndoLys-C digest of photolabeled δ subunit by Tricine-gel SDS-PAGE and then rpHPLC (Fig. 4C), sequence analysis of the major peak of 3H established the presence of the fragment beginning at δMet-257 (Fig. 4D). The peak of 3H release in cycle 13 in the presence of Carb indicated agonist-dependent photolabeling of δVal-269, the position in δM2 equivalent to βVal-261. In photolabelings with [3H]F4N3Bzoxy-AP at 254 nm, we also determined that βVal-261 and δVal-269 were photolabeled in an agonist-dependent manner and at similar efficiency as for photolabeling at 365 nm (Table 1).
      Figure thumbnail gr4
      Figure 4State-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 1Efficiency of [3H]F4N3Bzoxy-AP photolabeling of amino acids in the Torpedo nAChR transmembrane domain
      Labeled residuesPhotolabeling efficiency
      365 nm control/+Carb254 nm control/+Carb
      cpm/pmol
      βVal-261 (M2–13′)<0.1/1.4 ± 0.9 (3)<0.1/5.0 ± 2.6 (9)
      δVal-269 (M2–13′)<0.05/0.4 ± 0.2 (3)<0.05/0.23 ± 0.08 (5)
      γTrp-453 (M4)7 ± 4 (2)/6 (1)ND
      αCys-222 (M1) (αLeu-223)
      Because αLeu-223 immediately follows αCys-222, which is photolabeled more efficiently, the efficiency of photolabeling of αLeu-223 cannot be calculated reliably.
      ND0.7 ± 0.2 (2)/4.3 ± 1.7 (3)
      a Because αLeu-223 immediately follows αCys-222, which is photolabeled more efficiently, the efficiency of photolabeling of αLeu-223 cannot be calculated reliably.

      Promegestone inhibition of photolabeling in βM2 and δM2

      To examine the effects of promegestone on [3H]F4N3Bzoxy-AP photolabeling, membranes equilibrated with Carb in the absence or presence of 100 μm promegestone were irradiated at 254 nm, and the subunit fragments beginning at the N termini of βM2 and δM2 were isolated for sequence analysis. For the fragment beginning at βMet-249, promegestone reduced the peak of 3H release in cycle 13 (βVal-261) by ∼40% (Fig. 5A). For photolabeling within δM2, the fragment beginning at δMet-257 was isolated at a high level (I0 = 310 pmol, both conditions). In the presence of Carb there were peaks of 3H release at cycle 13, 16, and 20, consistent with photolabeling of the channel-lining residues δM2–13′, -16′, and -20′ (δVal-269, δLeu-272, and δGln-276) (Fig. 5B). Promegestone reduced photolabeling of δVal-269 by ∼50%.
      Figure thumbnail gr5
      Figure 5Promegestone 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.

      [3H]F4N3Bzoxy-AP photolabels residues in αM1

      Based upon the photoincorporation seen at the subunit level (Fig. 3D), irradiation at 254 nm resulted in pharmacologically-specific photolabeling in the nAChR α subunit. To identify photolabeling in the α subunit, we took advantage of the fact that in gel digestion of the α subunit with V8 protease results in the formation of three large, nonoverlapping subunit fragments that are readily resolved by SDS-PAGE (
      • White B.H.
      • Cohen J.B.
      Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine.
      ,
      • Blanton M.P.
      • Cohen J.B.
      Identifying the lipid–protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.
      ), including one of 20 kDa (αV8–20) that begins at αSer-173 and extends through the M1–M2–M3 helices and another of 10 kDa (αV8–10) that begins at αAsn-339 in the cytoplasmic domain and extends through the M4 helix. Furthermore, digestion of αV8–20 with EndoLys-C generates fragments readily separated by rpHPLC, one beginning at αHis-186 that contains αM1 and a second beginning at αMet-243, the N terminus of αM2, that extends through αM3 (
      • Pratt M.B.
      • Husain S.S.
      • Miller K.W.
      • Cohen J.B.
      Identification of sites of incorporation in the nicotinic acetylcholine receptor of a photoactivatible general anesthetic.
      ). When an EndoLys-C digest of αV8–20 isolated from nAChRs photolabeled at 254 nm in the presence of Carb was fractionated by rpHPLC (Fig. 6A), the major peak of 3H eluted at ∼70% organic solvent B where the fragment containing αM1 is known to elute (
      • Pratt M.B.
      • Husain S.S.
      • Miller K.W.
      • Cohen J.B.
      Identification of sites of incorporation in the nicotinic acetylcholine receptor of a photoactivatible general anesthetic.
      ,
      • Yu Z.
      • Cohen J.B.
      Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).
      ), with little 3H eluting in the more hydrophobic fractions where the fragment containing αM2 elutes. Sequence analysis of fractions containing the peak of 3H (Fig. 6B) established the presence of the αHis-186 fragment (I0 = 4 pmol), with no release of 3H above background in 15 cycles of Edman degradation. Similarly, the fragment beginning at αMet-243 (I0 = 1 pmol), which eluted at ∼85% organic solvent B, was sequenced without any 3H release above background in 15 cycles of Edman degradation that extended to αM2–15′ (Fig. 6C). These results indicated that the 3H incorporation in αV8–20 was likely to be within αM1.
      Figure thumbnail gr6
      Figure 6State-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.
      To identify amino acids photolabeled in αM1, α subunits were isolated from nAChRs photolabeled at 254 nm in the absence or presence of Carb and in the presence of Carb and promegestone. During sequencing of the fragment beginning at αIle-210 (I0 = 60 pmol, each condition), isolated by rpHPLC from tryptic digests of nAChRs (
      • Hamouda A.K.
      • Stewart D.S.
      • Chiara D.C.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Cohen J.B.
      Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.
      ,
      • Yu Z.
      • Cohen J.B.
      Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).
      ), there was a peak of 3H release in cycles 13/14 consistent with photolabeling of αCys-222/αLeu-223 (Fig. 6D). These residues were photolabeled at higher efficiency in the presence of Carb than in the absence (Table 1), and promegestone reduced the agonist-enhanced labeling by ∼50%. To confirm photolabeling of αCys-222/αLeu-223, a fragment beginning at αGln-208 was generated for sequencing by isolating the labeled αHis-186 fragments from α subunit EndoLys-C digests and treating them with cyanogen bromide to cleave at the C terminus of αMet-207 (
      • Yu Z.
      • Cohen J.B.
      Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).
      ,
      • Hamouda A.K.
      • Kimm T.
      • Cohen J.B.
      Physostigmine and galanthamine bind in the presence of agonist at the canonical and noncanonical subunit interfaces of a nicotinic acetylcholine receptor.
      ). When the αGln-208 fragments were sequenced (Fig. 6E), the peak of Carb-enhanced 3H release in cycle 15 confirmed labeling of αCys-222. Sequence analysis of fragments beginning at αIle-210 from an independent labeling experiment (+Carb and +Carb + PMG) provided additional evidence that αLeu-223 was photolabeled as well as αCys-222, because in this case the peak of 3H release was in cycle 14 rather than 13 (Fig. 6F).

      [3H]F4N3Bzoxy-AP photolabeling in M4 and M3 helices

      The nAChR M4 helices are most broadly exposed to lipid, with amino acids from the M1 and M3 helices also exposed at the lipid interface (
      • Blanton M.P.
      • Cohen J.B.
      Identifying the lipid–protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.
      ,
      • Unwin N.
      Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution.
      ). Photolabeling studies with [3H]promegestone, which forms a reactive ketyl radical upon irradiation at 312 nm, identified photolabeling of residues in αM4 (αCys-412 and αCys-418), βM4 (βTyr-441 and βCys-447), and γM4 (γCys-451 and γTrp-453) that are exposed at the nAChR–lipid interface and also photolabeled by small hydrophobic probes (
      • Blanton M.P.
      • Xie Y.
      • Dangott L.J.
      • Cohen J.B.
      The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid–protein interface.
      ). Any [3H]promegestone photolabeling of residues in βM2 or δM2, if it occurred, was at <10% the efficiency of any of the photolabeled cysteines. To identify photolabeling in αM4, we isolated by rpHPLC the fragment beginning at αTyr-401, the αM4 N terminus, from trypsin digests of αV8–10 isolated from nAChRs photolabeled at 254 and at 365 nm. No peaks of 3H release above background were detected during sequence analyses of these fragments through 20 cycles of Edman degradation (Fig. 7, A and B), which indicated that labeling, if it occurred, was at <0.3 cpm/pmol, i.e. at <5% the efficiency of labeling of αCys-222 but possibly at the labeling efficiency of δVal-269 (δM2–13′).
      Figure thumbnail gr7
      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 (
      • Blanton M.P.
      • Cohen J.B.
      Identifying the lipid–protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.
      ), 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 (). 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 ().
      Photolabeling in γM4 was characterized by a strategy similar to that used for αM4. The γ-subunits were digested in gel with V8-protease to produce fragments of ∼14 kDa (γV8–14) beginning at γLeu-373 and γIle-413, and EndoLys-C digests of those fragments were fractionated by rpHPLC. For nAChRs photolabeled at 365 nm, sequencing of the fragment beginning at γAla-450, the N terminus of γM4, revealed a peak of 3H release in cycle 4, corresponding to photolabeling of γTrp-453 in the absence or presence of Carb at a photolabeling efficiency 4-fold higher than that of βVal-261 (βM2–13′) (Fig. 7C and Table 1). In contrast, for nAChRs irradiated at 254 nm, photolabeling of γTrp-453, if it occurred, was at <10% the efficiency of βVal-261. This preferential photolabeling at 365 nm compared with 254 nm of γTrp-453, the only Trp in the nAChR TMD, may be the source of the increased γ subunit labeling seen after irradiation at 365 nm compared with 254 nm (Fig. 3).
      Multiple lipid-exposed residues in the nAChR M3 helices have been photolabeled by small hydrophobic photoprobes (
      • Jayakar S.S.
      • Dailey W.P.
      • Eckenhoff R.G.
      • Cohen J.B.
      Identification of propofol-binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog.
      ,
      • Blanton M.P.
      • Cohen J.B.
      Identifying the lipid–protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.
      ), whereas other residues in γM3 contributing to a binding pocket at the γ-α subunit interface have been photolabeled by photoreactive etomidate and barbiturate analogs (
      • Hamouda A.K.
      • Stewart D.S.
      • Chiara D.C.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Cohen J.B.
      Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.
      ,
      • Nirthanan S.
      • Garcia 3rd., G.
      • Chiara D.C.
      • Husain S.S.
      • Cohen J.B.
      Identification of binding sites in the nicotinic acetylcholine receptor for TDBzl-etomidate, a photoreactive positive allosteric effector.
      ,
      • Hamouda A.K.
      • Stewart D.S.
      • Husain S.S.
      • Cohen J.B.
      Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.
      ). For nAChRs photolabeled at 365 or 254 nm in the presence of Carb, we characterized [3H]F4N3Bzoxy-AP photolabeling in γM3 and δM3 by sequencing for 30 cycles of Edman degradation fragments that begin at γThr-276 and δThr-281 and extend through the M3 helices. The protocol used (
      • Hamouda A.K.
      • Stewart D.S.
      • Husain S.S.
      • Cohen J.B.
      Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.
      ) allowed efficient recovery of the fragments (I0 = 30–90 pmol), but at either labeling wavelength, there were no peaks of 3H release above background, which indicated that any labeling, if it occurred, was at <0.3 cpm/pmol.

      [3H]F4N3Bzoxy-AP photolabeling in the δ subunit helix bundle

      No photolabeling was detected of the residues in δM1 (δTyr-228 and δPhe-232), δM2 (δM2–18′), or the δM2–M3 loop (δIle-288) within the intrasubunit-binding site near the extracellular end of the δ subunit helix bundles that are photolabeled in an agonist-dependent manner by general anesthetics (halothane and photoreactive analogs of etomidate and propofol (
      • Jayakar S.S.
      • Dailey W.P.
      • Eckenhoff R.G.
      • Cohen J.B.
      Identification of propofol-binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog.
      ,
      • Hamouda A.K.
      • Stewart D.S.
      • Husain S.S.
      • Cohen J.B.
      Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.
      ,
      • Chiara D.C.
      • Dangott L.J.
      • Eckenhoff R.G.
      • Cohen J.B.
      Identification of nicotinic acetylcholine receptor amino acids photolabeled by the volatile anesthetic halothane.
      )), a convulsant barbiturate (
      • Yu Z.
      • Cohen J.B.
      Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).
      ), and small hydrophobic nAChR inhibitors (
      • Arevalo E.
      • Chiara D.C.
      • Forman S.A.
      • Cohen J.B.
      • Miller K.W.
      Gating-enhanced accessibility of hydrophobic sites within the transmembrane region of the nicotinic acetylcholine receptor’s δ-subunit–a time-resolved photolabeling study.
      ,
      • Garcia 3rd., G.
      • Chiara D.C.
      • Nirthanan S.
      • Hamouda A.K.
      • Stewart D.S.
      • Cohen J.B.
      [3H]Benzophenone photolabeling identifies state-dependent changes in nicotinic acetylcholine receptor structure.
      ). Any photolabeling of δM2–18′, if it occurred, was at less than 5% the level of δM2–13′ (Fig. 5B), and based upon sequencing the fragment beginning at δPhe-208 (I0 = 120 pmol), any labeling of δPhe-232 or δCys-236 was at less than 10% the level of labeling of βM2–13′ or αCys-232.

      Discussion

      In this report, we use F4N3Bzoxy-AP, a photoreactive analog of alphaxalone and allopreganolone that is a potent general anesthetic and GABAAR PAM (
      • Savechenkov P.Y.
      • Chiara D.C.
      • Desai R.
      • Stern A.T.
      • Zhou X.
      • Ziemba A.M.
      • Szabo A.L.
      • Zhang Y.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.
      ), to identify the locations of steroid-binding sites in the Torpedo nAChR in its native membrane environment. Many endogenous (glucocorticoids and progesterone) and synthetic (alphaxalone and promegestone) steroids act as noncompetitive antagonists of Torpedo and vertebrate skeletal muscle and neuronal nAChRs, but it is uncertain whether they act from the lipid interface or from sites within the nAChR (
      • Ke L.
      • Lukas R.J.
      Effects of steroid exposure on ligand binding and functional activities of diverse nicotinic acetylcholine receptor subtypes.
      ,
      • Arias H.R.
      • Bhumireddy P.
      • Bouzat C.
      Molecular mechanisms and binding site locations for noncompetitive antagonists of nicotinic acetylcholine receptors.
      ). Promegestone, which potently inhibited Torpedo nAChR responses and the binding of a desensitized state selective channel blocker, photolabeled amino acids at the nAChR–lipid interface without detectable photolabeling of amino acids in the ion channel (
      • Blanton M.P.
      • Xie Y.
      • Dangott L.J.
      • Cohen J.B.
      The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid–protein interface.
      ). F4N3Bzoxy-AP also inhibits the binding of a channel blocker in the nAChR-desensitized state but not in the resting, closed channel state. In contrast to the photolabeling results for promegestone, we find that [3H]F4N3Bzoxy-AP photolabels residues in the ion channel as well as at the lipid interface, a result providing direct evidence of an ion channel–binding site for steroids. The locations of the photolabeled amino acids in a homology model of the Torpedo nAChR structure (Fig. 8) establish that [3H]F4N3Bzoxy-AP binds to three distinct sites in the nAChR TMD: (i) in the ion channel, photolabeling βVal-261 and δVal-269 in an agonist-dependent manner; (ii) an intra-subunit site accessible from the lipid, identified by agonist-dependent photolabeling of αCys-232/αLeu-223, near the middle of the TMD; and (iii) at the lipid interface, photolabeling γTrp-453 near the cytoplasmic surface of the TMD. Based upon computational docking (Fig. 8), F4N3Bzoxy-AP is predicted to bind in the ion channel between positions M2–13′ and M2–20′ and between the α subunit M1 and M4 helices, extending from αCys-222 to the extracellular end of TMD.
      Figure thumbnail gr8
      Figure 8F4N3Bzoxy-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 (
      • Unwin N.
      Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution.
      )), 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 (
      • Blanton M.P.
      • Xie Y.
      • Dangott L.J.
      • Cohen J.B.
      The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid–protein interface.
      ), and by [125I]TID, which also labels αThr-422 and αVal-425 (orange) but not γTrp-453 or αHis-408 (
      • Blanton M.P.
      • Cohen J.B.
      Identifying the lipid–protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.
      ); (ii) αVal-218 (green) in αM1, photolabeled by a convulsant barbiturate [3H]S-mTFD-MPPB (
      • Yu Z.
      • Cohen J.B.
      Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).
      ); (iii) the amino acids (cyan) in αM1 (αLeu-231) and γM3 (γMet-299) photolabeled by the anesthetic barbiturate [3H]R-mTFD-MPAB (
      • Hamouda A.K.
      • Stewart D.S.
      • Chiara D.C.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Cohen J.B.
      Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.
      ); 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 (
      • Blanton M.P.
      • Cohen J.B.
      Identifying the lipid–protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.
      ). C and D, locations of F4N3Bzoxy-AP (molecular volume = 448 Å3) 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 Å3 and αM1/αM4, volume = 596 Å3).

      Steroid binding in the ion channel

      The agonist-enhanced photolabeling of βVal-261 and δVal-269 provides direct evidence that a steroid can bind in proximity to position M2–13′ in the ion channel. Based upon the [3H]TCP- and [3H]tetracaine-binding assays, F4N3Bzoxy-AP binds in the ion channel in the desensitized state (IC50 = 30 μm) with >10-fold higher affinity than in the closed channel state. As the structural changes in the ion channel associated with channel gating involve only small twists or tilts of the M2 helices, the residues that contribute to the lumen of the channel are the same in the closed, open, and desensitized states (
      • Cymes G.D.
      • Ni Y.
      • Grosman C.
      Probing ion-channel pores one proton at a time.
      ). The hydrophobic side chains of M2–13′, along with those at M2–9′, contribute to a hydrophobic plug preventing ion permeation in the closed channel state (
      • White B.H.
      • Cohen J.B.
      Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist.
      ,
      • Miyazawa A.
      • Fujiyoshi Y.
      • Unwin N.
      Structure and gating mechanism of the acetylcholine receptor pore.
      ). Small charged (tetracaine) and uncharged (TID, benzophenone, and AziPm) drugs bind at that level preferentially in the absence of agonist (
      • Gallagher M.J.
      • Cohen J.B.
      Identification of amino acids of the Torpedo nicotinic acetylcholine receptor contributing to the binding site for the noncompetitive antagonist [3H]tetracaine.
      ,
      • White B.H.
      • Cohen J.B.
      Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist.
      ,
      • Jayakar S.S.
      • Dailey W.P.
      • Eckenhoff R.G.
      • Cohen J.B.
      Identification of propofol-binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog.
      ,
      • Garcia 3rd., G.
      • Chiara D.C.
      • Nirthanan S.
      • Hamouda A.K.
      • Stewart D.S.
      • Cohen J.B.
      [3H]Benzophenone photolabeling identifies state-dependent changes in nicotinic acetylcholine receptor structure.
      ). Similar to F4N3Bzoxy-AP, bulkier photoreactive etomidate and barbiturate analogs bind near M2–13′ in the channel with higher affinity in the desensitized state (
      • Hamouda A.K.
      • Stewart D.S.
      • Chiara D.C.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Cohen J.B.
      Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.
      ,
      • Yu Z.
      • Cohen J.B.
      Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).
      ,
      • Hamouda A.K.
      • Stewart D.S.
      • Husain S.S.
      • Cohen J.B.
      Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.
      ).
      Because more limited quantities of fragments containing αM2 or γM2 could be isolated for sequencing than βM2 or δM2, it remains possible that there is also unidentified labeling in those fragments at the same efficiency as δVal-269. The large quantities of βM2 and δM2 fragments allowed identification of agonist-dependent photolabeling in βM2 and δM2, even though it comprised a smaller fraction of subunit labeling than that seen for the α subunit (Fig. 3). There was, however, agonist-enhanced PCP-inhibitable α subunit labeling in nAChRs photolabeled at 254 nm, with the agonist-enhanced labeling inhibitable by 300 μm promegestone. At the amino acid level, promegestone at 100 μm reduced photolabeling of βVal-261 and δVal-269 by ∼50%. Because promegestone is more potent than F4N3Bzoxy-AP as an inhibitor of [3H]TCP binding (
      • Blanton M.P.
      • Xie Y.
      • Dangott L.J.
      • Cohen J.B.
      The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid–protein interface.
      ), ∼90% inhibition would be predicted if the two steroids bound in a mutually exclusive fashion. However, even PCP and chlorpromazine, two positively charged drugs, can bind simultaneously at different levels in the ion channel (
      • Chiara D.C.
      • Hamouda A.K.
      • Ziebell M.R.
      • Mejia L.A.
      • Garcia 3rd., G.
      • Cohen J.B.
      [3H]Chlorpromazine photolabeling of the Torpedo nicotinic acetylcholine receptor identifies two state-dependent binding sites in the ion channel.
      ). Further studies are necessary to determine whether the inhibition of photolabeling is allosteric rather than competitive.

      F4N3Bzoxy-AP binding at the nAChR–lipid interface

      The photolabeling of γTrp-453 in γM4 provides evidence that F4N3Bzoxy-AP interacts with the same region of γM4 near the cytoplasmic end of the TMD as promegestone and a tricyclic aromatic probe, diazofluorene, which photolabel the same residue (
      • Blanton M.P.
      • Xie Y.
      • Dangott L.J.
      • Cohen J.B.
      The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid–protein interface.
      ,
      • Blanton M.P.
      • Dangott L.J.
      • Raja S.K.
      • Lala A.K.
      • Cohen J.B.
      Probing the structure of the nicotinic acetylcholine receptor ion channel with the uncharged photoactivable compound [3H]diazofluorene.
      ), and TID, which photolabels γCys-451 (
      • Blanton M.P.
      • Cohen J.B.
      Identifying the lipid–protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.
      ). However, it was surprising not to find labeling of any positions in αM4, which is photolabeled at αCys-412 and other residues by promegestone, diazofluorene, TID, and many general anesthetics (Fig. 8C). The lack of labeling of αCys-412 cannot result from an inability of F4N3Bzoxy-AP to form a stable adduct with cysteines, because αCys-222 in αM1 was one of the residues labeled most efficiently. One explanation for this unexpected result is that when F4N3Bzoxy-AP binds in proximity to αM4, the photoreactive azide incorporated at the steroid 11 position is oriented toward the lipid rather than the nAChR.
      In the nAChR structure, the photolabeled residues in αM1 (αCys-222/αLeu-223) are predicted to be accessible from the lipid, and computational docking predicts that F4N3Bzoxy-AP will intercalate between αM1 and αM4 in the outer half of the TMD (Fig. 8C). This is the location where cholesterol is predicted to be enriched in the outer leaflet of the lipid bilayer, based upon cryo-EM analyses of lipid distribution in the tubular vesicles formed from Torpedo nAChR-rich membranes (
      • Unwin N.
      Segregation of lipids near acetylcholine-receptor channels imaged by cryo-EM.
      ). As interactions between the M4 and M1/M3 helices are important for channel function (
      • Baenziger J.E.
      • Domville J.A.
      • Therien J.P.
      The role of cholesterol in the activation of nicotinic acetylcholine receptors.
      ), our results indicate that binding of F4N3Bzoxy-AP or other steroids at this site can perturb cholesterol–nAChR interactions important for nAChR conformational transitions. Photolabeling of αCys-222/αLeu-223 was state-dependent, as evidenced by enhanced labeling in the presence of agonist, a result also seen for a convulsant barbiturate that binds in the same region, photolabeling αVal-218 (
      • Yu Z.
      • Cohen J.B.
      Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).
      ). Consistent with this, mutations of αVal-218, αPro-221, and αCys-222 indicate that structural changes in this region are important determinants of nAChR gating and desensitization (
      • Purohit P.
      • Gupta S.
      • Jadey S.
      • Auerbach A.
      Functional anatomy of an allosteric protein.
      ,
      • Purohit P.
      • Chakraborty S.
      • Auerbach A.
      Function of the M1 π-helix in endplate receptor activation and desensitization.
      ). The location of this intrasubunit-binding site for steroids between αM1 and αM4 identified by F4N3Bzoxy-AP is similar to a binding site for steroids identified by photolabeling in a prokaryotic pLGIC with a cation-selective ion channel, the homomeric Gloeobacter ligand-gated ion channel (GLIC) (
      • Cheng W.W.L.
      • Chen Z.-W.
      • Bracamontes J.R.
      • Budelier M.M.
      • Krishnan K.
      • Shin D.J.
      • Wang C.
      • Jiang X.
      • Covey D.F.
      • Akk G.
      • Evers A.S.
      Mapping two neurosteroid-modulatory sites in the prototypic pentameric ligand-gated ion channel GLIC.
      ). In contrast, in a homomeric chimeric receptor containing a GABAAR α subunit TMD, a binding site for pregnenolone sulfate, an inhibitory steroid, was identified between the M4 and M3 helices near the cytoplasmic end of the TMD (
      • Laverty D.
      • Thomas P.
      • Field M.
      • Andersen O.J.
      • Gold M.G.
      • Biggin P.C.
      • Gielen M.
      • Smart T.G.
      Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.
      ).

      Experimental procedures

      Materials

      nAChR-rich membranes, containing 0.8–1.1 nmol of [3H]ACh-binding sites per mg of protein, were isolated from Torpedo californica electric organs (Aquatic Research Consultants, San Pedro, CA) as described (
      • Middleton R.E.
      • Cohen J.B.
      Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label.
      ) and stored in 38% (w/v) sucrose at −80 °C until use. F4N3Bzoxy-AP and [3H]F4N3Bzoxy-AP (45 Ci/mmol) were synthesized previously (
      • Savechenkov P.Y.
      • Chiara D.C.
      • Desai R.
      • Stern A.T.
      • Zhou X.
      • Ziemba A.M.
      • Szabo A.L.
      • Zhang Y.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.
      ). [3H]Acetylcholine (ACh, 30 Ci/mmol) was synthesized by esterification of choline and [3H]acetic anhydride. Promegestone and [3H]tenocyclidine ([3H]TCP, 58 Ci/mmol) were from PerkinElmer Life Sciences. [3H]Tetracaine (30 Ci/mmol) was from Sibtech (Newington, CT). Proadifen, Carb, PCP, and cyanogen bromide were from Sigma, and o-phthalaldehyde was from Alfa Aesar. Staphylococcus aureus endopeptidase Glu-C (V8 protease) was from MP Biomedicals (Solon, OH), l-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin from Worthington, and Lysobacter enzymogenes endoproteinase Lys-C (EndoLys-C) from Roche Diagnostics.

      Radioligand binding assays

      Centrifugation equilibrium binding assays of [3H]ACh, [3H]TCP, and [3H]tetracaine to Torpedo nAChR-rich membranes were performed in Torpedo saline buffer (in mm: 250 NaCl, 5 KCl, 3 CaCl2, 2 MgCl2, and 5 sodium phosphate, pH 7.0) as described (
      • Yu Z.
      • Cohen J.B.
      Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).
      ). Binding assays were performed at the following final concentrations: for [3H]ACh, 40 nm ACh-binding sites, 4 nm radioligand, and 0.5 mm diisopropylphosphofluoridate to inhibit acetylcholinesterase; for [3H]TCP and [3H]tetracaine, 500 nm ACh-binding sites and 10 nm radioligand. For [3H]TCP binding, membranes were equilibrated with the agonist Carb (1 mm) to stabilize nAChRs in the desensitized state. For [3H]tetracaine binding, membranes were equilibrated with 5 μm α-bungarotoxin to stabilize nAChRs in the resting state. Nonspecific binding of [3H]ACh, [3H]TCP, or [3H]tetracaine was determined in the presence of 100 μm Carb, PCP, or tetracaine, respectively. Stock solutions of F4N3Bzoxy-AP, alphaxalone, and promegestone were prepared at 60 mm in methanol, and all samples contained methanol at a final concentration of 0.5% (v/v). For each experiment, fx, the specifically bound 3H (cpmtotal − cpmnonspecific), was normalized to f0, the specifically bound 3H in the absence of competitor. Individual experiments were carried out with duplicate samples, and data from independent experiments were combined for analysis. The concentration-dependent inhibition of [3H]TCP binding was fit to the equation, fx/f0 = 1/(1 + x/IC50), where IC50 is the total ligand concentration producing the half-maximal inhibition of binding. The numbers of independent experiments are noted in the figure legends.

      [3H]F4N3Bzoxy-AP photolabeling and gel electrophoresis

      Conditions for photolabeling were identified by characterizing the absorption and photolysis characteristics of F4N3Bzoxy-AP in methanol. At the absorption maximum of 264 nm, the F4N3Bzoxy-AP extinction coefficient was 17,550 ± 450 m−1 cm−1. Photolysis with a 254-nm lamp (Spectronics EF160C) caused a progressive shift of the maximum absorption from 264 to 285 nm with a t½ of 20 s, associated with the formation of a broad secondary peak with an absorption maximum of ∼340 nm. Photolysis with a 365-nm lamp (Spectronics EN-280L) decreased the absorption maximum with a t½ of 17 min. Based upon these results, nAChR-rich membranes equilibrated with [3H]F4N3Bzoxy-AP were irradiated for 2 and 30 min with the 254- and 365-nm lamps, respectively. Before irradiation, Torpedo nAChR-rich membranes (2 μm ACh sites; 2.5 mg of protein/ml in Torpedo saline buffer supplemented with 1 mm oxidized GSH as an aqueous scavenger) were incubated at 4 °C with [3H]F4N3Bzoxy-AP for 30 min and then for 30 min in the absence or presence of other ligands. nAChRs were photolabeled on analytical or preparative scales using 0.1 or 10 mg of protein per condition, respectively. After photolabeling, membrane polypeptides were resolved by Tris-glycine SDS-PAGE on gels composed of 8% polyacrylamide, 0.33% bisacrylamide, and membrane polypeptides were visualized with GelCodeTM Blue Safe Protein Stain (Thermo Fisher Scientific). Invitrogen SeeBlue Plus2 pre-stained standards were used as molecular mass markers.
      For analytical photolabelings (3 μm [3H]F4N3Bzoxy-AP), samples were run in triplicate, with stained subunit bands excised from two sets for 3H quantification by liquid scintillation counting, and the third set was analyzed by fluorography using Amplify (GE Healthcare). For identification of photolabeled amino acids, nAChR-rich membranes were photolabeled on a preparative scale in five experiments with [3H]F4N3Bzoxy-AP at 0.4–0.8 μm, using three different membrane purifications: two with irradiation at 365 nm in the absence and presence of Carb, and three with irradiation at 254 nm (+Carb; −Carb/+Carb/+Carb + 100 μm promegestone; +Carb/+Carb + 100 μm promegestone). For the preparative photolabelings, bands containing the nAChR α, β, γ, and δ subunits were excised from the stained gels, and subunits were eluted passively for at least 3 days at room temperature in a buffer containing (in mm): 100 NH4HCO3, 2.5 dl-DTT, 0.1% SDS, pH 8.4. Thereafter, eluted materials were concentrated to a final volume of ∼400 μl by centrifugal filtration at room temperature using Vivaspin 15 Mr 5000 concentrators (Vivascience, Stonehouse, UK), precipitated by 75% acetone overnight at −20 °C, and resuspended in digestion buffer (in mm: 15 Tris, 0.5 EDTA, 0.1% SDS, pH 8.1). For most preparative photolabelings, 25% of α and γ subunit gel bands were eluted directly, and 75% of those gel bands were subjected to in-gel digestion with V8 protease (200 μg) on 15% polyacrylamide, 0.76% bisacrylamide mapping gels. The resultant subunit fragments (αV8–20, αV8–10, γV8–24, and γV8–14) (
      • White B.H.
      • Cohen J.B.
      Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine.
      ,
      • Blanton M.P.
      • Cohen J.B.
      Identifying the lipid–protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.
      ) were recovered from gel bands by passive elution, concentrated, precipitated, and resuspended in digestion buffer.

      Proteolytic digestions

      All enzymatic digestions were performed at room temperature. After photolabelings on a preparative scale, ∼50% of eluted α and β subunits and 100% of αV8–10, each in digestion buffer, were diluted 5-fold with 50 mm NH4HCO3, pH 8.1, supplemented with 0.5% Genapol to reduce the SDS concentration to 0.02%, and then digested with 200 μg of TPCK-treated trypsin in the presence of 0.4 mm CaCl2 overnight (β subunit) or for 3 days (α subunit and αV8–10). αV8–20, γV8–24, and γV8–14 and aliquots of α and δ subunits (∼50%) were digested using 0.3–0.5 units of EndoLys-C for 2 weeks. To characterize photolabeling in the M3 helices (
      • Garcia 3rd., G.
      • Chiara D.C.
      • Nirthanan S.
      • Hamouda A.K.
      • Stewart D.S.
      • Cohen J.B.
      [3H]Benzophenone photolabeling identifies state-dependent changes in nicotinic acetylcholine receptor structure.
      ), aliquots of β (40%), γ (25%), and δ (40%) subunits were digested with 200 μg of V8 protease for 3 days in digestion buffer. Small-pore Tricine SDS-polyacrylamide gels (16.5% T, 6% C) (
      • Arevalo E.
      • Chiara D.C.
      • Forman S.A.
      • Cohen J.B.
      • Miller K.W.
      Gating-enhanced accessibility of hydrophobic sites within the transmembrane region of the nicotinic acetylcholine receptor’s δ-subunit–a time-resolved photolabeling study.
      ,
      • Schägger H.
      • von Jagow G.
      Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
      ) were used to fractionate the trypsin and EndoLys-C digests of β and δ subunits, respectively. The subunit fragments recovered from those Tricine gel bands by passive elution and the other subunit digests were then fractionated by rpHPLC.

      rpHPLC and sequence analysis

      rpHPLC was performed as described (
      • Hamouda A.K.
      • Stewart D.S.
      • Husain S.S.
      • Cohen J.B.
      Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.
      ) using an Agilent 1100 binary rpHPLC system, a Brownlee Aquapore BU-300 column, and a mobile phase containing the aqueous solvent A (0.08% TFA in distilled water) and organic solvent B (3:2 acetonitrile/isopropyl alcohol supplemented with 0.05% TFA). Proteolytic digests were eluted at a flow rate of 0.2 ml/min using a nonlinear gradient, with organic solvent B increasing from 25 to 100% over 90 min with fractions collected every 2.5 min, and 3H elution was determined by liquid scintillation counting of 10% of each fraction.
      With the exception of rpHPLC fractions containingαM4, which were loaded onto polyvinylidene difluoride membrane filters at room temperature using Applied Biosystems ProSorbTM sample preparation cartridges, all rpHPLC fractions containing 3H-labeled peptides were drop-loaded at 45 °C onto Applied Biosystems Micro TFA glass fiber filters. After loading on filters, samples were treated with Biobrene Plus to stabilize the peptides and then subjected to Edman degradation sequencing on an Applied Biosystems Procise 492 protein sequencer. For some samples, as detailed in the figure legends, sequencing was interrupted at a predetermined cycle, and the filter was treated with o-phthalaldehyde to prevent further sequencing of any peptides not containing a proline at that cycle (
      • Middleton R.E.
      • Cohen J.B.
      Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label.
      ,
      • Brauer A.W.
      • Oman C.L.
      • Margolies M.N.
      Use of o-phthalaldehyde to reduce background during automated Edman degradation.
      ). As one method to characterize [3H]F4N3Bzoxy-AP photolabeling in αM1, samples containing the fragment beginning at αHis-186 were sequenced for 18 cycles, and filters were then treated with cyanogen bromide as described (
      • Hamouda A.K.
      • Kimm T.
      • Cohen J.B.
      Physostigmine and galanthamine bind in the presence of agonist at the canonical and noncanonical subunit interfaces of a nicotinic acetylcholine receptor.
      ,
      • Scott M.G.
      • Crimmins D.L.
      • McCourt D.W.
      • Tarrand J.J.
      • Eyerman M.C.
      • Nahm M.H.
      A simple in situ cyanogen bromide cleavage method to obtain internal amino acid sequence of proteins electroblotted to polyvinyldifluoride membranes.
      ) to cleave at the C-terminal side of αMet-207 before αM1.
      During N-terminal sequencing, either 1/6 or 2/3 of the material from each cycle of Edman degradation was used for phenylthiohydantoin (PTH)-derivative determination, and 5/6 (or 1/3) was collected to measure 3H release by scintillation counting. The amount of PTH-derivative released in a given sequencing cycle (in picomoles) was determined by comparing the peak height of the derivative in the chromatogram to the height of its standard peak. I0, the initial amount of the peptide in the sample (in picomoles), was determined from the amounts of PTH-derivative in each cycle by using SigmaPlot 11 to the equation Ix = I0 Rx, where Ix is the background-subtracted mass of the amino acid at cycle x, and R is the average repetitive yield of the Edman degradation. For samples containing multiple fragments, only PTH-derivatives unique to the fragment of interest were used in the fit, and amino acid derivatives whose amounts cannot be estimated accurately (His, Trp, Ser, Arg, and Cys) were omitted from the fits. When 2/3 of the material was used for PTH-derivative determination, E(x), the photolabeling efficiency (cpm/pmol) of an amino acid residue in cycle x, was calculated according to the equation (2(cpmx − cpm(x − 1)))/(I0 Rx), where cpmx is the 3H release in cpm at cycle x. When 1/6 of the material was used for PTH-derivative determination, E(x) = ((cpmx − cpm(x − 1)))/(5 I0 Rx).

      Computational docking

      A T. californica nAChR homology model was constructed based on the cryo-EM–derived structure of Torpedo marmorata nAChR (PDB code 2BG9) (
      • Unwin N.
      Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution.
      ) using the Create Homology Model tool in Discovery Studio 2017 (Accelrys Inc., San Diego), with modifications introduced to correct for the previously identified error in assignment of amino acids in the M2 and M3 helices (
      • Hibbs R.E.
      • Gouaux E.
      Principles of activation and permeation in an anion-selective Cys-loop receptor.
      ,
      • Mnatsakanyan N.
      • Jansen M.
      Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors.
      ). To preserve the side-chain orientations of residues conserved between species, the nonconserved residues in each subunit (3, 17, and 11 in α, β, and δ, respectively; in γ, an added N-terminal Glu and eight residues) were mutated individually using the Build and Edit Protein Tool. To correct the placement of the M2 and M3 residues, the M1–M2 loops were shortened by four residues and the Create Homology Model tool was used to align αThr-237–αSer-302 of the sequence with αGlu-241–αHis-306 from the structure, along with the equivalent alignments for the other subunits. The full structural model was minimized using CHARMm with the Generalized Born Implicit Membrane solvent model for 12 cycles (Smart Minimized method) to detect inappropriate residue placements and to reduce high-energy interactions (final energy, −66,779 kcal/mol).
      F4N3Bzoxy-AP was docked using 12-Å radius binding-site spheres centered as follows: 1) in the ion channel at the level of βVal-261 and δVal-269; and 2) at the lipid–α subunit interface at the level of αCys-222 and αLeu-223. Each sphere was seeded with 12 distributed replicas of F4N3Bzoxy-AP with the CDOCKER module used to generate 40 molecular dynamics–induced alterations for each replica, and then each altered structure was configured into 30 different starting orientations. The docking results for binding in the ion channel and at the lipid–protein interface between the αM1 and αM4 helices are shown as Connolly surface representations defined by a 1.4-Å diameter probe of the ensemble of 12 solutions with the lowest CDOCKER interaction energies. Similar docking results were obtained when F4N3Bzoxy-AP was docked in these sites of a T. californica homology nAChR model (
      • Yu Z.
      • Cohen J.B.
      Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).
      ) based upon the crystal structure of an (α4)2(β2)3 nAChR (Protein Data Bank entry 5KXI (
      • Morales-Perez C.L.
      • Noviello C.M.
      • Hibbs R.E.
      X-ray structure of the human α4β2 nicotinic receptor.
      ).

      Author contributions

      Z. Y. and J. B. C. conceptualization; Z. Y. data curation; Z. Y. formal analysis; Z. Y. and D. C. C. investigation; Z. Y. and J. B. C. methodology; Z. Y. writing-original draft; Z. Y., D. C. C., and J. B. C. writing-review and editing; P. Y. S. and K. S. B. resources; J. B. C. supervision; J. B. C. funding acquisition; J. B. C. validation.

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