Journal: Molecular Pharmacology

Article Title: Desformylflustrabromine (dFBr) and [ 3 H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

doi: 10.1124/mol.115.098913

Figure Lengend Snippet: dFBr inhibition of Torpedo nAChR is enhanced in the presence of agonist and by mutations in the ion channel favoring the open state. (A) Representative traces showing the effect of dFBr on ACh-induced current responses when 10 µM ACh was applied for 10 seconds, followed by co-application of 10 µM ACh and dFBr for 10 seconds, and then 10 seconds of 10 µM ACh alone. (B) The concentration dependence of dFBr inhibition when co-applied with ACh as in (A), with the current measured at the end of dFBr co-application normalized to the current just before (⬤, IC50 = 0.12 ± 0.01 µM), compared with a parallel experiment quantifying dFBr inhibition of peak ACh responses without prior ACh exposure (○, dashed line, fit from Fig. 2 for data from 10 oocytes). (C) Representative traces showing the effect of preapplication of 1 µM dFBr for 10 seconds on nAChR sensitivity to ACh. The ACh responses were inhibited by ∼15 ± 3% (N = 6). (D) dFBr inhibition of mutant nAChRs containing leucine to serine substitutions at position M2-9′ within the ion channel, quantified from the peak current elicited by 10 µM ACh with co-application of dFBr normalized to the current in the absence of dFBr. IC50 values for wild type, α, β, γ, and δ M2-9′ mutants were 1.0 ± 0.07, 0.2 ± .04, 0.3 ± 0.03, 0.1 ± 0.02, and 0.5 ± 0.06 µM, respectively. For each dFBr concentration in (B) and (D), average ± S.E. of several replicas from at least three oocytes were plotted and fit to a single-site model.

Article Snippet: The locations of the photolabeled amino acids in the ion channel and in the ECD are shown in nAChR homology models in and , along with the orientations of dFBr in these sites predicted by computational docking. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 10. caption a7 dFBr binding site in the Torpedo muscle-type nAChR ion channel. (A and B) Side view (A) and view from within the ion channel toward the α - β - δ subunits (B) of a nAChR homology model based on the X-ray structure of the mouse serotonin 5-HT3R (PDB ID 4PIR ) ( Hassaine et al., 2014 ) and (C) a homology model based on the cryo-electron microscopy structure of Torpedo marmorata nAChR (PDB ID 2BG9 ) ( Unwin, 2005 ).

Techniques: Inhibition, Concentration Assay, Mutagenesis

Journal: Molecular Pharmacology

Article Title: Desformylflustrabromine (dFBr) and [ 3 H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

doi: 10.1124/mol.115.098913

Figure Lengend Snippet: dFBr binds with high affinity to a site in the nAChR ion channel (desensitized state). The effects of dFBr on the equilibrium binding of [3H]ACh, [3H]PCP, and [3H]tetracaine (A) and the effect of dFBr, PCP, and tetracaine on the equilibrium binding of [3H]dFBr (B) to the Torpedo nAChR in the presence of α-BgTx (□, ▽, △) or Carb (⬤, ▴) were determined using centrifugation assays. (A) For each dFBr concentration, the specific binding of the radioligand was calculated from the total and nonspecific binding, normalized to the specific binding in the absence of dFBr, and fit to a single-site model using eq. 3. dFBr enhanced [3H]ACh binding (125% at 30 µM), inhibited [3H]PCP binding (+Carb) with IC50 = 3.6 ± 0.2 µM, and inhibited [3H]PCP and [3H]tetracaine binding in the presence of α-BgTx with IC50 values of 49 ± 6 and 63 ± 11 µM, respectively. (B) For each drug concentration, bound [3H]dFBr (in cpm/ml) was converted to nM [3H]dFBr. Inhibition curves were fit to a single-site model (eq. 1) or for inhibition by nonradioactive dFBr (+Carb) to a two-site model (eq. 2). For PCP (+Carb), IC50 = 0.9 ± 0.1 µM and Bns = 1.4 ± 0.1 nM, respectively. For dFBr (+α-BgTX), IC50 = 380 ± 70 µM, Bspec = 1.1 ± 0.08 nM, and Bns = 0.36 ± 0.09 nM. For inhibition by dFBr (+Carb), the high-affinity component of binding was fixed as the total binding [(+Carb) − (+α-BgTx)], the low-affinity component was equal to Bspec(+α-BgTx), and Bns was the same as in the presence of α-BgTx. With these assumptions, the calculated IC50 values for the high- and low-affinity components of binding were 4.1 ± 0.4 and 460 ± 30 µM, respectively.

Article Snippet: The locations of the photolabeled amino acids in the ion channel and in the ECD are shown in nAChR homology models in and , along with the orientations of dFBr in these sites predicted by computational docking. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 10. caption a7 dFBr binding site in the Torpedo muscle-type nAChR ion channel. (A and B) Side view (A) and view from within the ion channel toward the α - β - δ subunits (B) of a nAChR homology model based on the X-ray structure of the mouse serotonin 5-HT3R (PDB ID 4PIR ) ( Hassaine et al., 2014 ) and (C) a homology model based on the cryo-electron microscopy structure of Torpedo marmorata nAChR (PDB ID 2BG9 ) ( Unwin, 2005 ).

Techniques: Binding Assay, Centrifugation, Concentration Assay, Inhibition

Journal: Molecular Pharmacology

Article Title: Desformylflustrabromine (dFBr) and [ 3 H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

doi: 10.1124/mol.115.098913

Figure Lengend Snippet: Photoincorporation of [3H]dFBr into Torpedo nAChR-rich membranes. Membranes were photolabeled on an analytical scale with 0.5 µM [3H]dFBr in the absence and presence of nAChR ligands. Polypeptides were resolved by SDS-PAGE on duplicate gels that were stained with Coomassie blue (A, lane 1). One gel was prepared for fluorography (A, lanes 2–4), and polypeptide bands were excised from the second gel for 3H determination by liquid scintillation counting (B). Drug additions for the fluorograph in (A): lane 2, no other ligand added; lane 3, +1 mM Carb; lane 4, +1 mM Carb and 100 µM PCP. The electrophoretic mobilities of the nAChR α, β, γ, and δ subunits, rapsyn (Rn), the Na+/K+-ATPase α subunit (90K) and calectrin (37K) are indicated on the left.

Article Snippet: The locations of the photolabeled amino acids in the ion channel and in the ECD are shown in nAChR homology models in and , along with the orientations of dFBr in these sites predicted by computational docking. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 10. caption a7 dFBr binding site in the Torpedo muscle-type nAChR ion channel. (A and B) Side view (A) and view from within the ion channel toward the α - β - δ subunits (B) of a nAChR homology model based on the X-ray structure of the mouse serotonin 5-HT3R (PDB ID 4PIR ) ( Hassaine et al., 2014 ) and (C) a homology model based on the cryo-electron microscopy structure of Torpedo marmorata nAChR (PDB ID 2BG9 ) ( Unwin, 2005 ).

Techniques: SDS Page, Staining

Journal: Molecular Pharmacology

Article Title: Desformylflustrabromine (dFBr) and [ 3 H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

doi: 10.1124/mol.115.098913

Figure Lengend Snippet: Agonist-enhanced [3H]dFBr photolabeling in βM2, and δM2. Torpedo nAChR-rich membranes were photolabeled at preparative scale with 0.5 µM [3H]dFBr in the absence (○, ⬜) or presence (⬤, ▪) of 1 mM Carb. (A and B) Elution of peptides (solid line) and 3H (○, ⬤) during rpHPLC purifications of an ∼8 kDa β subunit fragment isolated by from a trypsin digest (A) and an ∼14 kDa δ subunit fragment isolated from an EndoLys-C digest (B). (C and D) 3H (○, ⬤) and phenylthiohydantoin amino acids (⬜, ▪) released during sequence analyses of rpHPLC fractions 30 to 31 from (A) and fractions 27–29 from (B), respectively. (C) The only sequence detected began at the N-terminus of βM2 (βMet249; 5 pmol each condition), and the major peak of 3H release in cycle 9 indicates photolabeling of βLeu257 (βM2-9; −Carb/+Carb, 2/50 cpm/pmol). (D) The only sequence detected began at the N-terminus of δM2 (δMet257; 10 pmol each condition). The peaks of 3H release in cycles 2, 5, 6, 9, and 16 indicate photolabeling (−Carb/+Carb, in cpm/pmol) at δSer258 (δM2-2; 1/7), δIle261 (δM2-5; 1/14), δSer262 (δM2-6; 2/17), δLeu265 (δM2-9; 0.5/14), and δLeu272 (δM2-16; 1/12).

Article Snippet: The locations of the photolabeled amino acids in the ion channel and in the ECD are shown in nAChR homology models in and , along with the orientations of dFBr in these sites predicted by computational docking. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 10. caption a7 dFBr binding site in the Torpedo muscle-type nAChR ion channel. (A and B) Side view (A) and view from within the ion channel toward the α - β - δ subunits (B) of a nAChR homology model based on the X-ray structure of the mouse serotonin 5-HT3R (PDB ID 4PIR ) ( Hassaine et al., 2014 ) and (C) a homology model based on the cryo-electron microscopy structure of Torpedo marmorata nAChR (PDB ID 2BG9 ) ( Unwin, 2005 ).

Techniques: Isolation, Sequencing

Journal: Molecular Pharmacology

Article Title: Desformylflustrabromine (dFBr) and [ 3 H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

doi: 10.1124/mol.115.098913

Figure Lengend Snippet: Isolation of [3H]dFBr-photolabeled α subunit peptides. The band for nAChR α subunit from the photolabeling experiment described in Fig. 6 was excised and transferred to a 15% acrylamide mapping gel and subjected to in-gel digestion with V8 protease to produce large nAChR α subunit fragments shown schematically in (A). rpHPLC fractionations of EndoLys-C digests of (B) αV8-20, which begins at αSer173, and (C) αV8-18, which begins at αThr52. Elution of peptides (solid line) and 3H (○-Carb, ⬤+Carb) were monitored. rpHPLC fractions 27–29 and 30–32 from the V8-20 digest and fractions 17–19 from the V8-18 digest were pooled for sequence analysis (Fig. 8).

Article Snippet: The locations of the photolabeled amino acids in the ion channel and in the ECD are shown in nAChR homology models in and , along with the orientations of dFBr in these sites predicted by computational docking. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 10. caption a7 dFBr binding site in the Torpedo muscle-type nAChR ion channel. (A and B) Side view (A) and view from within the ion channel toward the α - β - δ subunits (B) of a nAChR homology model based on the X-ray structure of the mouse serotonin 5-HT3R (PDB ID 4PIR ) ( Hassaine et al., 2014 ) and (C) a homology model based on the cryo-electron microscopy structure of Torpedo marmorata nAChR (PDB ID 2BG9 ) ( Unwin, 2005 ).

Techniques: Isolation, Sequencing

Journal: Molecular Pharmacology

Article Title: Desformylflustrabromine (dFBr) and [ 3 H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

doi: 10.1124/mol.115.098913

Figure Lengend Snippet: [3H]dFBr photolabels γTyr105, γTyr111, and δTyr212 in the presence of agonist. 3H (○, ⬤) and phenylthiohydantoin amino acids (⬜, ▪) released during sequence analyses of a γ subunit fragment beginning at γVal102 (∼8 pmol each condition) (A) and a δ subunit fragment beginning at δPhe206 (∼4 pmol each condition) (B), each isolated as described in Materials and Methods from the nAChR photolabeling of Fig. 6 (−Carb (○, ⬜), +Carb (⬤, ▪)). (A) The major peak of 3H release at cycle 10 indicates [3H]dFBr photolabeling of γTyr111 (−Carb/+Carb, 18/6 cpm/pmol). The small peak of Carb-enhanced 3H release at cycle 4 indicates photolabeling of γTyr105 (−Carb/+Carb, 0.2/1.4 cpm/pmol). (B) During sequencing of the fragment beginning at δPhe206, recovered from rpHPLC fractions 23–25 in Fig. 6B, the peak of Carb-enhanced 3H release at cycle 7 indicates photolabeling of δTyr212 (-Carb/+Carb, 0.2/1.4 cpm/pmol). OPA, o-phthalaldehyde.

Article Snippet: The locations of the photolabeled amino acids in the ion channel and in the ECD are shown in nAChR homology models in and , along with the orientations of dFBr in these sites predicted by computational docking. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 10. caption a7 dFBr binding site in the Torpedo muscle-type nAChR ion channel. (A and B) Side view (A) and view from within the ion channel toward the α - β - δ subunits (B) of a nAChR homology model based on the X-ray structure of the mouse serotonin 5-HT3R (PDB ID 4PIR ) ( Hassaine et al., 2014 ) and (C) a homology model based on the cryo-electron microscopy structure of Torpedo marmorata nAChR (PDB ID 2BG9 ) ( Unwin, 2005 ).

Techniques: Sequencing, Isolation

Journal: Molecular Pharmacology

Article Title: Desformylflustrabromine (dFBr) and [ 3 H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

doi: 10.1124/mol.115.098913

Figure Lengend Snippet: dFBr binding site in the Torpedo muscle-type nAChR ion channel. (A and B) Side view (A) and view from within the ion channel toward the α-β-δ subunits (B) of a nAChR homology model based on the X-ray structure of the mouse serotonin 5-HT3R (PDB ID 4PIR) (Hassaine et al., 2014) and (C) a homology model based on the cryo-electron microscopy structure of Torpedo marmorata nAChR (PDB ID 2BG9) (Unwin, 2005). The nAChR α, β, γ, and δ subunits are colored in gold, blue, green, and violet, respectively. Amino acids photolabeled by [3H]dFBr within the α/β/δM2 helices are colored in green and identified by their position numbers from the N-terminus of the M2 helices. In each model, the ensemble of the 100 lowest energy dFBr docking solutions within the channel is shown as a Connolly surface representation colored by atom (C, black; H, white; Br, red), defining volumes of 905 Å3 (B) and 821 Å3 (C) compared with the dFBr molecular volume of 241 Å3.

Article Snippet: The locations of the photolabeled amino acids in the ion channel and in the ECD are shown in nAChR homology models in and , along with the orientations of dFBr in these sites predicted by computational docking. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 10. caption a7 dFBr binding site in the Torpedo muscle-type nAChR ion channel. (A and B) Side view (A) and view from within the ion channel toward the α - β - δ subunits (B) of a nAChR homology model based on the X-ray structure of the mouse serotonin 5-HT3R (PDB ID 4PIR ) ( Hassaine et al., 2014 ) and (C) a homology model based on the cryo-electron microscopy structure of Torpedo marmorata nAChR (PDB ID 2BG9 ) ( Unwin, 2005 ).

Techniques: Binding Assay, Cryo-Electron Microscopy

Journal: Molecular Pharmacology

Article Title: Desformylflustrabromine (dFBr) and [ 3 H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

doi: 10.1124/mol.115.098913

Figure Lengend Snippet: dFBr binding sites in the Torpedo nAChR ECD. Views of the homology model, the Torpedo nAChR ECD based on the x-ray structure of the L-AChBP in the presence of Carb (PDB ID 1UV6) (Celie et al., 2004) with the α, β, γ, and δ subunits colored in gold, blue, green, and violet, respectively. A view from the top (A) and side views from the exterior (B and D) are shown with dFBr, in space-filling representation, docked at three distinct sites in the presence of the agonist Carb (yellow). Sites I and II are at the agonist-binding canonical interface near entry to the ACh binding site (Site I; A, D, and E) and in the vestibule of the ion channel (Site II; A, D, and F). Site III (A, B, and C) is at the δ-β subunit interface. Views from the exterior (C and E) and a view from the ion channel vestibule (F) showing dFBr docked in its predicted lowest energy orientations in Site I (E), Site II (F), and Site III (C).

Article Snippet: The locations of the photolabeled amino acids in the ion channel and in the ECD are shown in nAChR homology models in and , along with the orientations of dFBr in these sites predicted by computational docking. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 10. caption a7 dFBr binding site in the Torpedo muscle-type nAChR ion channel. (A and B) Side view (A) and view from within the ion channel toward the α - β - δ subunits (B) of a nAChR homology model based on the X-ray structure of the mouse serotonin 5-HT3R (PDB ID 4PIR ) ( Hassaine et al., 2014 ) and (C) a homology model based on the cryo-electron microscopy structure of Torpedo marmorata nAChR (PDB ID 2BG9 ) ( Unwin, 2005 ).

Techniques: Binding Assay