Dataset Information

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

ABSTRACT: Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of ?4?2 and ?2?2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and ?7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (????) and Torpedo (????) nAChR expressed in Xenopus oocytes with IC50 values of ? 1 ?M. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([(3)H]phencyclidine; IC50 = 4 ?M) than in the resting state ([(3)H]tetracaine; IC50 = 60 ?M), whereas it bound with only very low affinity to the ACh binding sites ([(3)H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [(3)H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [(3)H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical ?-? interface containing the transmitter binding sites and at the noncanonical ?-? subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [(3)H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity.

SUBMITTER: Hamouda AK

PROVIDER: S-EPMC4468644 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Publications

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

Hamouda Ayman K AK Wang Ze-Jun ZJ Stewart Deirdre S DS Jain Atul D AD Glennon Richard A RA Cohen Jonathan B JB

Molecular pharmacology 20150413 1

Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo (αβγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼ 1 μM. dFBr also inhibited the equilibrium binding ...[more]

PMID: 25870334

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Project description:Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in ?-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the ?-? subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 ?M, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the ?-? subunit interface, photolabeling ?M2-10 (?Ser-252) and ?Met-295 and ?Met-299 within ?M3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the ? subunit M2-M3 loop (?Ile-288) and the ? subunit transmembrane helix bundle (?Phe-232 and ?Cys-236 within ?M1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the ?-? subunit interface and/or within ? subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the ?-? subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate).

Dataset Information

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

Publications

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

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