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

摘要

Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of alpha 4 beta 2 and alpha 2b beta 2 nAChRs that, at concentrations >1 mu M, also inhibits these receptors and alpha 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 humanmuscle (alpha beta epsilon delta) andTorpedo (alpha beta gamma delta) nAChR expressed in Xenopus oocytes with IC50 values of similar to 1 mu M. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([H-3]phencyclidine; IC50 = 4 mu M) than in the resting state ([H-3]tetracaine; IC50 = 60 mu M), whereas it boundwith only very low affinity to the ACh binding sites ([H-3]ACh, IC50 = 1 mu M). Upon irradiation at 312 nm, [H-3]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, [H-3]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 alpha-gamma interface containing the transmitter binding sites and at the noncanonical delta-beta subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [H-3]dFBr is a photoaffinity probe with broad amino acid side chain reactivity.