title: Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.
abstract: Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human α7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design.
authors.affiliation: From the Departments of Anesthesiology.
authors.forename: Tommy S
authors.initials: TS
authors.lastname: Tillman
authors.affiliation: Structural Biology.
authors.forename: Frances J D
authors.initials: FJ
authors.lastname: Alvarez
authors.affiliation: From the Departments of Anesthesiology.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Reinert
authors.affiliation: Structural Biology.
authors.forename: Chuang
authors.initials: C
authors.lastname: Liu
authors.affiliation: Pharmacology and Chemical Biology, and.
authors.forename: Dawei
authors.initials: D
authors.lastname: Wang
authors.affiliation: From the Departments of Anesthesiology, Structural Biology, Pharmacology and Chemical Biology, and.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Pharmacology and Chemical Biology, and.
authors.forename: Kunhong
authors.initials: K
authors.lastname: Xiao
authors.affiliation: Structural Biology.
authors.forename: Peijun
authors.initials: P
authors.lastname: Zhang
authors.affiliation: From the Departments of Anesthesiology, Pharmacology and Chemical Biology, and Computational & Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260 ptang@pitt.edu.
authors.forename: Pei
authors.initials: P
authors.lastname: Tang
mesh_terms: D000818:Animals; D002038:Bungarotoxins / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007555:Isoxazoles / Q000737:chemistry / Q000494:pharmacology; D008012:Lidocaine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010622:Phencyclidine / Q000737:chemistry / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014981:Xenopus; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C508388:1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea; D002038:Bungarotoxins; D029968:Escherichia coli Proteins; D007555:Isoxazoles; D018832:Molecular Chaperones; D010671:Phenylurea Compounds; D011994:Recombinant Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; C006072:QX-222; D008012:Lidocaine; D010622:Phencyclidine
keywords: Cys-loop receptor; electron microscopy (EM); ion channel; nicotinic acetylcholine receptors (nAChR); pLGICs; pentameric ligand-gated ion channels; recombinant protein expression; α7 nAChR; α7nAChR