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J Biol Chem
2016 Aug 26;29135:18276-82. doi: 10.1074/jbc.M116.729970.
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Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.
Tillman TS
,
Alvarez FJ
,
Reinert NJ
,
Liu C
,
Wang D
,
Xu Y
,
Xiao K
,
Zhang P
,
Tang P
.
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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.
Alkondon,
Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons.
1997, Pubmed
Alkondon,
Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons.
1997,
Pubmed
Althoff,
X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors.
2014,
Pubmed
Anderson,
The process of structure-based drug design.
2003,
Pubmed
Aztiria,
Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems.
2000,
Pubmed
Bocquet,
X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation.
2009,
Pubmed
Bondarenko,
NMR structures of the human α7 nAChR transmembrane domain and associated anesthetic binding sites.
2014,
Pubmed
,
Xenbase
Braat,
The GABAA Receptor as a Therapeutic Target for Neurodevelopmental Disorders.
2015,
Pubmed
Cain,
Beyond gene expression: the impact of protein post-translational modifications in bacteria.
2014,
Pubmed
Cheng,
Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.
2015,
Pubmed
Connolly,
Alpha 4-2 beta 2 and other nicotinic acetylcholine receptor subtypes as targets of psychoactive and addictive drugs.
1992,
Pubmed
,
Xenbase
Couturier,
A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX.
1990,
Pubmed
,
Xenbase
Csárdi,
Accounting for experimental noise reveals that mRNA levels, amplified by post-transcriptional processes, largely determine steady-state protein levels in yeast.
2015,
Pubmed
Cuevas,
Local anaesthetic blockade of neuronal nicotinic ACh receptor-channels in rat parasympathetic ganglion cells.
1994,
Pubmed
Dascal,
Voltage clamp recordings from Xenopus oocytes.
2001,
Pubmed
,
Xenbase
Dineley,
Nicotinic ACh receptors as therapeutic targets in CNS disorders.
2015,
Pubmed
Dineley,
Amino acid determinants of alpha 7 nicotinic acetylcholine receptor surface expression.
2000,
Pubmed
Drisdel,
Neuronal alpha-bungarotoxin receptors are alpha7 subunit homomers.
2000,
Pubmed
Drisdel,
The role of palmitoylation in functional expression of nicotinic alpha7 receptors.
2004,
Pubmed
Du,
Glycine receptor mechanism elucidated by electron cryo-microscopy.
2015,
Pubmed
Fabian-Fine,
Ultrastructural distribution of the alpha7 nicotinic acetylcholine receptor subunit in rat hippocampus.
2001,
Pubmed
Fryer,
Noncompetitive functional inhibition at diverse, human nicotinic acetylcholine receptor subtypes by bupropion, phencyclidine, and ibogaine.
1999,
Pubmed
Gill,
Agonist activation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.
2011,
Pubmed
,
Xenbase
Green,
Ion-channel assembly.
1995,
Pubmed
Grønlien,
Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes.
2007,
Pubmed
,
Xenbase
Haas,
Optimization and use of peptide mass measurement accuracy in shotgun proteomics.
2006,
Pubmed
Hassaine,
X-ray structure of the mouse serotonin 5-HT3 receptor.
2014,
Pubmed
Hibbs,
Principles of activation and permeation in an anion-selective Cys-loop receptor.
2011,
Pubmed
Hilf,
X-ray structure of a prokaryotic pentameric ligand-gated ion channel.
2008,
Pubmed
Hilf,
Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.
2009,
Pubmed
Huang,
Crystal structure of human glycine receptor-α3 bound to antagonist strychnine.
2015,
Pubmed
Hurst,
A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization.
2005,
Pubmed
,
Xenbase
Kahsai,
Monitoring protein conformational changes and dynamics using stable-isotope labeling and mass spectrometry.
2014,
Pubmed
Labriola,
Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment.
2013,
Pubmed
Li,
Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.
2011,
Pubmed
Lynch,
Glycine receptors: a new therapeutic target in pain pathways.
2006,
Pubmed
Marley,
A method for efficient isotopic labeling of recombinant proteins.
2001,
Pubmed
Miledi,
Microtransplantation of neurotransmitter receptors from cells to Xenopus oocyte membranes: new procedure for ion channel studies.
2006,
Pubmed
,
Xenbase
Millar,
Assembly and trafficking of nicotinic acetylcholine receptors (Review).
2008,
Pubmed
Miller,
Crystal structure of a human GABAA receptor.
2014,
Pubmed
Molinari,
Up-regulation of human alpha7 nicotinic receptors by chronic treatment with activator and antagonist ligands.
1998,
Pubmed
Moraga-Cid,
Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure.
2015,
Pubmed
Morales,
Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.
1995,
Pubmed
,
Xenbase
Mowrey,
Insights into distinct modulation of α7 and α7β2 nicotinic acetylcholine receptors by the volatile anesthetic isoflurane.
2013,
Pubmed
Nobles,
Distinct phosphorylation sites on the β(2)-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin.
2011,
Pubmed
Peng,
High-affinity epibatidine binding of functional, human alpha7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells.
2005,
Pubmed
Qin,
Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis.
2008,
Pubmed
Rakhilin,
alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.
1999,
Pubmed
Rangwala,
Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors.
1997,
Pubmed
Scheres,
RELION: implementation of a Bayesian approach to cryo-EM structure determination.
2012,
Pubmed
Tang,
EMAN2: an extensible image processing suite for electron microscopy.
2007,
Pubmed
Thompson,
The 5-HT3 receptor as a therapeutic target.
2007,
Pubmed
Tillman,
ELIC-α7 Nicotinic acetylcholine receptor (α7nAChR) chimeras reveal a prominent role of the extracellular-transmembrane domain interface in allosteric modulation.
2014,
Pubmed
Unwin,
Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
2005,
Pubmed
Wu,
Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain.
2016,
Pubmed
Zdanowski,
Role of α7 nicotinic receptor in the immune system and intracellular signaling pathways.
2015,
Pubmed
de Marco,
Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.
2005,
Pubmed
