Advertisement

Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli*

Open AccessPublished:July 06, 2016DOI:https://doi.org/10.1074/jbc.M116.729970
      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.

      Introduction

      Human Cys-loop receptors are promising therapeutic targets for various neurological disorders and diseases (
      • Dineley K.T.
      • Pandya A.A.
      • Yakel J.L.
      Nicotinic ACh receptors as therapeutic targets in CNS disorders.
      • Braat S.
      • Kooy R.F.
      The GABAA Receptor as a Therapeutic Target for Neurodevelopmental Disorders.
      • Lynch J.W.
      • Callister R.J.
      Glycine receptors: a new therapeutic target in pain pathways.
      • Thompson A.J.
      • Lummis S.C.
      The 5-HT3 receptor as a therapeutic target.
      ). Structure-based drug design for these receptors requires their high-resolution structures (
      • Anderson A.C.
      The Process of Structure-Based Drug Design.
      ). Although Cys-loop receptors contain only four major receptor types, including nicotinic acetylcholine receptors (nAChRs),
      The abbreviations used are:
      nAChR
      nicotinic acetylcholine receptor
      pLGIC
      pentameric ligand-gated ion channel
      ECD
      extracellular domain
      TMD
      transmembrane domain
      EM
      electron microscopy.
      serotonin 5-HT3 receptors, glycine receptors, and GABAA receptors, each receptor type often has multiple subtypes that form numerous functional distinct receptors. Among the human Cys-loop receptors, high-resolution structures have been obtained for only the β3 GABAA and α3 glycine receptors (
      • Miller P.S.
      • Aricescu A.R.
      Crystal structure of a human GABAA receptor.
      ,
      • Huang X.
      • Chen H.
      • Michelsen K.
      • Schneider S.
      • Shaffer P.L.
      Crystal structure of human glycine receptor-α3 bound to antagonist strychnine.
      ). Structures for other eukaryotic Cys-loop receptors include the mouse serotonin 5-HT3A receptor (
      • Hassaine G.
      • Deluz C.
      • Grasso L.
      • Wyss R.
      • Tol M.B.
      • Hovius R.
      • Graff A.
      • Stahlberg H.
      • Tomizaki T.
      • Desmyter A.
      • Moreau C.
      • Li X.D.
      • Poitevin F.
      • Vogel H.
      • Nury H.
      X-ray structure of the mouse serotonin 5-HT3 receptor.
      ), the zebrafish α1 glycine receptor (
      • Du J.
      • Lü W.
      • Wu S.
      • Cheng Y.
      • Gouaux E.
      Glycine receptor mechanism elucidated by electron cryo-microscopy.
      ), the Caenorhabditis elegans GluCl (
      • Hibbs R.E.
      • Gouaux E.
      Principles of activation and permeation in an anion-selective Cys-loop receptor.
      ,
      • Althoff T.
      • Hibbs R.E.
      • Banerjee S.
      • Gouaux E.
      X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors.
      ) and the muscle-type nicotinic acetylcholine receptor (nAChR) from Torpedo marmorota (
      • Unwin N.
      Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
      ). The dichotomy between the small number of available structures and the relatively large receptor population in the superfamily indicates the technical difficulties for structural determination of these receptors. One of the greatest challenges for structural determination of Cys-loop receptors and similarly complex human membrane proteins is the production of a large quantity of well-folded functional proteins.
      The α7 nAChR (α7nAChR) is one of the most abundant nAChR subtypes found in the brain (
      • Couturier S.
      • Bertrand D.
      • Matter J.M.
      • Hernandez M.C.
      • Bertrand S.
      • Millar N.
      • Valera S.
      • Barkas T.
      • Ballivet M.
      A neuronal nicotinic acetylcholine receptor subunit (α7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX.
      ,
      • Fabian-Fine R.
      • Skehel P.
      • Errington M.L.
      • Davies H.A.
      • Sher E.
      • Stewart M.G.
      • Fine A.
      Ultrastructural distribution of the α7 nicotinic acetylcholine receptor subunit in rat hippocampus.
      ). It is also expressed in a wide variety of non-neuronal tissues (
      • Zdanowski R.
      • Krzyzowska M.
      • Ujazdowska D.
      • Lewicka A.
      • Lewicki S.
      Role of α7 nicotinic receptor in the immune system and intracellular signaling pathways.
      ). It has been implicated in diverse biological functions and is an important target for therapeutics (
      • Dineley K.T.
      • Pandya A.A.
      • Yakel J.L.
      Nicotinic ACh receptors as therapeutic targets in CNS disorders.
      ). α7nAChR mostly forms a homo-pentameric ligand-gated ion channel (pLGIC) that conducts calcium and other cations, though heteromeric α7β2-nAChR has also been found in both heterologous expression systems and native neurons (
      • Wu J.
      • Liu Q.
      • Tang P.
      • Mikkelsen J.D.
      • Shen J.
      • Whiteaker P.
      • Yakel J.L.
      Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain.
      ,
      • Mowrey D.D.
      • Liu Q.
      • Bondarenko V.
      • Chen Q.
      • Seyoum E.
      • Xu Y.
      • Wu J.
      • Tang P.
      Insights into distinct modulation of α7 and α7β2 nicotinic acetylcholine receptors by the volatile anesthetic isoflurane.
      ). Structures of the extracellular domain (ECD) and transmembrane domain (TMD) of α7nAChR have been determined separately by x-ray crystallography (
      • Li S.X.
      • Huang S.
      • Bren N.
      • Noridomi K.
      • Dellisanti C.D.
      • Sine S.M.
      • Chen L.
      Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.
      ) and NMR (
      • Bondarenko V.
      • Mowrey D.D.
      • Tillman T.S.
      • Seyoum E.
      • Xu Y.
      • Tang P.
      NMR structures of the human α7 nAChR transmembrane domain and associated anesthetic binding sites.
      ), but the full-length human α7nAChR has not previously been obtained from any species in a form suitable for structure determination (
      • Aztiria E.M.
      • Sogayar M.C.
      • Barrantes F.J.
      Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems.
      ,
      • Dineley K.T.
      • Patrick J.W.
      Amino acid determinants of α7 nicotinic acetylcholine receptor surface expression.
      ).
      Currently available structures for Cys-loop receptors were obtained from proteins expressed in mammalian (
      • Miller P.S.
      • Aricescu A.R.
      Crystal structure of a human GABAA receptor.
      ,
      • Hassaine G.
      • Deluz C.
      • Grasso L.
      • Wyss R.
      • Tol M.B.
      • Hovius R.
      • Graff A.
      • Stahlberg H.
      • Tomizaki T.
      • Desmyter A.
      • Moreau C.
      • Li X.D.
      • Poitevin F.
      • Vogel H.
      • Nury H.
      X-ray structure of the mouse serotonin 5-HT3 receptor.
      ) and insect (
      • Huang X.
      • Chen H.
      • Michelsen K.
      • Schneider S.
      • Shaffer P.L.
      Crystal structure of human glycine receptor-α3 bound to antagonist strychnine.
      ,
      • Du J.
      • Lü W.
      • Wu S.
      • Cheng Y.
      • Gouaux E.
      Glycine receptor mechanism elucidated by electron cryo-microscopy.
      • Hibbs R.E.
      • Gouaux E.
      Principles of activation and permeation in an anion-selective Cys-loop receptor.
      • Althoff T.
      • Hibbs R.E.
      • Banerjee S.
      • Gouaux E.
      X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors.
      ,
      • Moraga-Cid G.
      • Sauguet L.
      • Huon C.
      • Malherbe L.
      • Girard-Blanc C.
      • Petres S.
      • Murail S.
      • Taly A.
      • Baaden M.
      • Delarue M.
      • Corringer P.J.
      Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure.
      ) cell lines. We chose an alternate approach: to produce functional full-length human α7nAChR in E. coli. Production of recombinant proteins in E. coli is fast and inexpensive relative to other expression systems. In addition, E. coli may produce a more homogeneous population of purified proteins due to its limited ability for post-translational modifications (
      • Cain J.A.
      • Solis N.
      • Cordwell S.J.
      Beyond gene expression: the impact of protein post-translational modifications in bacteria.
      ). In contrast, the native pathway for α7nAChR expression in mammalian cells involves subcellular trafficking through multiple subcellular compartments, where specific chaperone proteins and post-translational modifications aid folding and assembly. Thus, α7nAChR purified from mammalian or insect cells contain a mixture of receptors with different post-translational modifications representing various stages of maturation, including glycosylation, palmitoylation, and alternate disulfide conformations (
      • Millar N.S.
      • Harkness P.C.
      Assembly and trafficking of nicotinic acetylcholine receptors (Review).
      • Drisdel R.C.
      • Manzana E.
      • Green W.N.
      The role of palmitoylation in functional expression of nicotinic α7 receptors.
      • Green W.N.
      • Millar N.S.
      Ion-channel assembly.
      ). These modifications were reported to be essential for the proper assembly and navigation of functional α7nAChR to the mammalian cell surface (
      • Millar N.S.
      • Harkness P.C.
      Assembly and trafficking of nicotinic acetylcholine receptors (Review).
      ). We hypothesize that these modifications are not needed for the proper folding and pentameric assembly of α7nAChR in the simpler expression environment of E. coli.
      Here we report that α7nAChR purified from E. coli retains signature properties of native α7nAChR, including the ability to assemble into pentameric structures, to bind specific ligands, and to form functional ion channels that can be activated by agonists, inhibited by channel blockers, and enhanced by α7nAChR-specific positive allosteric modulators. The study demonstrates that E. coli is capable of producing human Cys-loop receptors. It also suggests that the post-translational machinery may not be as essential as previously thought for expressing functional complex membrane proteins.

      Discussion

      Among all the members of the Cys-loop superfamily of ligand gated ion channels, α7nAChR is known to be particularly difficult to express (
      • Aztiria E.M.
      • Sogayar M.C.
      • Barrantes F.J.
      Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems.
      ,
      • Dineley K.T.
      • Patrick J.W.
      Amino acid determinants of α7 nicotinic acetylcholine receptor surface expression.
      ). Its functional expression in mammalian cells is cell-type dependent, which has been linked to the requirement for specific chaperone proteins and post-translational modifications (
      • Millar N.S.
      • Harkness P.C.
      Assembly and trafficking of nicotinic acetylcholine receptors (Review).
      ). Even in permissive cells, it has been estimated that only 62% of the total α7nAChR protein present in the cell is in a mature functional form (
      • Peng J.H.
      • Fryer J.D.
      • Hurst R.S.
      • Schroeder K.M.
      • George A.A.
      • Morrissy S.
      • Groppi V.E.
      • Leonard S.S.
      • Lukas R.J.
      High-affinity epibatidine binding of functional, human α7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells.
      ). This may be a problem inherent to heterologous expression in eukaryotic cells, not only for α7nAChR but also for other homologous receptors. Indeed, those eukaryotic Cys-loop channels for which structures have been successfully determined have all been mutagenized or processed in vitro in an effort to improve monodispersity of the final product (
      • Miller P.S.
      • Aricescu A.R.
      Crystal structure of a human GABAA receptor.
      • Huang X.
      • Chen H.
      • Michelsen K.
      • Schneider S.
      • Shaffer P.L.
      Crystal structure of human glycine receptor-α3 bound to antagonist strychnine.
      • Hassaine G.
      • Deluz C.
      • Grasso L.
      • Wyss R.
      • Tol M.B.
      • Hovius R.
      • Graff A.
      • Stahlberg H.
      • Tomizaki T.
      • Desmyter A.
      • Moreau C.
      • Li X.D.
      • Poitevin F.
      • Vogel H.
      • Nury H.
      X-ray structure of the mouse serotonin 5-HT3 receptor.
      • Du J.
      • Lü W.
      • Wu S.
      • Cheng Y.
      • Gouaux E.
      Glycine receptor mechanism elucidated by electron cryo-microscopy.
      • Hibbs R.E.
      • Gouaux E.
      Principles of activation and permeation in an anion-selective Cys-loop receptor.
      • Althoff T.
      • Hibbs R.E.
      • Banerjee S.
      • Gouaux E.
      X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors.
      ,
      • Moraga-Cid G.
      • Sauguet L.
      • Huon C.
      • Malherbe L.
      • Girard-Blanc C.
      • Petres S.
      • Murail S.
      • Taly A.
      • Baaden M.
      • Delarue M.
      • Corringer P.J.
      Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure.
      ). Production of functional human α7nAChR in E. coli suggests that specific chaperone proteins and post-translational modifications are not required for channel function, but instead are a consequence of sub-cellular trafficking in eukaryotic cells. Post-translational modification in E. coli is limited (
      • Cain J.A.
      • Solis N.
      • Cordwell S.J.
      Beyond gene expression: the impact of protein post-translational modifications in bacteria.
      ), which may improve homogeneity of the protein expression. Our results show that the E. coli chaperones induced by osmotic and cold shock as well as the chemical chaperone choline are sufficient for producing large quantities of well-folded α7nAChR.
      In addition to the expression conditions, the oligomeric state of α7nAChR was sensitive to purification procedures. Under optimal conditions, the pentameric form was stable for 2 or 3 days at 4 °C. However, the isolated pentamer fraction had a tendency to aggregate under conditions of low ionic strength or detergent concentration; or to dissociate to smaller oligomeric structures under conditions of high ionic strength or detergent concentration. The tendency to form multiple oligomeric structures may be an intrinsic property of α7nAChR. Metabolically labeled α7nAChR obtained from mammalian PC12 cell culture through microscale purification was found to form multiple oligomeric structures (
      • Drisdel R.C.
      • Green W.N.
      Neuronal α-bungarotoxin receptors are α7 subunit homomers.
      ). A mutated construct of the zebra finch α7nAChR expressed in HEK293F cells also showed micro-aggregation by negative-stain EM (
      • Cheng H.
      • Fan C.
      • Zhang S.W.
      • Wu Z.S.
      • Cui Z.C.
      • Melcher K.
      • Zhang C.H.
      • Jiang Y.
      • Cong Y.
      • Xu H.E.
      Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.
      ). Our study suggests that homogenous α7nAChR can be obtained by carefully controlling the purification conditions and time window.
      E. coli readily expresses the bacterial homologues of Cys-loop receptors, such as GLIC (
      • Bocquet N.
      • Nury H.
      • Baaden M.
      • Le Poupon C.
      • Changeux J.P.
      • Delarue M.
      • Corringer P.J.
      X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation.
      ,
      • Hilf R.J.
      • Dutzler R.
      Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.
      ) and ELIC (
      • Hilf R.J.
      • Dutzler R.
      X-ray structure of a prokaryotic pentameric ligand-gated ion channel.
      ). Because of the traditional wisdom that post-translational modification is essential for producing functional eukaryotic channel proteins, using E. coli to express mammalian Cys-loop receptors is almost uncharted territory. Our results challenge that traditional wisdom and suggest that careful manipulation of expression conditions can allow production of functional human α7nAChR in E. coli. It is likely that other members of the Cys-loop receptor superfamily can also be produced from E. coli following similar protocols. Given the pharmaceutical interest in these receptors as therapeutic targets, perhaps their expression in E. coli should be revisited.

      Author Contributions

      T. S. T. conducted most of the experiments and analyzed the results. F. J. D. A., C. L., and P. Z. performed the electron microscopy experiments. N. J. R. performed electrophysiology measurements. D. W. and K. X. performed the mass spectroscopy analysis. Y. X. and P. T. designed the project. T. S. T. and P. T. wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.

      References

        • Dineley K.T.
        • Pandya A.A.
        • Yakel J.L.
        Nicotinic ACh receptors as therapeutic targets in CNS disorders.
        Trends Pharmacol. Sci. 2015; 36: 96-108
        • Braat S.
        • Kooy R.F.
        The GABAA Receptor as a Therapeutic Target for Neurodevelopmental Disorders.
        Neuron. 2015; 86: 1119-1130
        • Lynch J.W.
        • Callister R.J.
        Glycine receptors: a new therapeutic target in pain pathways.
        Curr. Opin. Investig. Drugs. 2006; 7: 48-53
        • Thompson A.J.
        • Lummis S.C.
        The 5-HT3 receptor as a therapeutic target.
        Expert. Opin. Ther. Targets. 2007; 11: 527-540
        • Anderson A.C.
        The Process of Structure-Based Drug Design.
        Chem. Biol. 2003; 10: 787-797
        • Miller P.S.
        • Aricescu A.R.
        Crystal structure of a human GABAA receptor.
        Nature. 2014; 512: 270-275
        • Huang X.
        • Chen H.
        • Michelsen K.
        • Schneider S.
        • Shaffer P.L.
        Crystal structure of human glycine receptor-α3 bound to antagonist strychnine.
        Nature. 2015; 526: 277-280
        • Hassaine G.
        • Deluz C.
        • Grasso L.
        • Wyss R.
        • Tol M.B.
        • Hovius R.
        • Graff A.
        • Stahlberg H.
        • Tomizaki T.
        • Desmyter A.
        • Moreau C.
        • Li X.D.
        • Poitevin F.
        • Vogel H.
        • Nury H.
        X-ray structure of the mouse serotonin 5-HT3 receptor.
        Nature. 2014; 512: 276-281
        • Du J.
        • Lü W.
        • Wu S.
        • Cheng Y.
        • Gouaux E.
        Glycine receptor mechanism elucidated by electron cryo-microscopy.
        Nature. 2015; 526: 224-229
        • Hibbs R.E.
        • Gouaux E.
        Principles of activation and permeation in an anion-selective Cys-loop receptor.
        Nature. 2011; 474: 54-60
        • Althoff T.
        • Hibbs R.E.
        • Banerjee S.
        • Gouaux E.
        X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors.
        Nature. 2014; 512: 333-337
        • Unwin N.
        Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
        J. Mol. Biol. 2005; 346: 967-989
        • Couturier S.
        • Bertrand D.
        • Matter J.M.
        • Hernandez M.C.
        • Bertrand S.
        • Millar N.
        • Valera S.
        • Barkas T.
        • Ballivet M.
        A neuronal nicotinic acetylcholine receptor subunit (α7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX.
        Neuron. 1990; 5: 847-856
        • Fabian-Fine R.
        • Skehel P.
        • Errington M.L.
        • Davies H.A.
        • Sher E.
        • Stewart M.G.
        • Fine A.
        Ultrastructural distribution of the α7 nicotinic acetylcholine receptor subunit in rat hippocampus.
        J. Neurosci. 2001; 21: 7993-8003
        • Zdanowski R.
        • Krzyzowska M.
        • Ujazdowska D.
        • Lewicka A.
        • Lewicki S.
        Role of α7 nicotinic receptor in the immune system and intracellular signaling pathways.
        Cent. Eur. J. Immunol. 2015; 40: 373-379
        • Wu J.
        • Liu Q.
        • Tang P.
        • Mikkelsen J.D.
        • Shen J.
        • Whiteaker P.
        • Yakel J.L.
        Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain.
        Trends Pharmacol. Sci. 2016; 37: 562-574
        • Mowrey D.D.
        • Liu Q.
        • Bondarenko V.
        • Chen Q.
        • Seyoum E.
        • Xu Y.
        • Wu J.
        • Tang P.
        Insights into distinct modulation of α7 and α7β2 nicotinic acetylcholine receptors by the volatile anesthetic isoflurane.
        J. Biol. Chem. 2013; 288: 35793-35800
        • Li S.X.
        • Huang S.
        • Bren N.
        • Noridomi K.
        • Dellisanti C.D.
        • Sine S.M.
        • Chen L.
        Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.
        Nat. Neurosci. 2011; 14: 1253-1259
        • Bondarenko V.
        • Mowrey D.D.
        • Tillman T.S.
        • Seyoum E.
        • Xu Y.
        • Tang P.
        NMR structures of the human α7 nAChR transmembrane domain and associated anesthetic binding sites.
        Biochim. Biophys. Acta. 2014; 1838: 1389-1395
        • Aztiria E.M.
        • Sogayar M.C.
        • Barrantes F.J.
        Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems.
        Neurochem. Res. 2000; 25: 171-180
        • Dineley K.T.
        • Patrick J.W.
        Amino acid determinants of α7 nicotinic acetylcholine receptor surface expression.
        J. Biol. Chem. 2000; 275: 13974-13985
        • Moraga-Cid G.
        • Sauguet L.
        • Huon C.
        • Malherbe L.
        • Girard-Blanc C.
        • Petres S.
        • Murail S.
        • Taly A.
        • Baaden M.
        • Delarue M.
        • Corringer P.J.
        Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure.
        Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 2865-2870
        • Cain J.A.
        • Solis N.
        • Cordwell S.J.
        Beyond gene expression: the impact of protein post-translational modifications in bacteria.
        J. Proteom. 2014; 97: 265-286
        • Millar N.S.
        • Harkness P.C.
        Assembly and trafficking of nicotinic acetylcholine receptors (Review).
        Mol. Membr. Biol. 2008; 25: 279-292
        • Drisdel R.C.
        • Manzana E.
        • Green W.N.
        The role of palmitoylation in functional expression of nicotinic α7 receptors.
        J. Neurosci. 2004; 24: 10502-10510
        • Green W.N.
        • Millar N.S.
        Ion-channel assembly.
        Trends Neurosci. 1995; 18: 280-287
        • Tillman T.S.
        • Seyoum E.
        • Mowrey D.D.
        • Xu Y.
        • Tang P.
        ELIC-α7 Nicotinic acetylcholine receptor (α7nAChR) chimeras reveal a prominent role of the extracellular-transmembrane domain interface in allosteric modulation.
        J. Biol. Chem. 2014; 289: 13851-13857
        • de Marco A.
        • Vigh L.
        • Diamant S.
        • Goloubinoff P.
        Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.
        Cell Stress Chaperones. 2005; 10: 329-339
        • Molinari E.J.
        • Delbono O.
        • Messi M.L.
        • Renganathan M.
        • Arneric S.P.
        • Sullivan J.P.
        • Gopalakrishnan M.
        Up-regulation of human α7 nicotinic receptors by chronic treatment with activator and antagonist ligands.
        Eur. J. Pharmacol. 1998; 347: 131-139
        • Rangwala F.
        • Drisdel R.C.
        • Rakhilin S.
        • Ko E.
        • Atluri P.
        • Harkins A.B.
        • Fox A.P.
        • Salman S.S.
        • Green W.N.
        Neuronal α-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors.
        J. Neurosci. 1997; 17: 8201-8212
        • Peng J.H.
        • Fryer J.D.
        • Hurst R.S.
        • Schroeder K.M.
        • George A.A.
        • Morrissy S.
        • Groppi V.E.
        • Leonard S.S.
        • Lukas R.J.
        High-affinity epibatidine binding of functional, human α7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells.
        J. Pharmacol. Exp. Ther. 2005; 313: 24-35
        • Grønlien J.H.
        • Håkerud M.
        • Ween H.
        • Thorin-Hagene K.
        • Briggs C.A.
        • Gopalakrishnan M.
        • Malysz J.
        Distinct profiles of α7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes.
        Mol. Pharmacol. 2007; 72: 715-724
        • Gill J.K.
        • Savolainen M.
        • Young G.T.
        • Zwart R.
        • Sher E.
        • Millar N.S.
        Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 5867-5872
        • Rakhilin S.
        • Drisdel R.C.
        • Sagher D.
        • McGehee D.S.
        • Vallejo Y.
        • Green W.N.
        α-bungarotoxin receptors contain α7 subunits in two different disulfide-bonded conformations.
        J. Cell Biol. 1999; 146: 203-218
        • Alkondon M.
        • Pereira E.F.
        • Cortes W.S.
        • Maelicke A.
        • Albuquerque E.X.
        Choline is a selective agonist of α7 nicotinic acetylcholine receptors in the rat brain neurons.
        Eur. J. Neurosci. 1997; 9: 2734-2742
        • Labriola J.M.
        • Pandhare A.
        • Jansen M.
        • Blanton M.P.
        • Corringer P.J.
        • Baenziger J.E.
        Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment.
        J. Biol. Chem. 2013; 288: 11294-11303
        • Miledi R.
        • Palma E.
        • Eusebi F.
        Microtransplantation of neurotransmitter receptors from cells to Xenopus oocyte membranes: new procedure for ion channel studies.
        Methods Mol. Biol. 2006; 322: 347-355
        • Morales A.
        • Aleu J.
        • Ivorra I.
        • Ferragut J.A.
        • Gonzalez-Ros J.M.
        • Miledi R.
        Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8468-8472
        • Csárdi G.
        • Franks A.
        • Choi D.S.
        • Airoldi E.M.
        • Drummond D.A.
        Accounting for experimental noise reveals that mRNA levels, amplified by post-transcriptional processes, largely determine steady-state protein levels in yeast.
        PLoS Genetics. 2015; 11: e1005206
        • Cuevas J.
        • Adams D.J.
        Local anaesthetic blockade of neuronal nicotinic ACh receptor-channels in rat parasympathetic ganglion cells.
        Br. J. Pharmacol. 1994; 111: 663-672
        • Connolly J.
        • Boulter J.
        • Heinemann S.F.
        α4–2 β2 and other nicotinic acetylcholine receptor subtypes as targets of psychoactive and addictive drugs.
        Br. J. Pharmacol. 1992; 105: 657-666
        • Fryer J.D.
        • Lukas R.J.
        Noncompetitive functional inhibition at diverse, human nicotinic acetylcholine receptor subtypes by bupropion, phencyclidine, and ibogaine.
        J. Pharmacol. Exp. Ther. 1999; 288: 88-92
        • Hurst R.S.
        • Hajós M.
        • Raggenbass M.
        • Wall T.M.
        • Higdon N.R.
        • Lawson J.A.
        • Rutherford-Root K.L.
        • Berkenpas M.B.
        • Hoffmann W.E.
        • Piotrowski D.W.
        • Groppi V.E.
        • Allaman G.
        • Ogier R.
        • Bertrand S.
        • Bertrand D.
        • Arneric S.P.
        A novel positive allosteric modulator of the α7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization.
        J. Neurosci. 2005; 25: 4396-4405
        • Drisdel R.C.
        • Green W.N.
        Neuronal α-bungarotoxin receptors are α7 subunit homomers.
        J. Neurosci. 2000; 20: 133-139
        • Cheng H.
        • Fan C.
        • Zhang S.W.
        • Wu Z.S.
        • Cui Z.C.
        • Melcher K.
        • Zhang C.H.
        • Jiang Y.
        • Cong Y.
        • Xu H.E.
        Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.
        Acta Pharmacol. Sin. 2015; 36: 1013-1023
        • Bocquet N.
        • Nury H.
        • Baaden M.
        • Le Poupon C.
        • Changeux J.P.
        • Delarue M.
        • Corringer P.J.
        X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation.
        Nature. 2009; 457: 111-114
        • Hilf R.J.
        • Dutzler R.
        Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.
        Nature. 2009; 457: 115-118
        • Hilf R.J.
        • Dutzler R.
        X-ray structure of a prokaryotic pentameric ligand-gated ion channel.
        Nature. 2008; 452: 375-379
        • Qin H.
        • Hu J.
        • Hua Y.
        • Challa S.V.
        • Cross T.A.
        • Gao F.P.
        Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis.
        BMC Biotechnol. 2008; 8: 51
        • Marley J.
        • Lu M.
        • Bracken C.
        A method for efficient isotopic labeling of recombinant proteins.
        J. Biomol. NMR. 2001; 20: 71-75
        • Kahsai A.W.
        • Rajagopal S.
        • Sun J.
        • Xiao K.
        Monitoring protein conformational changes and dynamics using stable-isotope labeling and mass spectrometry.
        Nat. Protoc. 2014; 9: 1301-1319
        • Nobles K.N.
        • Xiao K.
        • Ahn S.
        • Shukla A.K.
        • Lam C.M.
        • Rajagopal S.
        • Strachan R.T.
        • Huang T.Y.
        • Bressler E.A.
        • Hara M.R.
        • Shenoy S.K.
        • Gygi S.P.
        • Lefkowitz R.J.
        Distinct phosphorylation sites on the β(2)-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin.
        Sci. Signal. 2011; 4: ra51
        • Haas W.
        • Faherty B.K.
        • Gerber S.A.
        • Elias J.E.
        • Beausoleil S.A.
        • Bakalarski C.E.
        • Li X.
        • Villén J.
        • Gygi S.P.
        Optimization and use of peptide mass measurement accuracy in shotgun proteomics.
        Mol. Cell Proteomics. 2006; 5: 1326-1337
        • Tang G.
        • Peng L.
        • Baldwin P.R.
        • Mann D.S.
        • Jiang W.
        • Rees I.
        • Ludtke S.J.
        EMAN2: an extensible image processing suite for electron microscopy.
        J. Struct. Biol. 2007; 157: 38-46
        • Scheres S.H.
        RELION: implementation of a Bayesian approach to cryo-EM structure determination.
        J. Struct. Biol. 2012; 180: 519-530
      1. Dascal, N., (2001) Voltage clamp recordings from Xenopus oocytes. Current Protocols in Neuroscience/ editorial board, Jacqueline N. Crawley et al., Chapter 6, Unit 6 12