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• W2103770311 abstract "The N-terminal extracellular region (amino acids 1–209) of the α-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% α-helical and 45% β-structure. The fragment bound α-[3H]bungarotoxin in 1:1 stoichiometry with a K D value of 0.5 nm as determined from kinetic measurements (4 nm from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 × 105m−1s−1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with α-[3H]bungarotoxin yielding the following K I values: acetylcholine, 69 μm; nicotine, 0.42 μm; anatoxin-a, 3 μm; tubocurarine, 400 μm; and methyllycaconitine, 0.12 μm. The results demonstrate that the N-terminal extracellular region of the nAChR α-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor. The N-terminal extracellular region (amino acids 1–209) of the α-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% α-helical and 45% β-structure. The fragment bound α-[3H]bungarotoxin in 1:1 stoichiometry with a K D value of 0.5 nm as determined from kinetic measurements (4 nm from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 × 105m−1s−1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with α-[3H]bungarotoxin yielding the following K I values: acetylcholine, 69 μm; nicotine, 0.42 μm; anatoxin-a, 3 μm; tubocurarine, 400 μm; and methyllycaconitine, 0.12 μm. The results demonstrate that the N-terminal extracellular region of the nAChR α-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor. nicotinic acetylcholine receptor acetylcholine recombinant fragment of the N-terminal extracellular region (amino acids 1–209) of the α-subunit of the nAChR circular dichroism isopropyl-β-thiogalactoside polymerase chain reaction nuclear magnetic resonance. So far, most of our knowledge on the three-dimensional structure of neuroreceptors is based on a combination of electronmicroscopical, biochemical and immunological data obtained for the nicotinic acetylcholine receptor (nAChR)1 from the electric ray Torpedo (1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar, 2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar). These studies have elucidated the overall dimensions of the receptor protein (3Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (715) Google Scholar), its position with respect to the surrounding lipid bilayer, the locations of functional domains and amino acid residues belonging to the integral ion channel (4Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (356) Google Scholar, 5Dreger M. Krauss M. Herrmann A. Hucho F. Biochemistry. 1997; 36: 839-847Crossref PubMed Scopus (40) Google Scholar), its gating structures (6Sakmann B. EMBO J. 1992; 11: 2002-2016Crossref PubMed Scopus (5) Google Scholar, 7Changeux J.P. Galzi J.L. Devillers T.A. Bertrand D. Q. Rev. Biophys. 1992; 25: 395-432Crossref PubMed Scopus (154) Google Scholar), and the binding sites for several classes of ligands (1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar, 2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar, 7Changeux J.P. Galzi J.L. Devillers T.A. Bertrand D. Q. Rev. Biophys. 1992; 25: 395-432Crossref PubMed Scopus (154) Google Scholar, 8Bertrand D. Changeux J.-P. Semin. Neurosci. 1995; 7: 75-90Crossref Scopus (94) Google Scholar, 9Conti-Fine B.M. Maelicke A. Reinhardt-Maelicke S. Chiapinelli V. McLane K.E. Ann. N. Y. Acad. Sci. 1995; 757: 133-152Crossref PubMed Scopus (10) Google Scholar, 10Schrattenholz A. Roth U. Godovac-Zimmermann J. Maelicke A. Biochemistry. 1997; 36: 13333-13340Crossref PubMed Scopus (11) Google Scholar, 11Schrattenholz A. Godovac-Zimmermann J. Schäfer H.J. Albuquerque E.X. Maelicke A Eur. J. Biochem. 1993; 216: 671-677Crossref PubMed Scopus (74) Google Scholar, 12Middleton R.E. Cohen J.B. Biochemistry. 1991; 30: 6987-6997Crossref PubMed Scopus (176) Google Scholar). However, a high resolution three-dimensional structure of the nAChR or any other neuroreceptor is still missing. If available, it could provide a molecular correlate for the recognition function of the receptor and thereby also for rational drug design.Prompted by the fact that all attempts to crystallize the detergent-solubilized whole nAChR protein have been unsuccessful for the past more than 20 years, we have begun to overexpress selected domains of the receptor in bacterial expression systems. If successfully renatured, the domains that are not transmembranous should be water-soluble and thus should provide a better material for protein crystallization than the detergent-solubilized whole receptor protein. Moreover, if small enough in size, such fragments should be suited for multidimensional NMR analysis (13Fraenkel Y. Shalev D.E. Gershoni J.M. Navon G. Crit. Rev. Biochem. Mol. Biol. 1996; 31: 273-301Crossref PubMed Scopus (17) Google Scholar). As presented here for the N-terminal extracellular region of the nAChR α-subunit, we attempted and achieved expression as a water-soluble globular protein that displays ligand binding properties comparable to or better than those of the SDS gel-isolated α-subunit. To achieve appropriate renaturation, we developed a protocol for the complete unfolding (by means of chaotropic agents and disulfide-reducing agents) and refolding (by means of an oxido shuffling system and l-arginine as structure-stabilizing agent) of the expressed polypeptide (14Rudolph R. Tscheche H. Modern Methods in Protein and Nucleic Acid Research. Walter de Gruyter, Berlin1990: 149-171Google Scholar). The experimental conditions were such that self-organization of the unfolded protein was favored at the expense of (unwanted) aggregation and denaturation. In this way, we are able to prepare large quantities of the functional ligand binding domain from inclusion bodies of transformed Escherichia coli bacteria. Our results confirm that the N-terminal extracellular domain indeed harbors major elements of the ligand recognition function of the nAChR, as has long been suggested on the basis of affinity labeling and immunological studies (for recent reviews, see Refs. 1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar, 2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar, 8Bertrand D. Changeux J.-P. Semin. Neurosci. 1995; 7: 75-90Crossref Scopus (94) Google Scholar, 9Conti-Fine B.M. Maelicke A. Reinhardt-Maelicke S. Chiapinelli V. McLane K.E. Ann. N. Y. Acad. Sci. 1995; 757: 133-152Crossref PubMed Scopus (10) Google Scholar, 15Léna C. Changeux J.P. Trends Neurosci. 1993; 16: 181-186Abstract Full Text PDF PubMed Scopus (173) Google Scholar, and 16Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar).DISCUSSIONThe Torpedo nAChR has been established as the prototypic model for fast ligand-gated ion channels in the central and peripheral nervous system due to its abundance in electric organs of electric fish (Torpedo and Electrophorus), which made it accessible to detailed biochemical and biophysical studies. To date, it has been impossible to obtain high resolution structural information at the atomic level due to resistance of the receptor protein to crystallization attempts (for recent reviews, see Refs. 1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar,2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar, 8Bertrand D. Changeux J.-P. Semin. Neurosci. 1995; 7: 75-90Crossref Scopus (94) Google Scholar, 9Conti-Fine B.M. Maelicke A. Reinhardt-Maelicke S. Chiapinelli V. McLane K.E. Ann. N. Y. Acad. Sci. 1995; 757: 133-152Crossref PubMed Scopus (10) Google Scholar, 15Léna C. Changeux J.P. Trends Neurosci. 1993; 16: 181-186Abstract Full Text PDF PubMed Scopus (173) Google Scholar, and 16Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar).Therefore, attention has focused on the expression of relevant functional domains of the nAChR (such as the large extracellular loops of nicotinic α-subunits, which contain most of the determinants for binding of agonists and competitive antagonists) in heterologous systems, always with the hope of finding a general method to obtain substantial amounts of material, suitable for structural analysis at the atomic level by NMR or x-ray/crystallography.Large scale expression of recombinant proteins in E. colioften results in confinement of the desired protein in so-called inclusion bodies, which concentrate the heterologous protein in a denatured and aggregated conformation. Recently, methods have been developed to restore the functional native conformation of recombinant proteins by ensuring conditions that allow correct refolding and disulfide bond formation starting from solubilized denatured material (14Rudolph R. Tscheche H. Modern Methods in Protein and Nucleic Acid Research. Walter de Gruyter, Berlin1990: 149-171Google Scholar, 28Rudolph R. Lilie H. FASEB J. 1996; 10: 49-56Crossref PubMed Scopus (568) Google Scholar, 29Sadana A. Biotechnol. Bioeng. 1995; 48: 481-489Crossref PubMed Scopus (17) Google Scholar, 43Chaudhuri J.B. Ann. N. Y. Acad. Sci. 1994; 721: 374-385Crossref PubMed Scopus (34) Google Scholar).Using an oxido shuffling system with reduced and oxidized glutathione and l-arginine as a stabilizing agent, we have been able to obtain large amounts of an α-nAChR fragment that is soluble in aqueous buffers without the use of detergents and contains the first 209 N-terminal amino acids, including relevant binding sites for agonists and competitive antagonists, as demonstrated by radioligand binding experiments. The affinity for α-bungarotoxin (K D value, 0.5 nm) is lower than for whole membrane-bound receptor (0.01 nm) (26Maelicke A. Fulpius B.W. Klett R.P. Reich E. J. Biol. Chem. 1977; 252: 4811-4830Abstract Full Text PDF PubMed Google Scholar, 39Maelicke A. Reich E. Cold Spring Harbor Symp. Quant. Biol. 1976; 40: 231-235Crossref PubMed Scopus (7) Google Scholar), but higher than for detergent-solubilized isolated α-subunit (100 nm) (40Haggerty J.G. Frohner S.C. J. Biol. Chem. 1981; 256: 8294-8297Abstract Full Text PDF PubMed Google Scholar, 41Gershoni J.M. Hawrot E. Lentz T.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4973-4977Crossref PubMed Scopus (85) Google Scholar). These differences in affinity probably arise from the fact that in native nAChR, ligand binding sites are composed of discontinuous subsites, which contribute to the actual ligand binding domain. Most of these determinants reside within α-subunits, although there is also some contribution from neighboring (mainly β) subunits. Moreover, evidence from photoaffinity labeling experiments seemed to indicate location of toxin binding sites at the interface of subunits (for recent reviews, see Refs. 1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar, 2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar, and 16Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar). The missing interaction of ligands with these additional subsites could be the explanation for the 40-fold lower affinity of α-bungarotoxin to α nAChR1–209 fragment compared with the native pentameric receptor. On the other hand, the affinity of α-bungarotoxin to isolated and solubilized nAChR α-subunit is even 200-fold weaker when compared with α nAChR1–209 fragment, probably because the use of detergents is disturbing crucial secondary structure elements (see also Table II).In native nAChR, there are different agonist-binding states, namely low and high affinity states. Events leading to opening of the receptor intrinsic ion channel involve sequential binding of two molecules of agonist to the two α-subunits at relatively low affinity (EC50 of 0.1–10 μm, depending upon agonist used), followed by conformational changes that are associated with an approximately 100–1000-fold increase in affinity for nicotine, for example, and a desensitized state of the receptor, which can no longer be activated by agonist (for recent reviews, see Refs. 1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar, 2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar, 15Léna C. Changeux J.P. Trends Neurosci. 1993; 16: 181-186Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 16Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar, and 55Edelstein S.J. Schaad O. Henry E. Bertrand D. Changeux J.P. Biol. Cybern. 1996; 75: 361-379Crossref PubMed Scopus (112) Google Scholar).K I values for displacement of α-bungarotoxin from the renatured αnAChR1–209 fragment by small nicotinic ligands are as follows: acetylcholine (69 μm), nicotine (0.42 μm), anatoxin-a (3 μm),d-tubocurarine (400 μm), and methyllycaconitine (0.12 μm); these values are very close to those reported from equilibrium binding studies employing entireTorpedo receptor (26Maelicke A. Fulpius B.W. Klett R.P. Reich E. J. Biol. Chem. 1977; 252: 4811-4830Abstract Full Text PDF PubMed Google Scholar) or the isolated Torpedoα-subunit (40Haggerty J.G. Frohner S.C. J. Biol. Chem. 1981; 256: 8294-8297Abstract Full Text PDF PubMed Google Scholar, 41Gershoni J.M. Hawrot E. Lentz T.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4973-4977Crossref PubMed Scopus (85) Google Scholar). Only in the case of acetylcholine was the affinity obtained for the αnAChR1–209 fragment significantly lower. These results again would imply that high affinity binding states for ACh might result from conformational changes recruiting additional subsites from other subunits, possibly at interfaces between subunits (see Table II for summary and comparison of K I and K D values).Secondary structure determination of the renatured α nAChR1–209 fragment by CD spectroscopy indicated 15% α-helical and 45% β-strand structure. Whereas the value for β-strand structure is consistent with data obtained for the membrane-bound and solubilized whole Torpedo nAChR by CD measurements (30Mielke D.L. Wallace B.A. J. Biol. Chem. 1988; 263: 3177-3182Abstract Full Text PDF PubMed Google Scholar, 31Wu C.-S.C. Sun X.H. Yang J.T. J. Protein Chem. 1990; 9: 119-126Crossref PubMed Scopus (13) Google Scholar, 32Moore W.M. Holladay L.A. Puett D. Brady R.N. FEBS Lett. 1974; 45: 145-149Crossref PubMed Scopus (43) Google Scholar), results from Fourier transformed infrared and Raman spectroscopy (33Yager P. Chang E.L. Williams R.W. Dalziel A.W. Biophys. J. 1984; 45: 26-28Abstract Full Text PDF PubMed Scopus (26) Google Scholar,34Méthot N. McCarthy M.P. Baenzinger J.E. Biochemistry. 1994; 33: 7709-7717Crossref PubMed Scopus (46) Google Scholar) and theoretical modeling (35Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 36Ortells M.O. Proteins. 1997; 29: 391-398Crossref PubMed Scopus (13) Google Scholar) (Table I) show that 15% α-helical structure is lower than the values of approximately 30 (37Unwin N. J. Mol. Biol. 1996; 257: 586-596Crossref PubMed Scopus (99) Google Scholar) and 20% (35Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholar) that have been obtained in two studies published so far explicitly concerning the extracellular N-terminal domain of nicotinic α-subunits. The first study used cryoelectron microscopy (36Ortells M.O. Proteins. 1997; 29: 391-398Crossref PubMed Scopus (13) Google Scholar), and the differences observed could be explained in terms of subtle conformational changes due to interactions at interfaces to other subunits; the theoretical model makes a prediction (20% α-helix) that is nearer to the value we obtained (15%) than to the 30% α-helix derived from cryoelectron microscopy. Taken together, the biophysical data suggest that the renaturation procedure resulted in a α nAChR1–209 fragment that appeared to be structurally homogenous and to display a secondary structure composition that is consistent with previous predictions.Expression of recombinant proteins in E. coli results in the absence of posttranslational modifications, like N-glycosylation, which are not part of the synthetic repertoire of bacteria. Native α-subunits of Torpedo nAChR are glycosylated on Asn 141, glycosylation does not affect ligand binding properties but rather seems to play an important role in transport of the different types of subunits to the plasma membrane and in the assembly of the receptor pentamer (see, e.g. Ref. 56Gehle V.M. Sumikawa K. Brain. Res. Mol. Brain Res. 1991; 11: 17-25Crossref PubMed Scopus (58) Google Scholar and, for reviews, Refs. 1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar, 2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar,15Léna C. Changeux J.P. Trends Neurosci. 1993; 16: 181-186Abstract Full Text PDF PubMed Scopus (173) Google Scholar, and 16Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar).Thus, the expression of the extracellular ligand binding domain of the α-subunit of Torpedo nAChR in E. coli and subsequent renaturation to a native conformation resulted in a water-soluble protein with ligand-binding properties very similar to the native receptor. The biochemical parameters confirm the homogeneity and functionality of the protein, which presents an example for the production and characterization of ligand binding domains of other nAChR, namely neuronal subtypes, which are not available from natural sources in high amounts, or from other members of the multigene superfamily of five-subunit ionotropic receptors, like γ-aminobutyric acidA, glycine, 5-hydroxytryptamine3 or glutamate receptors.To date, only the expression of short nicotinic receptor-fragment peptides, comprising mainly the assumed acetylcholine-binding site around the two adjacent cysteines 192 and 193, has been achieved in E. coli (41Gershoni J.M. Hawrot E. Lentz T.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4973-4977Crossref PubMed Scopus (85) Google Scholar), and a NMR solution structure of such peptides in complex with α-bungarotoxin has been published (44Basus V.J. Song G Hawrot E. Biochemistry. 1993; 32: 12290-12298Crossref PubMed Scopus (95) Google Scholar). Recently, there were entries in the Protein Data Bank at Brookhaven National Laboratory of the NMR solution structures of the M2 membrane-spanning domains (which are assumed to contribute to the ion channel pore) of rat nAChR and human glutamate receptor of N-methyl-d-aspartate-subtype NR1, which have been expressed in E. coli and reconstituted in artificial micelles (Protein Data Bank accession numbers 1A11 and 2NR1). In summary, expression in E. coli and reconstitution of functional domains of neurotransmitter receptors appears to be an attractive possibility to obtain detailed structural information in the absence of successful crystallization of the whole receptor protein. Alternative strategies include recent successful attempts to express the N-terminal domain of mouse muscle α-nAChR in the membranes of Xenopus oocytes or Chinese hamster ovary cells (53West Jr., A.P. Bjorkman P.J. Dougherty D.A. Lester H.A. J. Biol. Chem. 1997; 272: 25468-25473Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and rat α7 nAChR in Xenopus oocytes (54Wells G.B. Anand R. Wang F. Lindstrom J. J. Biol. Chem. 1998; 273: 964-973Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Solubility and yield remained problematic, although these systems have advantages with respect to posttranslational modifications.Also, for drug screening, large scale expression of neuroreceptor ligand binding domains offers attractive perspectives: pathological alterations of ligand-gated neuroreceptor ion channels are implicated in diseases or pathological conditions such as endogenous depression (γ-aminobutyric acidA receptors and 5-hydroxytryptamine3 receptors) (47Broekkamp C.L. Leysen D. Peeters B.W. Pinder R.M. J. Med. Chem. 1995; 38: 4615-4633Crossref PubMed Scopus (103) Google Scholar), stroke and amyotrophic lateral sclerosis (excitotoxicity and glutamate receptors) (48Shaw P.J. Ince P.G. J. Neurol. 1997; 244: S3-S14Crossref PubMed Google Scholar), schizophrenia (nAChR and γ-aminobutyric acidAreceptors) (49 and 50), myasthenia gravis, morbus Alzheimer, morbus Parkinson, and Chagas's disease (nAChR) (51Léna C. Changeux J.P. Curr. Opin. Neurobiol. 1997; 7: 674-682Crossref PubMed Scopus (87) Google Scholar, 52Gotti C. Fornasari D. Clementi F. Prog. Neurobiol. 1997; 53: 199-237Crossref PubMed Scopus (402) Google Scholar).Altogether, this work demonstrates the feasibility of large scale production of functional ligand binding domains of neuroreceptors in a prokaryotic expression system for structural investigations applying NMR or x-ray crystallography, at the same time offering possibilities with regard to pharmacological screening systems. So far, most of our knowledge on the three-dimensional structure of neuroreceptors is based on a combination of electronmicroscopical, biochemical and immunological data obtained for the nicotinic acetylcholine receptor (nAChR)1 from the electric ray Torpedo (1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar, 2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar). These studies have elucidated the overall dimensions of the receptor protein (3Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (715) Google Scholar), its position with respect to the surrounding lipid bilayer, the locations of functional domains and amino acid residues belonging to the integral ion channel (4Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (356) Google Scholar, 5Dreger M. Krauss M. Herrmann A. Hucho F. Biochemistry. 1997; 36: 839-847Crossref PubMed Scopus (40) Google Scholar), its gating structures (6Sakmann B. EMBO J. 1992; 11: 2002-2016Crossref PubMed Scopus (5) Google Scholar, 7Changeux J.P. Galzi J.L. Devillers T.A. Bertrand D. Q. Rev. Biophys. 1992; 25: 395-432Crossref PubMed Scopus (154) Google Scholar), and the binding sites for several classes of ligands (1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar, 2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar, 7Changeux J.P. Galzi J.L. Devillers T.A. Bertrand D. Q. Rev. Biophys. 1992; 25: 395-432Crossref PubMed Scopus (154) Google Scholar, 8Bertrand D. Changeux J.-P. Semin. Neurosci. 1995; 7: 75-90Crossref Scopus (94) Google Scholar, 9Conti-Fine B.M. Maelicke A. Reinhardt-Maelicke S. Chiapinelli V. McLane K.E. Ann. N. Y. Acad. Sci. 1995; 757: 133-152Crossref PubMed Scopus (10) Google Scholar, 10Schrattenholz A. Roth U. Godovac-Zimmermann J. Maelicke A. Biochemistry. 1997; 36: 13333-13340Crossref PubMed Scopus (11) Google Scholar, 11Schrattenholz A. Godovac-Zimmermann J. Schäfer H.J. Albuquerque E.X. Maelicke A Eur. J. Biochem. 1993; 216: 671-677Crossref PubMed Scopus (74) Google Scholar, 12Middleton R.E. Cohen J.B. Biochemistry. 1991; 30: 6987-6997Crossref PubMed Scopus (176) Google Scholar). However, a high resolution three-dimensional structure of the nAChR or any other neuroreceptor is still missing. If available, it could provide a molecular correlate for the recognition function of the receptor and thereby also for rational drug design. Prompted by the fact that all attempts to crystallize the detergent-solubilized whole nAChR protein have been unsuccessful for the past more than 20 years, we have begun to overexpress selected domains of the receptor in bacterial expression systems. If successfully renatured, the domains that are not transmembranous should be water-soluble and thus should provide a better material for protein crystallization than the detergent-solubilized whole receptor protein. Moreover, if small enough in size, such fragments should be suited for multidimensional NMR analysis (13Fraenkel Y. Shalev D.E. Gershoni J.M. Navon G. Crit. Rev. Biochem. Mol. Biol. 1996; 31: 273-301Crossref PubMed Scopus (17) Google Scholar). As presented here for the N-terminal extracellular region of the nAChR α-subunit, we attempted and achieved expression as a water-soluble globular protein that displays ligand binding properties comparable to or better than those of the SDS gel-isolated α-subunit. To achieve appropriate renaturation, we developed a protocol for the complete unfolding (by means of chaotropic agents and disulfide-reducing agents) and refolding (by means of an oxido shuffling system and l-arginine as structure-stabilizing agent) of the expressed polypeptide (14Rudolph R. Tscheche H. Modern Methods in Protein and Nucleic Acid Research. Walter de Gruyter, Berlin1990: 149-171Google Scholar). The experimental conditions were such that self-organization of the unfolded protein was favored at the expense of (unwanted) aggregation and denaturation. In this way, we are able to prepare large quantities of the functional ligand binding domain from inclusion bodies of transformed Escherichia coli bacteria. Our results confirm that the N-terminal extracellular domain indeed harbors major elements of the ligand recognition function of the nAChR, as has long been suggested on the basis of affinity labeling and immunological studies (for recent reviews, see Refs. 1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar, 2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar, 8Bertrand D. Changeux J.-P. Semin. Neurosci. 1995; 7: 75-90Crossref Scopus (94) Google Scholar, 9Conti-Fine B.M. Maelicke A. Reinhardt-Maelicke S. Chiapinelli V. McLane K.E. Ann. N. Y. Acad. Sci. 1995; 757: 133-152Crossref PubMed Scopus (10) Google Scholar, 15Léna C. Changeux J.P. Trends Neurosci. 1993; 16: 181-186Abstract Full Text PDF PubMed Scopus (173) Google Scholar, and 16Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar). DISCUSSIONThe Torpedo nAChR has been established as the prototypic model for fast ligand-gated ion channels in the central and peripheral nervous system due to its abundance in electric organs of electric fish (Torpedo and Electrophorus), which made it accessible to detailed biochemical and biophysical studies. To date, it has been impossible to obtain high resolution structural information at the atomic level due to resistance of the receptor protein to crystallization attempts (for recent reviews, see Refs. 1Maelicke A. Barnard E.A. Burgen A.S.V. Roberts G.C.K. Receptor Subunits and Complexes. Cambridge University Press, Cambridge, United Kingdom1992: 119-162Google Scholar,2Lindstrom J. Mol. Neurobiol. 1996; 15: 193-222Crossref Scopus (397) Google Scholar, 8Bertrand D. Changeux J.-P. Semin. Neurosci. 1995; 7: 75-90Crossref Scopus (94) Google Scholar, 9Conti-Fine B.M. Maelicke A. Reinhardt-Maelicke S. Chiapinelli V. McLane K.E. Ann. N. Y. Acad. Sci. 1995; 757: 133-152Crossref PubMed Scopus (10) Google Scholar, 15Léna C. Changeux J.P. Trends Neurosci. 1993; 16: 181-186Abstract Full Text PDF PubMed Scopus (173) Google Scholar, and 16Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar).Therefore, attention has focused on the expression of relevant functional domains of the nAChR (such as the large extracellular loops of nicotinic α-subunits, which contain most of the determinants for binding of agonists and competitive antagonists) in heterologous systems, always with the hope of" @default.
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