The functional architecture of the acetylcholine nicotinic receptor explored by affinity labelling and site-directed mutagenesis

Changeux, Jean‐Pierre; Galzi, Jean‐Luc; Devillers‐Thiéry, Anne; Bertrand, Daniel

doi:10.1017/s0033583500004352

Cited by 180 publications

(102 citation statements)

References 199 publications

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“…As a consequence of that discovery, the nAChR at the neuromuscular junction was extensively characterized. It is known to form a heteropentamer consisting of two ␣1-subunits, single ␤1-and ␦-subunits, and either a ␥-or an ⑀-subunit (Mishina et al, 1986;Changeux et al, 1992). In the fetal stage of development, mammalian muscle cells express nAChRs that consist of ␣1␤1␥␦-subunits, but, in late gestation, the ␥-subunit is replaced with the ⑀-subunit (␣1␤1⑀␦-subunits) (Mishina et al, 1986;Witzemann et al, 1987Witzemann et al, , 1989.…”

Section: Introductionmentioning

confidence: 99%

A Uniquely Selective Inhibitor of the Mammalian Fetal Neuromuscular Nicotinic Acetylcholine Receptor

Teichert¹,

Rivier²,

Torres³

et al. 2005

J. Neurosci.

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We have purified and characterized a novel conotoxin from the venom of Conus obscurus, which has the unique property of selectively and potently inhibiting the fetal form of the mammalian neuromuscular nicotinic acetylcholine receptor (nAChR) (␣1␤1␥␦-subunits). Although this conotoxin, ␣A-conotoxin OIVB (␣A-OIVB), is a high-affinity antagonist (IC 50 of 56 nM) of the fetal muscle nAChR, it has Ͼ1800-fold lower affinity for the adult muscle nAChR (␣1␤1⑀␦-subunits) and virtually no inhibitory activity at a high concentration on various neuronal nAChRs (IC 50 Ͼ 100 M in all cases). The peptide (amino acid sequence, CCGVONAACPOCVCNKTCG), with three disulfide bonds, has been chemically synthesized in a biologically active form. Although the neuromuscular nAChRs are perhaps the most extensively characterized of the receptors/ion channels of the nervous system, the precise physiological roles of the fetal form of the muscle nAChR are essentially unknown. ␣A-OIVB is a potentially important tool for delineating the functional roles of ␣1␤1␥␦ receptors in normal development, as well as in various adult tissues and in pathological states. In addition to its potential as a research tool, ␣A-OIVB may have some direct biomedical applications.

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Section: Introductionmentioning

confidence: 99%

A Uniquely Selective Inhibitor of the Mammalian Fetal Neuromuscular Nicotinic Acetylcholine Receptor

Teichert¹,

Rivier²,

Torres³

et al. 2005

J. Neurosci.

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“…In the pentameric complex of GABAR, there are two putative GABAbinding pockets at the interfaces of ␣ and ␤ subunits. Amino acids that participate in ligand binding have been identified by photoaffinity labeling and sequencing, as well as by site-directed mutagenesis (17)(18)(19)(20)(21)(22)(23). Loops of amino acids that form an acetylcholine-binding pocket were identified in the ␣ subunit of nAChR (24,25), and homologous residues involved in GABA binding were identified in ␤ subunits of GABAR (26,27).…”

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confidence: 99%

A Single M1 Residue in the β2 Subunit Alters Channel Gating of GABAA Receptor in Anesthetic Modulation and Direct Activation

Chang

Olcese

Olsen

2003

Journal of Biological Chemistry

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General anesthetics allosterically modulate the activity of neuronal ␥-aminobutyric acid, type A (GABA A ), receptors. Previous mutational studies from our laboratory and others have shown that the regions in transmembrane domain 1 (M1) and pre-M1 of ␣ and ␤ subunits in GABA receptors are essential for positive modulation of GABA binding and function by the intravenous (IV) general anesthetics. Mutation of ␤2Gly-219 to Phe corresponded in nearly eliminated the modulatory effect of IV anesthetics in ␣1/␤2/␥2S combination. However, the general anesthetics retained the ability to directly open the channel of mutant G219F, and the apparent affinity for GABA was increased, and desensitization rate was reduced. In this study, we made additional single mutations such as 219 Ser, Cys, Ile, Asp, Arg, Tyr, and Trp. The larger side chains of the replacement residues produced the greatest reduction in enhancement of GABA currents by IV anesthetics at clinical concentrations (Trp > Tyr ‫؍‬ Phe > Arg > Asp > Ile > Cys > Ser ‫؍‬ wild type). Compared with a 2-3-fold response in wild type, pentobarbital and propofol enhanced less than 0.5-fold; etomidate and alphaxalone modulation was reduced from more than 4-to 1-fold in G219F, G219Y, and G219W. A linear correlation was observed between the volume of the residue at position 219 and the loss of modulation. An identical correlation was found for the effect of modulation on left-shift in the GABA EC 50 value; furthermore, the same rank order of residues, related to size, was found for reduction in the maximal direct channel-gating by pentobarbital (1 mM) and etomidate (100 M) and for increased apparent affinity for direct gating by the IV anesthetics.Considerable evidence supports the hypothesis that general anesthesia can be produced, at least in part, by enhancing neuronal inhibition mediated by GABA A receptors (GABAR). 1 GABA and most volatile and general intravenous (IV) anesthetics bind to GABAR. These are ligand-gated channels that enhance the flow of chloride ions (Cl Ϫ ) into the postsynaptic neuron, thus increasing its resting potential and making it less likely to fire, i.e. the inhibitory effect in GABA synapses. GABAR are modulated positively by a wide variety of structurally diverse general anesthetics (1). In particular, volatile anesthetics (2, 3) and IV anesthetic agents such as the barbiturates (4, 5), propofol (6), etomidate (7), and neuroactive steroid anesthetics, like alphaxalone (8 -10), all enhance the function of GABAR at clinically relevant concentrations. In addition, IV and volatile anesthetics can activate GABAR directly in the absence of GABA (11). Anesthetics appear to allosterically modulate ligand-gated ion channel (LGIC) receptor function by binding to the receptor itself, but not at the agonist-binding site, and to affect the conformation of the membrane protein in a functional manner. Exactly how such modulatory sites affect agonist-regulated channel opening is a major contemporary question in pharmacology and neurobiology. Obviously, the struct...

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“…These studies have elucidated the overall dimensions of the receptor protein (3), its position with respect to the surrounding lipid bilayer, the locations of functional domains and amino acid residues belonging to the integral ion channel (4,5), its gating structures (6,7), and the binding sites for several classes of ligands (1,2,(7)(8)(9)(10)(11)(12). However, a high resolution three-dimensional structure of the nAChR or any other neuroreceptor is still missing.…”

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confidence: 99%

Expression and Renaturation of the N-terminal Extracellular Domain of Torpedo Nicotinic Acetylcholine Receptor α-Subunit

Schrattenholz¹,

Pfeiffer²,

Pejovic³

et al. 1998

Journal of Biological Chemistry

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The N-terminal extracellular region (amino acids 1-209) of the ␣-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% ␣-helical and 45% ␤-structure. The fragment bound ␣- 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 (1, 2). These studies have elucidated the overall dimensions of the receptor protein (3), its position with respect to the surrounding lipid bilayer, the locations of functional domains and amino acid residues belonging to the integral ion channel (4, 5), its gating structures (6, 7), and the binding sites for several classes of ligands (1, 2, 7-12). 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 (13). As presented here for the N-terminal extracellular region of the nAChR ␣-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 ␣-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 (14). 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. 1,2,8,9,15,and 16). EXPERIMENTAL PROCEDURE...

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The functional architecture of the acetylcholine nicotinic receptor explored by affinity labelling and site-directed mutagenesis

Cited by 180 publications

References 199 publications

A Uniquely Selective Inhibitor of the Mammalian Fetal Neuromuscular Nicotinic Acetylcholine Receptor

A Uniquely Selective Inhibitor of the Mammalian Fetal Neuromuscular Nicotinic Acetylcholine Receptor

A Single M1 Residue in the β2 Subunit Alters Channel Gating of GABAA Receptor in Anesthetic Modulation and Direct Activation

Expression and Renaturation of the N-terminal Extracellular Domain of Torpedo Nicotinic Acetylcholine Receptor α-Subunit

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