Interaction of [125I]-.alpha.-bungarotoxin with acetylcholine receptor from Torpedo californica

Blanchard, Steven G.; Quast, Ulrich; Reed, K.; Lee, Theodore; Schimerlik, Michael I.; Vandlen, Richard; Claudio, Toni; Strader, Catherine D.; Moore, Helen; Raftery, Michael A.

doi:10.1021/bi00577a005

Cited by 110 publications

(86 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The quotient of k off and k on , 0.5 nM, is lower than the K D value of 4 nM obtained from equilibrium binding experiments. The difference can be explained taking into account the second order kinetics of association: in the range of lower concentrations, on is slower than in the case of near-saturating concentrations (38,59), which results in apparently lower concentrations of bound radioligand.…”

Section: Resultsmentioning

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

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

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

View full text Add to dashboard Cite

show abstract

“…The initial specific activity was in the range of 40 -80 Ci/mmol. For competitive binding assays monoiodinated ␣-bungarotoxin was prepared by chromatography on CM-Sephadex essentially as described previously (24). The specific activity was adjusted by addition of unlabeled ␣-bungarotoxin to a range of 60 -120 Ci/mmol.…”

Section: Methodsmentioning

confidence: 99%

The Drosophila Acetylcholine Receptor Subunit Dα5 Is Part of an α-Bungarotoxin Binding Acetylcholine Receptor

Wu¹,

Ma²,

Pierzchala

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

The central nervous system of Drosophila melanogaster contains an ␣-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an ␣-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 mol of 125 I-␣-bungarotoxin binding sites/g of protein.The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl Nicotinic acetylcholine receptors (nAChRs)1 are key elements of fast cholinergic synaptic transmission (1, 2). They are part of a large superfamily of ligand-gated ion channels comprising 5HT 3 , GABA A , and GABA C , glycine, and invertebrate anionic glutamate receptors. nAChRs are widely distributed in both vertebrates and invertebrates. In addition to their occurrence in the highly specialized tissue of the electrical organ of certain species of fish (Torpedo californica, Electrophorus electricus), nAChRs are mostly found at the vertebrate neuromuscular junction and the nervous system of both vertebrates and invertebrates (3, 4).Structurally, nAChRs are formed by assembly of their subunits into a pentameric membrane protein complex. Subunits carrying two conserved adjacent cysteines in the amino-terminal domain in the vicinity of the acetylcholine binding site are classified as ␣-subunits, while the remainder of the subunit types are designated non-␣ or -␤. The individual subunits have a large amino-terminal domain, which includes the binding site for the ligand, followed by four membrane spanning domains M1-M4. The M2 segments of the five subunits form the lining of the ion channel, which is transiently permeable to cations (Na ϩ , K ϩ , Ca 2ϩ ) upon binding of the ligand acetylcholine (ACh). Between M3 and M4 a large intracellular loop exists, which contains several phosphorylation sites required for receptor stabilization (for reviews, see Refs. 3, 5, and 6).Biochemical analysis of receptors subunits composition in a variety of species as well the information obtained from the sequencing of the genome of humans, mice, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster indicates a large number of genes encoding nicotinic acetylcholine receptor subunits. Nicotinic acetylcholine receptors from muscle have an invariant subunit composition ((␣1) 2 (␤1)␦␥ (fetal) or ⑀). Neuronal nicotinic acetylcholine receptors are composed of ␣ and non-␣-subunits with a stoichoimetry of (␣) 2 (non-␣) 3 . The large number of genes encoding nAChR subunits in the nervous system can give rise to an array of functionally distinct nAChRs. Since the vertebrate nervous system also contains homomeric nAChRs (e.g. (␣ 7 ) 5 ) the entire range ...

show abstract

“…The concentration of a-BuTx sites was determined according to Schmidt and Raftery (42) with DEAE-cellulose filter discs and 125I-labeled a-BuTx (43). Protein concentration was determined by the method of Lowry et al (44), with bovine serum albumin as the standard.…”

Section: Methodsmentioning

confidence: 99%

Topographic studies of Torpedo acetylcholine receptor subunits as a transmembrane complex.

Strader¹,

Raftery²

1980

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The exposure of the four subunits of the acetylcholine receptor from Torpedo californica on both the extracellular and cytoplasmic faces of the postsynaptic membranes of the electroplaque cells has been investigated. Sealed membrane vesicles containing no protein components other than the receptor were isolated and were shown to have 95% of their synaptic surfaces facing the medium. The susceptibility of the four receptor subunits in these preparations to hydrolysis by trypsin both from the external and from the internal medium was used to investigate the exposure of the subunits on the synaptic and cytoplasmic surfaces of the membrane. It was shown by sodium dodecyl sulfate gel electrophoresis of the tryptic products that all four subunits are exposed on the extracellular surface to a similar degree. All four subunits are also exposed on the internal surface of the membrane, but the apparent degree of exposure varies with the subunit size, the larger subunits being more exposed. The results are discussed in terms of a possible topographic model of the receptor as a transmembrane protein complex.Excitable membrane vesicles highly enriched in the acetylcholine receptor (AcChoR) have been purified from the electric organs of several species of electric fish. These membrane preparations possess the properties of nicotinic postsynaptic membranes: they bind a-neurotoxins (1, 2) and cholinergic ligands (3, 4) and they possess distinct binding sites for local anesthetics (4-7) and the alkaloid histrionicotoxin (8, 9). Binding of cholinergic agonists results in the flux of inorganic cations through the vesicular membrane (10-13), and the demonstration of such flux through membrane preparations containing only the AcChoR protein (14,15) suggests that the AcChoR functions as a cation-translocating protein complex.Detergent-extracted, chromatographically purified AcChoR from Torpedo californica sediments both as a 9S monomer and as a 13.7S dimer (16). Except for one account (17), both species have been found to consist of four subunits of 40,000, 50,000, 60,000, and 65,000 daltons, with binding sites for a-neurotoxins, agonists, and some antagonists located on the 40,000-dalton subunit (18-30). These four subunits were found to possess a high incidence of amino acid sequence homology (31) and to exist in both membrane-bound and detergent-solubilized receptor as a pentameric complex with a stoichiometry of 2:1:1:1 (31, 32). This stoichiometry dictates a size of 255,000 daltons for the AcChoR monomer, in agreement with the experimentally determined values of 250,000-270,000 daltons (33-35). In light of this molecular definition of the AcChoR and of its defined function alluded to above, it is of interest to determine the topography of its subunits in the membrane and eventually to assign a specific function to each polypeptide.By use of antibodies to purified AcChoR, it has been found at the electron microscopic level by both thin-sectioning (36) and examination of replicas of intact and sheared membrane vesicles (37...

show abstract

Interaction of [125I]-.alpha.-bungarotoxin with acetylcholine receptor from Torpedo californica

Cited by 110 publications

References 27 publications

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

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

The Drosophila Acetylcholine Receptor Subunit Dα5 Is Part of an α-Bungarotoxin Binding Acetylcholine Receptor

Topographic studies of Torpedo acetylcholine receptor subunits as a transmembrane complex.

Contact Info

Product

Resources

About