Nonequivalence of .alpha.-bungarotoxin binding sites in the native nicotinic receptor molecule

Conti‐Tronconi, Bianca M.; Tang, Fengyan; Walgrave, Susan L.; Gallagher, Warren H.

doi:10.1021/bi00456a029

Cited by 31 publications

(22 citation statements)

References 62 publications

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“…This suggests that the number of detectable ␣-BTX-binding sites per pentameric ␣7 receptor is one, consistent with the third prediction above. Since, under certain experimental conditions, only one of the two ␣-BTXbinding sites of the muscle-type nAChR can be detected (39,40), it is possible that the ␣7 receptor consists of two or more ␣-BTX-binding sites, but only one is detected under our experimental conditions. In either case, the estimated molar ratio supports the current working hypothesis that the ␣-BTX-binding nAChR is a homo-oligomer of ␣7 subunits in the rat brain.…”

Section: Discussionmentioning

confidence: 93%

The α-Bungarotoxin-binding Nicotinic Acetylcholine Receptor from Rat Brain Contains Only the α7 Subunit

Chen

Patrick

1997

Journal of Biological Chemistry

202

124

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When expressed in Xenopus oocytes, the rat ␣7 subunit forms homo-oligomeric nicotinic acetylcholine receptors, which are blocked by ␣-bungarotoxin. Since the pharmacological and physiological properties of the ␣7 receptor expressed in oocytes are similar to those of the ␣-bungarotoxin-sensitive nicotinic currents recorded from neuronal preparations and the distribution patterns of ␣7 mRNA and ␣-bungarotoxin-binding sites in the rat brain are very similar, ␣7 is thought to be the main component of the ␣-bungarotoxin-binding nicotinic receptor in the mammalian brain. However, while ␣7 is found in purified ␣-bungarotoxin-binding complexes from rat brain or PC12 cells, other proteins copurify with it. Therefore, the question whether ␣7 forms a homo-oligomeric ␣-bungarotoxin-binding nicotinic receptor in the mammalian brain remains. We have developed and characterized affinity-purified polyclonal antibodies and used these antibodies in Western blot analyses of ␣-bungarotoxin-binding proteins purified from rat brains. We report here that our experimental data support the current working hypothesis that the ␣-bungarotoxin-binding nicotinic receptor is a homooligomer of ␣7 subunits in the rat brain.␣-Bungarotoxin (␣-BTX), 1 a snake toxin that binds to the muscle-type nicotinic acetylcholine receptor (nAChR), also binds to the surface of sympathetic ganglion cells, cultured PC12 cells (rat pheochromocytoma cells), and brain membranes. The ␣-BTX-binding sites in these preparations are distinct subpopulations of the neuronal nAChRs. This conclusion is based on the observations that 1) the ␣-BTX-sensitive nAChRs have a relatively low affinity for nicotine in comparison with the ␣-BTX-insensitive receptors (1); 2) the anatomical distribution of ␣-BTX-binding sites and nicotine-binding sites in the brain is highly distinct (2); 3) ␣-BTX binds to the surface of PC12 cells, but fails to block nicotinic agonist-mediated responses in these cells (3-5); and 4) ␣-BTX-sensitive and -insensitive nicotinic receptors in PC12 cells and in the brain can be distinguished immunologically (6, 7).The molecular basis for these differences have been revealed by cloning the genes encoding subunits of the neuronal nAChRs (for reviews, see Ref. 8). In the rat, 10 genes encoding neuronal nAChR subunits have been identified to date. Seven encode ␣ subunits (␣2-␣7 and ␣9), and three encode ␤ subunits (␤2-␤4). When expressed in Xenopus oocytes, only ␣7 and ␣9 form homo-oligomeric nAChRs, and activation of these receptors can be blocked by ␣-BTX (9, 10). Since in situ hybridization has shown the ␣9 subunit to be present in hair cells but not in the brain and the ␣9 receptor expressed in oocytes has unique pharmacological properties that are similar to those of the cholinergic receptor observed in vertebrate cochlear hair cells, the ␣9 receptor is thought to be involved primarily in the cholinergic efferent innervation of the cochlear hair cells (10). On the other hand, ␣7 mRNA is widely expressed in the rat brain and is thought to be the main componen...

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Section: Discussionmentioning

confidence: 93%

The α-Bungarotoxin-binding Nicotinic Acetylcholine Receptor from Rat Brain Contains Only the α7 Subunit

Chen

Patrick

1997

Journal of Biological Chemistry

202

124

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“…23), no detachment of [ 125 I]␣BTx from preformed AChRϪ[ 125 I]␣BTx complexes was detected under the crystallization and emulsification conditions.…”

Section: Emulsification Of the Lipidic Matrix Triggers The Formation mentioning

confidence: 95%

“…Two ␣BTx molecules can specifically bind one receptor heteropentamer. The affinity of the toxin for both sites of a detergent-soluble Torpedo AChR is similar and very high (23), displaying equilibrium dissociation constant (K d ) in the picomolar range. The half-life of the complex is Ͼ10 days (23).…”

Section: Purification Of Achr-␣btx Complexes For Crystallizationmentioning

confidence: 99%

“…The affinity of the toxin for both sites of a detergent-soluble Torpedo AChR is similar and very high (23), displaying equilibrium dissociation constant (K d ) in the picomolar range. The half-life of the complex is Ͼ10 days (23). Accordingly, no detachment of the toxin from the receptor was observed during the preparation of AChR-␣BTx complexes.…”

Section: Purification Of Achr-␣btx Complexes For Crystallizationmentioning

confidence: 99%

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Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid–detergent matrices

Paas

Cartaud

Recouvreur

et al. 2003

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

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Nicotinic acetylcholine receptors (AChRs) belong to a superfamily of oligomeric proteins that transduce electric signals across the cell membrane on binding of neurotransmitters. These receptors harbor a large extracellular ligand-binding domain directly linked to an ion-conducting channel-forming domain that spans the cell membrane 20 times and considerably extends into the cytoplasm. Thus far, none of these receptor channels has been crystallized in three dimensions. The crystallization of the AChR from Torpedo marmorata electric organs is challenged here in lipidic؊detergent matrices. Detergent-soluble AChR complexed with ␣-bungarotoxin (␣BTx), a polypeptidic competitive antagonist, was purified. The AChR-␣BTx complex was reconstituted in a lipidic matrix composed of monoolein bilayers that are structured in three dimensions. The ␣BTx was conjugated to a photo-stable fluorophore, enabling us to monitor the physical behavior of the receptor-toxin complex in the lipidic matrix under light stereomicroscope, and to freeze fracture regions containing the receptor-toxin complex for visualization under a transmission electron microscope. Conditions were established for forming 2D receptor-toxin lattices that are stacked in the third dimension. 3D AChR nanocrystals were thereby grown inside the highly viscous lipidic 3D matrix. Slow emulsification of the lipidic matrix converted these nanocrystals into 3D elongated thin crystal plates of micrometer size. The latter are stable in detergent-containing aqueous solutions and can currently be used for seeding and epitaxial growth, en route to crystals of appropriate dimensions for x-ray diffraction studies.nicotinic acetylcholine receptor ͉ crystallization ͉ structure T o date, the crystal structures of Ϸ50 integral membrane proteins have been determined at atomic resolution. This result is in stark contrast to their abundance, as they comprise one-third of all gene products. Inherent difficulties with highlevel expression, purification, and, particularly, crystallization of integral membrane proteins account for this situation (1). Receptor channels that selectively regulate transport of ions across the cell membrane are large membrane-spanning oligomers. Among them are the pentameric ligand-gated ion channels (also termed the Cys-loop receptors), which mediate and modulate fast cell-cell communication throughout the nervous system.The first and most extensively studied pentameric ligand-gated ion channel is the acetylcholine receptor (AChR; reviewed in refs. 2-4). The AChR isolated from the electric organs of Torpedo ray is available in milligram amounts suitable for crystallization experiments. It is a pseudosymmetrical glycoprotein of Ϸ290 kDa comprised of five subunits (2␣␤␥␦) and carries two ACh-binding sites at the ␣Ϫ␥ and ␣Ϫ␦ boundaries (2, 3). So far, it was possible to organize this oligomeric protein only in tubular 2D crystals within receptor-rich membrane fragments (5, 6). These crystals enabled the collection of electron diffraction data at 9-Å down to 4-Å r...

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“…The nAChR-binding activity of Oh-4, Oh-7, and their Trp-modified derivatives was determined by modification of the centrifugation assay described by Conti-Tronconi et al (1990). A constant amount of nAChR-enriched membrane fragments (12/.cg) and 3H-a-bungarotoxin (0.685 pmol) was incubated with increasing concentration of unlabeled Oh-4, Oh-7, or their modified derivatives.…”

Section: Receptor Binding Assaymentioning

confidence: 99%

Chemical modification of tryptophan residues in α-neurotoxins fromOphiophagus hannah (king cobra) venom

et al. 1995

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Two alpha-neurotoxins, Oh-4 and Oh-7, from the king cobra (Ophiophagus hannah) venom were subjected to Trp modification with 2-nitrophenylsulfenyl chloride (NPS-Cl). One major NPS derivative was isolated from the modified mixtures of Oh-4 and two from Oh-7 by HPLC. Amino acid analysis and sequence determination revealed that Trp-27 in Oh-4, and Trp-30 and Trp-26 and 30 in the two Oh-7 derivatives, were modified, respectively. Sulfenylation of Trp-27 in Oh-4 caused about 70% drop in lethal toxicity and nicotinic acetylcholine receptor-binding activity. Modification of Trp-30 in Oh-7 resulted in the decrease of lethal toxicity by 36% and binding activity by 61%. The activities were further lost when the conserved Trp-26 in Oh-7 was modified. Sulfenylation of the Trp residues did not significantly affect the secondary structure of the toxins as revealed by the CD spectra. These results indicate that the Trp residues in these two long alpha-neurotoxins may be involved in the receptor binding.

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Nonequivalence of .alpha.-bungarotoxin binding sites in the native nicotinic receptor molecule

Cited by 31 publications

References 62 publications

The α-Bungarotoxin-binding Nicotinic Acetylcholine Receptor from Rat Brain Contains Only the α7 Subunit

The α-Bungarotoxin-binding Nicotinic Acetylcholine Receptor from Rat Brain Contains Only the α7 Subunit

Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid–detergent matrices

Chemical modification of tryptophan residues in α-neurotoxins fromOphiophagus hannah (king cobra) venom

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