Crystalline arrays of membrane-bound acetylcholine receptor.

Kistler, Joerg; Stroud, Robert M.

doi:10.1073/pnas.78.6.3678

Cited by 84 publications

(52 citation statements)

References 24 publications

(26 reference statements)

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“…2 and 3). The spherical particles that compose these nanocrystals correspond to AChR-␣BTx complexes seen from a top view, as (i) only purified AChR-␣BTx complexes were used in the reconstitution step; (ii) these complexes were stably labeled with rhodamine, a fluorophore covalently attached to ␣BTx; and (iii) these particles display the shape and dimensions typical of the AChR, as previously visualized in native membrane preparations (5)(6)(7)26). Moreover, despite the low resolution of the applied freeze fracture͞transmission EM techniques, the pit surrounded by the walls of the receptor's extracellular domain was, in most cases, readily identified (e.g., Fig.…”

Section: Discussionmentioning

confidence: 99%

“…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 resolution (refs.…”

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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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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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“…Electron microscopy of negatively stained, purified, and membrane-bound preparations of receptor disclose ringlike particles or rosettes, 80 to 90 A in diameter, with a stain-filled central pit (38,(62)(63)(64)(65)(66) (Fig. lA).…”

Section: Morphology Of the Receptor Proteinmentioning

confidence: 98%

“…The two a chains, identified by biotinylated 1335 a-toxin (69,70), image analysis (68,71), or monoclonal antibody fragments (66), have been reported to make angles of 110°± 30Q (69,70), 144°± 4°(66), or 160°(68) and thus are not adjacent within the oligomer. On the basis of crosslinking experiments, the order oa y ot 8 p (36,69,70) has been proposed [see however (64)]. …”

Section: Morphology Of the Receptor Proteinmentioning

confidence: 99%

Acetylcholine Receptor: An Allosteric Protein

Changeux

Devillers‐Thiéry

Chemouilli

1984

Science

628

287

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The nicotine receptor for the neurotransmitter acetylcholine is an allosteric protein composed of four different subunits assembled in a transmembrane pentamer alpha 2 beta gamma delta. The protein carries two acetylcholine sites at the level of the alpha subunits and contains the ion channel. The complete sequence of the four subunits is known. The membrane-bound protein undergoes conformational transitions that regulate the opening of the ion channel and are affected by various categories of pharmacologically active ligands.

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“…Kistler & Stroud, 1981;Miyazawa et al, 1999). Tubes are built from tightly packed ribbons of receptor dimers, and intervening lipid molecules (Brisson & Unwin, 1984).…”

Section: Molecular Architecturementioning

confidence: 99%