Image analysis of the heavy form of the acetylcholine receptor from Torpedo marmorata

Bon, F.; Lebrun, Évelyne; Gomel, J.; Rapenbusch, Roland Van; Cartaud, Jean; Popot, Jean‐Luc; Changeux, Jean‐Pierre

doi:10.1016/0022-2836(84)90421-2

Cited by 43 publications

(21 citation statements)

References 67 publications

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“…Both in purified preparations or in situ, the heavy form looks, in agreement with the molecular weight studies (23,46), like a pair or doublet of light-form rosettes (68,74,75) (or, on the side, cylinders) juxtaposed at a center-to-center distance of 90 to 95 A (Fig. IA).…”

Section: Morphology Of the Receptor Proteinsupporting

confidence: 82%

“…The precise assignment of each of these peaks to specific subunits is not definitely settled except for the a subunit, which carries at least part of the a-bungarotoxin binding site. 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%

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Acetylcholine Receptor: An Allosteric Protein

Changeux

Devillers‐Thiéry

Chemouilli

1984

Science

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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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Section: Morphology Of the Receptor Proteinsupporting

confidence: 82%

Section: Morphology Of the Receptor Proteinmentioning

confidence: 99%

Acetylcholine Receptor: An Allosteric Protein

Changeux

Devillers‐Thiéry

Chemouilli

1984

Science

Self Cite

628

287

View full text Add to dashboard Cite

show abstract

“…Subunits 2 and 5, presumably the two a subunits, were more recessed. These observations agree very well with the reconstructed AChR image (22,24,25,30). A similar pentameric structure of AChR has been obtained by STM with isolated membranes from Torpedo electric organ (6); however, mechanisms for imaging relatively nonconductive biological samples tens of nanometers thick by STM are unknown.…”

Section: Methodssupporting

confidence: 84%

“…1B), or 1.9 x 109 AChR molecules per oocyte. For the 250-kDa multisubunit AChR, the minimum center-to-center distance between channels is 9-10 nm (19,(22)(23)(24)(25). The channels in closely packed areas (Fig.…”

Section: Methodsmentioning

confidence: 99%

Atomic force microscopy of cloned nicotinic acetylcholine receptor expressed in Xenopus oocytes.

Lal

Yu²

1993

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

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The nicotinic acetylcholine receptor (AChR) was expressed in Xenopus oocytes from in vitro transcribed mRNA and was imaged by atomic force microscopy. A characteristic pentameric structure of AChR was readily observed on the extracellular face of the cell membrane, with a central pore surrounded by protruding AChR subunits. These structures were seen only in mRNA-injected oocytes that also gave acetylcholine-induced membrane currents. The size of individual AChR channels, the angles between subunits, and the interchannel spacing were all compatible with the current model of AChR. In addition, localized patches of microscopic AChR clustering were observed, with packing density approaching that at the neuromuscular junction. These findings show the potential of studying cloned membrane proteins in oocytes for both their surface topography and their structurefunction relationship in native membrane without the need for crystallization.A large number of membrane proteins, including receptors, ion channels, and transporters, have been molecularly cloned, allowing characterization of their function. Based on the primary amino acid sequence of these proteins, their secondary structure and topology in the membrane have been proposed. However, their quaternary structure is mostly unknown. This is mainly due to the lack of a suitable method for three-dimensional imaging of membrane proteins: the existing techniques such as x-ray diffraction require crystallization, which is very difficult, if not impossible, for integral membrane proteins.Recently developed scanning probe microscopy, especially scanning tunneling microscopy (STM) and atomic force microscopy (AFM), can obtain three-dimensional images with nanometer resolution under physiological conditions (1-3). Images ofproteins, membranes, and nucleic acids have been obtained with . However, potential pitfalls of STM imaging of biological specimens exist (7), and image formation mechanisms are not known for relatively nonconducting biological materials of thickness greater than a few nanometers (2). This is because STM imaging relies on the probability of electron tunneling through the energy barrier, and such probability decreases exponentially with distance. AFM, on the other hand, is well suited for biological materials because it does not require electron-conductive materials, and has been used on many biological specimens (5,(8)(9)(10)(11)(12)(13)(14)(15), including macromolecules in noncrystalline form. However, membrane macromolecules studied by AFM were either purified or reconstituted in lipid vesicles. Such manipulations may alter the native topography of proteins due to either purification damage or altered lipid environment. In an attempt to image proteins in the intact cell membrane, we utilized Xenopus oocytes and expressed acetylcholine receptor (AChR) subunits from cDNA clones. We report here the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in a...

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“…The nAChR pentamer contains all the structural elements required for the physiological response: the binding sites for the cholinergic ligands, the ion channel and the mechanisms which ensure the various modes of coupling between these two categories of sites (Galzi et al, 1991b;Cockcroft et al, 1992;Ochoa et al, 1989Ochoa et al, , 1992. Electron microscopy of nAChR complexes with a bungarotoxin (Hamilton, McLaughlin & Karlin, 1977;Holtzman et al, 1982;Karlin et al, 1983;Bon et al, 1984;Kubalek et al, 1987) or with anti oz subunit antibodies (Fairclough et al, 1983) reveals that in the oligomet the two o~ subunits are not adjacent. Yet, the arrangement of the other subunits in the pentamer is still debated (Unwin, 1993).…”

mentioning

confidence: 99%