Biochemical properties of acetylcholine receptor subunits from Torpedo californica

Lindstrom, Jon; Merlie, John P.; Yogeeswaran, Ganesa

doi:10.1021/bi00588a003

Cited by 241 publications

(116 citation statements)

References 37 publications

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“…More At the ultrastructural level, high-resolution electron micrographs of the AcChoR rosettes show from four to six similar "subunits" arranged about an electron-dense core (18,(48)(49)(50). This is consistent with recent reports that the receptor molecule consists of four homologous subunits present in the ratio 2:1:1:1 (31,32), for a total of five similar subunits, all exposed on the extracellular surface of the membrane. The four subunits show a considerable amount of sequence homology at their amino termini (31); however, it is not known whether the homology continues through the carboxyl termini of all four polypeptides.…”

Section: Discussionsupporting

confidence: 88%

“…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)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(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)(34)(35).…”

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confidence: 99%

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

Strader¹,

Raftery²

1980

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

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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...

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Section: Discussionsupporting

confidence: 88%

mentioning

confidence: 99%

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

Strader¹,

Raftery²

1980

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

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“…Studies of heteromeric nACh receptors, in which the stoichiometry has been established (Reynolds and Karlin, 1978;Lindstrom et al, 1979;Raftery et al, 1980), concluded that each subunit of the receptor complex carrying this leucine mutation contributed an Ϸ10-fold increase in ACh sensitivity (Filatov and White, 1995;Labarca et al, 1995). For example, if two subunits in the nACh receptor complex are mutated, an Ϸ100-fold increase in ACh sensitivity was observed.…”

Section: Resultsmentioning

confidence: 99%

Stoichiometry of a Recombinant GABA_AReceptor

et al. 1996

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GABA is the main inhibitory neurotransmitter in the mammalian brain. The postsynaptic GABA A receptor/pore complex is presumed to be a pentamer typically composed of a combination of ␣, ␤, and ␥ subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABA A receptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat ␣1(␣L263S), ␤2(␣L259S), and ␥2(␣L274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., ␣ and ␣L263S), along with their wild-type counterparts (e.g., ␤ and ␥), in Xenopus laevis oocytes resulted in mixed populations of receptors with distinct GABA sensitivities. This is consistent with the interpretation that the leucine mutation increased the GABA sensitivity in proportion to the number of incorporated mutant subunits. The apparent number of incorporated subunits for each class (␣, ␤, and ␥) could then be determined from the number of components comprising the compound GABA dose-response relationships. Using this approach, we conclude that the recombinant ␣1␤2␥2 GABA A receptor is a pentamer composed of two ␣ subunits, two ␤ subunits, and one ␥ subunit.

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“…Their stoichiometry in the mature complex is a2Py8 (5)(6)(7). These subunits are synthesized with signal sequences (8)(9)(10) and are cotranslationally integrated into the RER membrane (1) by a mechanism that utilizes the recently purified (11,12) signal recognition particle (8).…”

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confidence: 99%

Identification of homo-oligomers as potential intermediates in acetylcholine receptor subunit assembly.

Anderson¹,

Blobel²

1983

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

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We have examined the sedimentation behavior, on sucrose density gradients, of acetylcholine receptor (AcChoR) subunits synthesized in vitro and integrated into heterologous rough microsomal membranes. In media containing nondenaturing detergents such as Triton X-100 or deoxycholate, the subunits appear to self-associate although, as previously reported, no heterologous interactions were detected. The sedimentation profiles assume a broad distribution in the region of 7-13 S. However, the peak fractions occupy the same region of the gradient as does native AcChoR, run in parallel. Such large homo-oligomers were not observed for another membrane protein, opsin, studied in the same way. This indicated that the associations are indeed between the AcChoR subunits and not simply between all newly synthesized membrane proteins. The homologous associations are interpreted to suggest a mechanism for maintaining the ionophore surfaces of the subunits in an energetically preferred, but metastable, configuration during the lengthy period of post-translational assembly.The oligomeric assembly of the acetylcholine receptor (AcChoR) subunits poses a unique problem in membrane protein biogenesis-that is, how four distinct transmembrane glycoproteins, translated from separate mRNAs (1), are assembled into a quaternary complex that forms a hydrophilic ion channel through the lipid bilayer. The problem is further complicated by the observation that AcChoR subunit assembly does not occur immediately upon insertion of the subunits into the rough endoplasmic reticulum (RER) membrane (1) but rather after a delay of at least 30 min, or perhaps even longer (2). This fact implies that (i) newly synthesized AcChoR subunits must be transported to a common subcellular site, perhaps the Golgi apparatus (2, 3), for assembly to occur, and (ii) the individual subunits must assume a stable configuration in the lipid bilayer of intracellular organelles, for a considerable time prior to formation of the ionophore complex.The AcChoR subunits are comprised by four polypeptidesa, ,3, y, and 6, of apparent molecular masses of 40, 50, 60, and 65 kilodaltons (kDa), respectively (4). Their stoichiometry in the mature complex is a2Py8 (5-7). These subunits are synthesized with signal sequences (8-10) and are cotranslationally integrated into the RER membrane (1) by a mechanism that utilizes the recently purified (11, 12) signal recognition particle (8). Although these subunits are not associated with one another at this stage in their biosynthesis (1), they exhibit a transmembrane topology that is similar, at least in its gross aspects, to that observed for their counterparts in the mature hetero-oligomer (13,14).In this study we have examined the sedimentation behavior of these early biosynthetic forms of the AcChoR subunits on sucrose density gradients. The subunits each appear to self-associate, forming homo-oligomers in the range of 7-13 S. Such homologous associations were not observed for another membrane protein, opsin, studied in an ident...

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Biochemical properties of acetylcholine receptor subunits from Torpedo californica

Cited by 241 publications

References 37 publications

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

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

Stoichiometry of a Recombinant GABA_AReceptor

Identification of homo-oligomers as potential intermediates in acetylcholine receptor subunit assembly.

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Biochemical properties of acetylcholine receptor subunits from Torpedo californica

Cited by 241 publications

References 37 publications

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

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

Stoichiometry of a Recombinant GABAAReceptor

Identification of homo-oligomers as potential intermediates in acetylcholine receptor subunit assembly.

Contact Info

Product

Resources

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Stoichiometry of a Recombinant GABA_AReceptor