The molecular cloning and characterisation of cDNA coding for the α subunit of the acetyl-choline receptor

Sumikawa, Katumi; Houghton, Michael; Smith, J.C.; Bell, L. D.; Richards, B. M.; Barnard, Eric A.

doi:10.1093/nar/10.19.5809

Cited by 176 publications

(58 citation statements)

References 26 publications

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“…The teams of Shosaku Numa (38 -40), Stephen Heinemann (41,42), and Eric Barnard (43), as well as Anne Devillers-Thiéry, and Jérôme Giraudat (44,45) in my laboratory, struggled to clone the complementary DNAs of the different subunits from electric organ and muscle and to establish their complete sequence. Experiments by Eric Barnard and Ricardo Miledi had demonstrated that messenger RNA extracted from the electric organ of Torpedo injected into Xenopus oocytes led to the synthesis and incorporation of functional AChRs into the membrane of the oocyte (46).…”

Section: The Pentameric Organization Of the Nicotinic Receptor And Thmentioning

confidence: 99%

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

Changeux

2012

Journal of Biological Chemistry

211

149

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A critical event in the history of biological chemistry was the chemical identification of the first neurotransmitter receptor, the nicotinic acetylcholine receptor. Disciplines as diverse as electrophysiology, pharmacology, and biochemistry joined together in a unified and rational manner with the common goal of successfully identifying the molecular device that converts a chemical signal into an electrical one in the nervous system. The nicotinic receptor has become the founding father of a broad family of pentameric membrane receptors, paving the way for their identification, including that of the GABA A receptors.It has been 42 years since the isolation of the nicotinic acetylcholine receptor (nAChR) 2 from fish electric organ, the first ligand-gated ion channel and the first ion channel ever identified; 25 years since the first GABA A and glycine receptor subunits were cloned and sequenced and concomitantly their homology with the nAChRs recognized; and 5 years since the discovery that closely homologous ligand-gated ion channels are present in prokaryotes (1). In this minireview, I briefly retrace the main steps in the discovery of the nAChR, the titular head of this receptor superfamily. The Concept of Receptor and the Chemical Identification of the Acetylcholine ReceptorThe English physiologist John Newport Langley, working with neuromuscular preparations, proposed in 1905 that muscle tissue possesses "a substance that combines with nicotine and curare . . . receives the stimulus and transmits it." He called the muscle entity the "receptive substance." In the subsequent 50 years, the concept of pharmacological receptors inspired three main lines of research: first, the pharmacological approach aimed at characterizing the specificity of the receptor site by using novel chemical ligands (e.g. the distinction between nicotinic and muscarinic AChRs by Sir Henry Dale); second, the electrophysiological approach exemplified by Bernard Katz and John Eccles aimed at understanding the ionic responses to endogenous neurotransmitter signals; and third, the chemical tradition aimed at the chemical identification of the receptor molecule(s).In the late 1960s, lipids, polysaccharides, proteins, and even nucleic acids were considered as potential receptors. The early independent efforts of Carlos Chagas, Eduardo de Robertis, and David Nachmansohn to identify the receptor for acetylcholine (ACh) in the electric organ of the fish Electrophorus electricus with radioactive ligands were abandoned because their tissue extracts lacked specificity (2). However, in the course of these studies, Nachmansohn recognized the extraordinarily rich content of nicotinic synapses in the electric organ (2). With Ernest Schoffeniels, he devised a method for preparing individual cells, or electroplaques, from the electric organ. This offered the opportunity to investigate, simultaneously, the electrophysiological, pharmacological, and biochemical characteristics of the response to ACh within the same biological system (2). At this time, th...

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Section: The Pentameric Organization Of the Nicotinic Receptor And Thmentioning

confidence: 99%

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

Changeux

2012

Journal of Biological Chemistry

211

149

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“…Several laboratories recently succeeded in cloning and sequencing some (6)(7)(8)(9) or all (10)(11)(12) of the cDNAs encoding the four subunits of Torpedo AcChoR. It was found that there is significantly more homology between the deduced protein sequences of the a-,8 and y-8 pairs than between any other combinations of subunits, suggesting that an initial duplication of the ancestral gene had given rise to two protogenes, one of which later yielded the y and 8 genes while the other gave rise to the a and p genes (12).…”

mentioning

confidence: 99%

Structure linkage, and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor.

Nef¹,

Mauron²,

Stalder³

et al. 1984

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

125

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We have cloned and sequenced a fragment of the chicken genome approximately 9 kilobases in length that comprises the genes encoding the 6 and 'y subunits of the nicotinic acetyicholine receptor. The two genes are homologous and have identical structures: both consist of 12 exons, some of which precisely correspond to predicted structural domains of the receptor subunits. The 8 and y subunit gehes are encoded by the same DNA strand and are very closely linked, there being only 740 base pairs between the last codon of 8 and the initiator codon of y. Blot analysis demonstrates that the genes we describe are unique in the genome. Comparison of the predicted protein sequence for the correspodding subunits of chicken and of the elasmobranch Torpedo reveals a high degree of conservation in Aome but not all of the protein domains.The nicotinic acetylcholine receptor (AcChok) mediates synaptic transmission at the vertebrate neuromuscular junction. Located in the folds of the postsynaptic membrane, the AcChoR is a cation channel whose opening is triggered by acetylcholine. AcChoR activation normally results in an influx of Na' that depolarizes the postsynaptic membrane and leads to muscle contraction. The AcChoR is the best understood of all ligand-gated ion channels due to its relative abundance in the electrocytes of Torpedo or Electrophorus, from which workable quantities of receptor can be purified. The receptor is a pentamer of four different subunits in the stoichiometty a2f8W. All subunits are glycosylated and span the membrane. They assemble into a barrel-shaped structure whose central cavity is thought to be the gatpd ion channel (for recent reviews see refs. 1-3). That the four subunits participate in the assembly of a single functional AcChoR molecule was elegantly verified by showing that all four mRNA species must be injected in Xenopus oocytes to allow detection of physiologically active receptor (4). Microsequencing of the amino ends of the four purified Torpedo subunits demonstrated that they were related and suggested that they had evolved from an ancestral gene by a series of duplications (5).Several laboratories recently succeeded in cloning and sequencing some (6-9) or all (10-12) of the cDNAs encoding the four subunits of Torpedo AcChoR. It was found that there is significantly more homology between the deduced protein sequences of the a-,8 and y-8 pairs than between any other combinations of subunits, suggesting that an initial duplication of the ancestral gene had given rise to two protogenes, one of which later yielded the y and 8 genes while the other gave rise to the a and p genes (12). Moreover, all subunits were shown to have four very hydrophobic stretches three of them closely grouped-of sufficient length to span the membrane. This finding suggested a model for the transmembrane insertion of the receptor whereby each subunit weaves four times through the membrane to yield an assembly of twenty transmembrane a helices, a subset of which constitutes the channel per se (8,9,12). A relate...

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“…Our recent studies have elucidated the primary structures of all four subunits of the Torpedo calijornica acetylcholine receptor by cloning and sequencing cDNAs encoding the respective subunit precursors [4 -61; cDNA sequences for the y subunit precursor of the T.caliJornica receptor [7] and the M subunit precursor of the Torpedo murmorata receptor [8,91 have also been reported by other groups. Conspicuous amino acid sequence homology is found among the four subunits of the T. caliJornica acetylcholine receptor, suggesting that the genes encoding them have been generated from a single common ancestor by gene duplications.…”

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confidence: 99%

Primary structure of gamma subunit precursor of calf-muscle acetylcholine receptor deduced from the cDNA sequence

et al. 1984

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Clones carrying cDNA sequences for the 7 subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has revealed the primary structure of this polypeptide, which consists of 519 amino acids including a hydrophobic prepeptide of 22 amino acids. The 7 subunit of the calf muscle acetylcholine receptor, like the M subunit of the calf as well as the human muscle receptor, shares features characteristic of all four subunits of the Torpedo electroplax receptor, such as the putative disulphide bridge corresponding to that in the c1 subunit proposed as being in close proximity to the acetylcholine binding site and the four putative, hydrophobic transmembrane segments M I -M4. Thus, the calf y subunit molecule apparently exhibits the same transmembrane topology as proposed for the fish receptor subunits. The degree of sequence homology between the calf and Torpedo subunits (56 %) is lower than that between the a subunits of the two species (81 %). Some regions of the 1 subunit molecule, including the region encompassing the putative disulphide bridge and the region containing the putative transmembrane segments M I , M2 and M3, are relatively well conserved between the two species.Much information has accumulated as to the nicotinic acetylcholine receptor from fish electric organ, which is a pentameric protein complex with a subunit structure of cc,fiyfi (for review see [ l -31). Our recent studies have elucidated the primary structures of all four subunits of the Torpedo calijornica acetylcholine receptor by cloning and sequencing cDNAs encoding the respective subunit precursors [4 -61; cDNA sequences for the y subunit precursor of the T.caliJornica receptor [7] and the M subunit precursor of the Torpedo murmorata receptor [8, 91 have also been reported by other groups. Conspicuous amino acid sequence homology is found among the four subunits of the T. caliJornica acetylcholine receptor, suggesting that the genes encoding them have been generated from a single common ancestor by gene duplications. Furthermore, work by our group has demonstrated that Enzymes. Alkaline phosphatase or orthophosphoric-monoester phosphohydrolase (alkaline optimum) (EC 3.1.3.1); DNA ligase or poly(deoxyribonuc1eotide) : poly(deoxyribonuc1eotide) ligase (AMPforming, NMN-forming) (EC 6.5

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The molecular cloning and characterisation of cDNA coding for the α subunit of the acetyl-choline receptor

Cited by 176 publications

References 26 publications

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

Structure linkage, and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor.

Primary structure of gamma subunit precursor of calf-muscle acetylcholine receptor deduced from the cDNA sequence

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