Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor

• Published in: The EMBO journal Vol. 9; no. 13; pp. 4391 - 4398
• Main Authors: Marshall, J., Buckingham, S. D., Shingai, R., Lunt, G. G., Goosey, M. W., Darlison, M. G., Sattelle, D. B., Barnard, E. A.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.12.1990
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Cloning, Molecular; Cockroaches; DNA - chemistry; Drosophila; Fundamental and applied biological sciences. Psychology; Gene expression; Grasshoppers - genetics; Ion Channels - metabolism; Mice; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Muscles - metabolism; Nervous System - metabolism; Rats; Receptors, Nicotinic - genetics; RNA - biosynthesis; Schistocerca gregaria; Sequence Homology, Nucleic Acid; Xenopus laevis; Insecta; Orthoptera; Amphibia; Electrophysiology; Nervous system; Ionic channel; Acrididae; Cholinergic receptor; Neuromediator; Schistocerca gregaria; Molecular cloning; Nicotinic receptor; Heterologous system; Xenopus laevis; Primary structure; Salientia; Gene expression; Vertebrata; Microinjection; Complementary DNA; Arthropoda; Acridoidea; Acetylcholine; Invertebrata; Oocyte
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1990.tb07889.x

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha‐bungarotoxin (alpha‐Bgt) and kappa‐bungarotoxin (kappa‐Bgt). Reversible block of the expressed insect AChR by mecamylamine, d‐tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha‐Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.