Expression and characterization of the chick nicotinic acetylcholine receptor alpha-subunit in insect cells using a baculovirus vector.
A baculovirus transfer vector was constructed containing an entire cDNA copy of the chick nicotinic acetylcholine receptor (nAChR) alpha-subunit under control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoter. Recombinant baculovirus was obtained by co-transfection of Spodoptera frugiperda cells with infectious, wild-type AcNPV DNA and the transfer vector. Polyhedrin-negative, recombinant viruses were identified which expressed the nAChR alpha-subunit. The insect cell-expressed alpha-subunit protein had a molecular mass of 42 kDa and was shown to be targeted to the plasma membrane by fluorescence microscopy and toxin-binding assays. The levels of expression were low, approximately 1-2% of cell proteins, when compared with the levels of natural polyhedrin protein. The expressed receptor alpha-subunit was recognised by polyclonal antisera raised against purified Torpedo nAChR alpha-subunit and carried the binding site for the snake venom toxin, alpha-bungarotoxin. Bound alpha-bungarotoxin was displaced in competition binding assays by alpha-cobra toxin, carbamylcholine and d-tubocurarine, and thus had a similar pharmacological profile to that obtained with authentic receptors in muscle cells and receptors expressed in other systems i.e. Xenopus oocytes and mammalian cells. We have also shown that when the chick nAChR alpha-subunit is expressed in the absence of other receptor subunits, unexpectedly high concentrations of nicotine (10 mM) were required to displace bound alpha-bungarotoxin.
School of Biological and Molecular Sciences, Oxford Polytechnic, Headington, England.
A E
AE
Atkinson
F G
FG
Earley
D J
DJ
Beadle
L A
LA
King
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009036:Moths; D015345:Oligonucleotide Probes; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D004247:DNA