Expression and characterization of the rat alpha 4 beta 2 neuronal nicotinic cholinergic receptor in baculovirus-infected insect cells.
Baculovirus expression systems have been developed to generate 1) a neuronal nicotinic cholinergic receptor comprising both the alpha 4 and beta 2 subunits and 2) the alpha 4 and beta 2 subunits individually. The presence of the alpha 4 and beta 2 genes in the various baculovirus-infected Sf9 cells was confirmed following polymerase chain reaction (PCR) of the extracted viral DNAs, gel electrophoresis, and double strand sequencing. Autofluorography, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of infected cell lysates radiolabeled with 35S-methionine and immunoprecipated with mAb 270 (specific for the beta 2 subunit), revealed the presence of characteristic 52-kD bands in beta 2- and alpha 4 beta 2 recombinant viral-infected cells, but not in control cells or cells infected with wild-type virus or recombinant virus containing alpha 4 alone. The 52-kD protein, which is specific for mAb 270, is known to be the beta 2 subunit of neuronal nAChRs. Specific [3H]methylcarbamylcholine binding was observed in cells infected with both alpha 4 or beta 2 but not with the alpha 4 or beta 2 genes alone. Scatchard analysis revealed a Bmax = 5.50 pmol/mg and a Kd = 1 nM. The degree of [3H]methylcarbamylcholine binding/mg membrane protein was 180-fold greater than that found in rat brain. The study demonstrates that the major neuronal nAChR, which comprises alpha 4 and beta 2 subunits and is present in very low abundance in mammalian brain, can be prepared by a baculovirus expression system in sufficient quantities for chemical and crystallographic structural analysis.
Wang
D X
DX
Department of Pharmacology, University of Rochester Medical Center, NY 14642, USA.
Abood
L G
LG
D000818:Animals; D016367:Baculoviridae; D002217:Carbachol / Q000031:analogs & derivatives / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005822:Genetic Vectors; D007700:Kinetics; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013462:Sulfur Radioisotopes; D014162:Transfection
D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; C053765:N-methylcarbamylcholine; D002217:Carbachol; D008715:Methionine