Expression and characterization of the rat α4 β2 neuronal nicotinic cholinergic receptor in baculovirus-infected insect cells

✍ Scribed by D.X. Wang; L.G. Abood

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 489 KB
• Volume: 44
• Category: Article
• ISSN: 0360-4012
• DOI: 10.1002/(sici)1097-4547(19960515)44:4<350::aid-jnr6>3.0.co;2-b

✦ Synopsis

Baculovirus expression systems have been developed

to generate 1) a neuronal nicotinic cholinergic receptor comprising both the a4 and 92 subunits and 2) the a4 and p2 subunits individually. The presence of the a4 and p2 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 sulfatepolyacrylamide gel electrophoresis of infected cell lysates radiolabeled with 35S-methionine and immunoprecipated with mAb 270 (specific for the 92 subunit), revealed the presence of characteristic 52-kD bands in 92-and a4 92 recombinant viralinfected cells, but not in control cells or cells infected with wild-type virus or recombinant virus containing a4 alone. The 52-kD protein, which is specific for mAb 270, is known to be the 92 subunit of neuronal nAChRs. Specific [3H]methylcarbamylcholine binding was observed in cells infected with both a4 or p2 but not with the a4 or 92 genes alone. Scatchard analysis revealed a B, , = 5.50 pmoVmg and a Kd = 1 nM. The degree of [3H]methylcarbamylcholine bindindmg membrane protein was 180-fold greater than that found in rat brain. The study demonstrates that the major neuronal nAChR, which comprises a4 and p2 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.