Expression and characterization of the rat ?4 ?2 neuronal nicotinic cholinergic receptor in baculovirus-infected insect cells

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Abstract
Baculovirus expression systems have been developed to generate 1) a neuronal nicotinic cholinergic receptor comprising both the α4 and β2 subunits and 2) the α4 and β2 subunits individually. The presence of the α4 β2 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 β2 subunit), revealed the presence of characteristic 52-kD bands in β2- and α4 β2 recombinant viral-infected cells, but not in control cells or cells infected with wild-type virus or recombinant virus containing α4 alone. The 52-kD protein, which is specific for mAb 270, is known to be the β2 subunit of neuronal nAChRs. Specific [3H]methylcarbamylcholine binding was observed in cells infected with both α4 or β2 but not with the α4 or β2 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 α4 and β2 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.