F. Gaymard et al., THE BACULOVIRUS INSECT CELL SYSTEM AS AN ALTERNATIVE TO XENOPUS OOCYTES - FIRST CHARACTERIZATION OF THE AKT1 K+ CHANNEL FROM ARABIDOPSIS-THALIANA/, The Journal of biological chemistry, 271(37), 1996, pp. 22863-22870
Two plant (Arabidopsis thaliana) K+ transport systems, KAT1 and AKT1,
have been expressed in insect cells (Sf9 cell line) using recombinant
baculoviruses. Microscopic observation after immunogold staining revea
led that the expressed AKT1 and KAT1 polypeptides were mainly associat
ed with internal membranes, but that a minute fraction was targeted to
the cell membrane, KAT1 was known, from earlier electrophysiological
characterization in Xenopus oocytes, to be an inwardly rectifying volt
age-gated channel highly selective for K+, while similar experiments h
ad failed to characterize AKT1. Insect cells expressing KAT1 displayed
an exogenous inwardly rectifying K+ conductance reminiscent of that d
escribed previously in Xenopus oocytes expressing KAT1. Under similar
conditions, cells expressing AKT1 showed a disturbed cell membrane ele
ctrical stability that precluded electrophysiological analysis. Use of
a baculovirus transfer vector designed so as to decrease the expressi
on level allowed the first electrophysiological characterization of AK
T1. The baculovirus system can thus be used as an alternative method w
hen expression in Xenopus oocytes is unsuccessful for electrophysiolog
ical characterization of the ion channel of interest. The plant AKT1 p
rotein has been shown in this way to be an inwardly rectifying voltage
gated channel highly selective for K+ ions and sensitive to cGMP.