THE BACULOVIRUS INSECT CELL SYSTEM AS AN ALTERNATIVE TO XENOPUS OOCYTES - FIRST CHARACTERIZATION OF THE AKT1 K+ CHANNEL FROM ARABIDOPSIS-THALIANA/

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.