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 revealed that the expressed AKT1 and KAT1 polypeptides were mainly associated 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 voltage-gated channel highly selective for K+, while similar experiments had failed to characterize AKT1. Insect cells expressing KAT1 displayed an exogenous inwardly rectifying K+ conductance reminiscent of that described previously in Xenopus oocytes expressing KAT1. Under similar conditions, cells expressing AKT1 showed a disturbed cell membrane electrical stability that precluded electrophysiological analysis. Use of a baculovirus transfer vector designed so as to decrease the expression level allowed the first electrophysiological characterization of AKT1. The baculovirus system can thus be used as an alternative method when expression in Xenopus oocytes is unsuccessful for electrophysiological characterization of the ion channel of interest. The plant AKT1 protein has been shown in this way to be an inwardly rectifying voltage-gated channel highly selective for K+ ions and sensitive to cGMP.
Gaymard
F
F
Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Ecole Nationale Supérieure Agronomique de Montpellier, Institut National de la Recherche Agronomique, CNRS URA 2133, 34060 Montpellier Cedex 1, France.
Cerutti
M
M
Horeau
C
C
Lemaillet
G
G
Urbach
S
S
Ravallec
M
M
Devauchelle
G
G
Sentenac
H
H
Thibaud
J B
JB
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D006152:Cyclic GMP / Q000494:pharmacology; D004594:Electrophysiology; D005822:Genetic Vectors; D010940:Plant Proteins / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D018411:Spodoptera / Q000166:cytology; D014981:Xenopus
D029681:Arabidopsis Proteins; C501210:KAT1 protein, Arabidopsis; D010940:Plant Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; C074500:AKT1 protein, Arabidopsis; D000255:Adenosine Triphosphate; D006152:Cyclic GMP