Functional expression of the plant K+ channel KAT1 in insect cells.
Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1, and KST1 have recently been identified on the molecular level. Among them, we focussed on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K+-selective (impermeable to Na+ and even NH4+) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as -60 to -80mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-traditional modification and assembly of a green inward rectifier.
Marten
I
I
Institut für Biophysik der Universität Hannover, Germany.
Gaymard
F
F
Lemaillet
G
G
Thibaud
J B
JB
Sentenac
H
H
Hedrich
R
R
D000818:Animals; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics; D029681:Arabidopsis Proteins; D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004553:Electric Conductivity; D015870:Gene Expression; D018408:Patch-Clamp Techniques; D010940:Plant Proteins / Q000235:genetics* / Q000502:physiology; D015221:Potassium Channels / Q000235:genetics* / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism*
D029681:Arabidopsis Proteins; C501210:KAT1 protein, Arabidopsis; D010940:Plant Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D002118:Calcium