Expression of functionally active ATP-sensitive K-channels in insect cells using baculovirus.
We have expressed active ATP-sensitive K-channels (K(ATP) channels) in Spodoptera frugiperda (Sf9) cells using a baculovirus vector. A high yield of active channels was obtained on co-infection with SUR1 and Kir6.2 engineered to contain N- and/or C-terminal tags to permit detection by Western blotting. Channel activity was sensitive to ATP, glibenclamide and diazoxide. Channel activity was also obtained on expression of a C-terminally truncated Kir6.2 (Kir6.2 deltaC26): these channels were blocked by ATP but were insensitive to sulphonylureas. In contrast to Xenopus oocytes and mammalian cells the full length Kir6.2 also gave rise to active channels in Sf9 cells when expressed alone. The highest yield of active K(ATP) channels was obtained on infection with a fusion protein containing SUR1 linked to Kir6.2 deltaC26 via a 6-amino acid linker.
Mikhailov
M V
MV
Nuffield Dept. of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, UK. michail.mikhailov@ndcb.ox.ac.uk
Proks
P
P
Ashcroft
F M
FM
Ashcroft
S J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004594:Electrophysiology / Q000379:methods; D005905:Glyburide / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology
D007328:Insulin; D015221:Potassium Channels; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012413:Rubidium; D005905:Glyburide