Expression of functionally active ATP‐sensitive K‐channels in insect cells using baculovirus

FEBS Letters

Ashcroft, Stephen J.H.¹  Proks, Peter²  Ashcroft, Frances M.²  Mikhailov, Michael V.¹ 

[1] Nuffield Dept. of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK;Laboratory of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK

关键词: Potassium channel;    ATP;    Sulphonylurea receptor;    Baculovirus;    Gene expression;   

DOI  :  10.1016/S0014-5793(98)00640-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have expressed active ATP-sensitive K-channels (K_ATP charmels) 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 (Kir6.2ΔC26): 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 Kir6.2ΔC26 via a 6-amino acid linker.

【 授权许可】

Unknown