Calnexin co-expression and the use of weaker promoters increase the expression of correctly assembled Shaker potassium channel in insect cells.
Voltage-gated potassium channels control the membrane potential of excitable cells. To understand their function, knowledge of their structure is essential. However, these channels are scarce in natural sources, and overexpression is necessary to generate material for structural studies. We have compared functional expression of the Drosophila Shaker H4 potassium channel in stable insect cell lines and in baculovirus-infected insect cells, using three different baculovirus promoters. Stable insect cell lines expressed correctly assembled channel, which was glycosylated and found predominantly at, or close to, the cell surface. In comparison, the majority of baculovirus-overexpressed Shaker was intracellular and incorrectly assembled. The proportion of functional Shaker increased, however, if the weaker basic protein promoter was used rather than the stronger p10 or polyhedrin promoters. In addition, co-expression of the molecular chaperone, calnexin, increased the quantity of correctly assembled channel protein, suggesting that calnexin can be used to increase the efficiency of channel expression in insect cells.
Higgins
Matthew K
MK
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. mkh20@cam.ac.uk
Demir
Mukerrem
M
Tate
Christopher G
CG
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D037281:Calnexin / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D029721:Drosophila Proteins; D005786:Gene Expression Regulation / Q000187:drug effects; D007313:Insecta; D018832:Molecular Chaperones / Q000494:pharmacology; D015221:Potassium Channels / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D051637:Shaker Superfamily of Potassium Channels; D014162:Transfection; D015854:Up-Regulation
D029721:Drosophila Proteins; D018832:Molecular Chaperones; D015221:Potassium Channels; C496929:Sh protein, Drosophila; D051637:Shaker Superfamily of Potassium Channels; D037281:Calnexin