Expression, purification, and evidence for the interaction of the two nucleotide-binding folds of the sulphonylurea receptor.
The ATP-sensitive potassium channel is made up of four pore forming Kir6.2 subunits, surrounded by four regulatory sulphonylurea receptor (SUR) subunits. The latter subunit contains two nucleotide-binding folds (NBFs) that confer the ability on the channel to sense changes in the metabolic status ([ATP]/[ADP]) of the cell and couple the changes to the membrane potential of the cell. In an attempt to better understand the mechanisms by which NBFs influence the activity of the channel, we have expressed the NBF domains with C-terminally added epitopes (FLAG to NBF1 and His(6) to NBF2) in Escherichia coli and the rabbit reticulocyte lysate system and examined the ability of these domains to interact with each other and with Kir6.2. Both NBFs could be expressed to high levels in E. coli and purified to homogeneity from inclusion bodies. Re-folding of the proteins proved to be unsuccessful. However, we were able to obtain small amounts of radio-labelled NBFs in a soluble state. Using co-immunoprecipitation, we demonstrate that the radio-labelled NBF1 and NBF2 interact with each other. Neither of the NBFs bound to Kir6.2 expressed in the presence of canine microsomes.
Hough
Emma
E
School of Biomedical Sciences, Leeds University, Leeds LS2 9JT, UK.
Mair
Lindsey
L
Mackenzie
William
W
Sivaprasadarao
Asipu
A
D018528:ATP-Binding Cassette Transporters; D004926:Escherichia coli; D008715:Methionine / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D015221:Potassium Channels / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D017510:Protein Folding; D011955:Receptors, Drug / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; D009711:Nucleotides; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D008715:Methionine