Interactions of the sulfonylurea receptor 1 subunit in the molecular assembly of beta-cell K(ATP) channels.
We have investigated protein interactions involved in pancreatic beta-cell ATP-sensitive potassium channel assembly. These channels, which are of key importance for control of insulin release, are a hetero-oligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits with two nucleotide-binding domains (NBD1 and NBD2). We divided SUR1 into two halves at Pro-1042. Expression of either the individual N- or C-terminal domain in a baculovirus expression system did not lead to glibenclamide binding activity, although studies with green fluorescent protein fusion proteins showed that both half-molecules were inserted into the plasma membrane. However, significant glibenclamide binding activity was observed when the half-molecules were co-expressed (even when NBD2 was deleted from the C-terminal half-molecule). Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity. We conclude that the glibenclamide-binding site includes amino acid residues from both halves of the molecule, that there is strong interaction between different regions of SUR1, that NBD2 is not essential for glibenclamide binding, and that interactions between Kir6.2 and SUR1 participate in ATP-sensitive potassium channel assembly. Investigation of NBD1-green fluorescent protein fusion protein distribution inside insect cells expressing C-terminal halves of SUR1 demonstrated strong interaction between NBD1 and NBD2. We also expressed and purified NBD1 from Escherichia coli. Purified NBD1 was found to exist as a tetramer indicating strong homomeric attractions and a possible role for NBD1 in SUR1 assembly.
Mikhailov
M V
MV
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom. michail.mikhailov@ndcb.ox.ac.uk
Ashcroft
S J
SJ
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D049452:Green Fluorescent Proteins; D007515:Islets of Langerhans / Q000378:metabolism*; D008164:Luminescent Proteins; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013453:Sulfonylurea Compounds / Q000737:chemistry / Q000378:metabolism; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; D008164:Luminescent Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D011994:Recombinant Proteins; D013453:Sulfonylurea Compounds; D064233:Sulfonylurea Receptors; D049452:Green Fluorescent Proteins