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 w ith two nucleotide-binding domains (NBD1 and NBD2), We divided SUR1 into tw o halves at Pro-1042, Expression of either the individual N- or C-terminal domain in a baculovirus expression system did not lead to glibenclamide bin ding activity, although studies with green fluorescent protein fusion prote ins 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-ter minal half-molecule). Simultaneous expression of Kir6.2 resulted in enhance d glibenclamide binding activity. We conclude that the glibenclamide-bindin g 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 Kir 6.2 and SUR1 participate in ATP-sensitive potassium channel assembly. Inves tigation of NBD1-green fluorescent protein fusion protein distribution insi de insect cells expressing C-terminal halves of SUR1 demonstrated strong in teraction between NBD1 and NBD2. We also expressed and purified NBD1 from E scherichia coli, Purified NBD1 was found to exist as a tetramer indicating strong homomeric attractions and a possible role for NBD1 in SUR1 assembly.