ATP hydrolysis mediated by SUR - NBFs

ATP-sensitive potassium (K_ATP) channels, composed of a heterooctamer of Kir6 and SUR (sulfonylurea receptor) subunits, are a link between cellular metabolism and membrane excitability. There is evidence in mice suggesting that the K_ATP in the pancreas is active over a larger range of metabolic conditions than those in the heart. We are testing the possibility that the differences in the SUR isotypes are at least partially responsible for these physiological differences between cardiac and pancreatic K_ATP. We expressed and purified Nucleotide Binding Fold 1 and 2 (NBF1 and NBF2) from both SUR1 and SUR2A as Maltose Biding Protein (MPB) -tagged constructs, and determined the in vitro ATPase activity using a colorimetric protocol. The results indicate that the level of activity is dependent on time, protein concentration, and concentration of Mg2+. The KM (mM-1) and Vmax (nmol/min/mg) values obtained for SUR1-NBF1, SUR1-NBF2, SUR2A-NBF1, SUR2A-NBF2 are 0.25 and 12.0, 0.31 and 23.6, 0.18 and 8.8, and 0.16 and 7.3, respectively. The data suggest that NBF2 of SUR1 has a higher ATPase activity than the other NBFs. Studies conducted on the structure of bacterial NBFs indicate that the NBFs function as dimers, and proposed experiments will lead to the study of dimer constructs, with the introduction of mutations to both monomers and dimers.