Functional clustering of mutations in the dimer interface of the nucleotide binding folds of the sulfonylurea receptor.
ATP-sensitive K(+) (K(ATP)) channels modulate their activity as a function of inhibitory ATP and stimulatory Mg-nucleotides. They are constituted by two proteins: a pore-forming K(+) channel subunit (Kir6.1, Kir6.2) and a regulatory sulfonylurea receptor (SUR) subunit, an ATP-binding cassette (ABC) transporter that confers MgADP stimulation to the channel. Channel regulation by MgADP is dependent on nucleotide interaction with the cytoplasmic nucleotide binding folds (NBF1 and NBF2) of the SUR subunit. Crystal structures of bacterial ABC proteins indicate that NBFs form as dimers, suggesting that NBF1-NBF2 heterodimers may form in SUR and other eukaryotic ABC proteins. We have modeled SUR1 NBF1 and NBF2 as a heterodimer, and tested the validity of the predicted dimer interface by systematic mutagenesis. Engineered cysteine mutations in this region have significant effects, both positive and negative, on MgADP stimulation of K(ATP) channels in excised patches and on macroscopic channel activity in intact cells. Additionally, the mutations cluster in the model structure according to their functional effect, such that patterns of alteration emerge. Of note, three gain-of-function mutations, leading to MgADP hyperstimulation of the channel, are located in the D-loop region at the center of the predicted dimer interface. Overall, the data support the idea that SUR1 NBFs assemble as heterodimers and that this interaction is functionally critical.
Masia
Ricard
R
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Nichols
Colin G
CG
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D018360:Crystallography, X-Ray / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry*; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011955:Receptors, Drug / Q000378:metabolism*; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; D009711:Nucleotides; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D000244:Adenosine Diphosphate; D003545:Cysteine