ABSTRACT
The ATP-sensitive potassium (K(ATP)) channel couples glucose metabolism to insulin secretion in pancreatic beta-cells. It comprises regulatory sulfonylurea receptor 1 and pore-forming Kir6.2 subunits. Binding and/or hydrolysis of Mg-nucleotides at the nucleotide-binding domains of sulfonylurea receptor 1 stimulates channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. We report here the first purification and functional characterization of sulfonylurea receptor 1. We also compared the ATPase activity of sulfonylurea receptor 1 with that of the isolated nucleotide-binding domains (fused to maltose-binding protein to improve solubility). Electron microscopy showed that nucleotide-binding domains purified as ring-like complexes corresponding to approximately 8 momomers. The ATPase activities expressed as maximal turnover rate [in nmol P(i).s(-1).(nmol protein)(-1)] were 0.03, 0.03, 0.13 and 0.08 for sulfonylurea receptor 1, nucleotide-binding domain 1, nucleotide-binding domain 2 and a mixture of nucleotide-binding domain 1 and nucleotide-binding domain 2, respectively. Corresponding K(m) values (in mm) were 0.1, 0.6, 0.65 and 0.56, respectively. Thus sulfonylurea receptor 1 has a lower K(m) than either of the isolated nucleotide-binding domains, and a lower maximal turnover rate than nucleotide-binding domain 2. Similar results were found with GTP, but the K(m) values were lower. Mutation of the Walker A lysine in nucleotide-binding domain 1 (K719A) or nucleotide-binding domain 2 (K1385M) inhibited the ATPase activity of sulfonylurea receptor 1 by 60% and 80%, respectively. Beryllium fluoride (K(i) 16 microm), but not MgADP, inhibited the ATPase activity of sulfonylurea receptor 1. In contrast, both MgADP and beryllium fluoride inhibited the ATPase activity of the nucleotide-binding domains. These data demonstrate that the ATPase activity of sulfonylurea receptor 1 differs from that of the isolated nucleotide-binding domains, suggesting that the transmembrane domains may influence the activity of the protein.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/metabolism , Multidrug Resistance-Associated Proteins/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/isolation & purification , Animals , Binding Sites , Carrier Proteins/genetics , Carrier Proteins/metabolism , Hydrolysis , Kinetics , Maltose-Binding Proteins , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/isolation & purification , Nucleotides/metabolism , Potassium Channels, Inwardly Rectifying , Protein Binding , Rats , Receptors, Drug , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sulfonylurea ReceptorsABSTRACT
ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/physiology , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/physiology , Potassium Channels/chemistry , Potassium Channels/physiology , Receptors, Drug/chemistry , Receptors, Drug/physiology , ATP-Binding Cassette Transporters/ultrastructure , Amino Acid Sequence , Animals , Cryoelectron Microscopy , Mice , Molecular Sequence Data , Potassium Channels/ultrastructure , Potassium Channels, Inwardly Rectifying/isolation & purification , Potassium Channels, Inwardly Rectifying/ultrastructure , Protein Structure, Tertiary , Rats , Receptors, Drug/ultrastructure , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/physiology , Recombinant Fusion Proteins/ultrastructure , Sulfonylurea ReceptorsSubject(s)
Cloning, Molecular/methods , Genetic Vectors , Nucleopolyhedroviruses/genetics , Polymerase Chain Reaction/methods , Viral Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Green Fluorescent Proteins , Insecta/virology , Luminescent Proteins/genetics , Molecular Sequence Data , Occlusion Body Matrix Proteins , Recombinant Fusion Proteins/analysis , Transfection/methods , Viral Structural Proteins , beta-Galactosidase/geneticsSubject(s)
ATP-Binding Cassette Transporters , Hypoglycemic Agents/analysis , Hypoglycemic Agents/metabolism , Potassium Channels, Inwardly Rectifying , Potassium Channels/metabolism , Receptors, Drug/metabolism , Animals , Carbamates/metabolism , Gliclazide/metabolism , Glyburide/metabolism , Kinetics , Piperidines/metabolism , Protein Binding , Rats , Spodoptera/metabolism , Sulfonylurea Receptors , Tolbutamide/metabolismABSTRACT
The role of the matrix (MA) domain of simian immunodeficiency virus (SIV) and bovine leukaemia virus (BLV) Gag in the assembly of virus-like particles (VLP) in insect cells has been investigated. Wild-type SIV and BLV Gag assembled to form discrete VLP structures typical of many retroviruses analysed by similar systems. When amino acids predicated by the three-dimensional structure to be at the interface of SIV MA monomers were deleted, VLP assembly was abolished consistent with a role for MA multimerization in assembly. When amino acids predicted to be in the analogous positions in BLV MA were mutated, however, VLP assembly was not affected. These data indicate that the models of assembly derived from one model retrovirus may not necessarily apply to more distantly related viruses despite the structural similarity present in equivalent Gag domains.
