RESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Hidrólise , Cinética , Proteínas Ligantes de Maltose , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/isolamento & purificação , Nucleotídeos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Ligação Proteica , Ratos , Receptores de Droga , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Receptores de SulfonilureiasRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Canais de Potássio/química , Canais de Potássio/fisiologia , Receptores de Droga/química , Receptores de Droga/fisiologia , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica , Camundongos , Dados de Sequência Molecular , Canais de Potássio/ultraestrutura , Canais de Potássio Corretores do Fluxo de Internalização/isolamento & purificação , Canais de Potássio Corretores do Fluxo de Internalização/ultraestrutura , Estrutura Terciária de Proteína , Ratos , Receptores de Droga/ultraestrutura , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Recombinantes de Fusão/ultraestrutura , Receptores de SulfonilureiasAssuntos
Clonagem Molecular/métodos , Vetores Genéticos , Nucleopoliedrovírus/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Fluorescência Verde , Insetos/virologia , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Proteínas de Matriz de Corpos de Inclusão , Proteínas Recombinantes de Fusão/análise , Transfecção/métodos , Proteínas Estruturais Virais , beta-Galactosidase/genéticaAssuntos
Transportadores de Cassetes de Ligação de ATP , Hipoglicemiantes/análise , Hipoglicemiantes/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Animais , Carbamatos/metabolismo , Gliclazida/metabolismo , Glibureto/metabolismo , Cinética , Piperidinas/metabolismo , Ligação Proteica , Ratos , Spodoptera/metabolismo , Receptores de Sulfonilureias , Tolbutamida/metabolismoRESUMO
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.