RESUMO
ATP-sensitive potassium (KATP) channels play a central role in glucose-stimulated insulin secretion (GSIS) by pancreatic beta-cells. Activity of these channels is determined by their open probability (Po) and the number of channels present in a cell. Glucose is known to reduce Po, but whether it also affects the channel density is unknown. Using INS-1 model beta-cell line, we show that the expression of K(ATP) channel subunits, Kir6.2 and SUR1, is high at low glucose, but declines sharply when the ambient glucose concentration exceeds 5mM. In response to glucose deprivation, channel synthesis increases rapidly by up-regulating translation of existing mRNAs. The effects of glucose deprivation could be mimicked by pharmacological activation of 5'-AMP-activated protein kinase with 5-aminoimidazole-4-carboxamide ribonucleotide and metformin. Pancreatic beta-cells which have lost their ability for GSIS do not show such changes implicating a possible (patho-)physiological link between glucose-regulated KATP channel expression and the capacity for normal GSIS.
Assuntos
Trifosfato de Adenosina/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/biossíntese , Glucose/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Proteínas Quinases Ativadas por AMP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Meios de Cultivo Condicionados , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Glucose/fisiologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/fisiologia , Canais de Potássio/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Ratos , Receptores de Droga/biossíntese , Receptores de SulfonilureiasRESUMO
The ATP-sensitive potassium channel is made up of four pore forming Kir6.2 subunits, surrounded by four regulatory sulphonylurea receptor (SUR) subunits. The latter subunit contains two nucleotide-binding folds (NBFs) that confer the ability on the channel to sense changes in the metabolic status ([ATP]/[ADP]) of the cell and couple the changes to the membrane potential of the cell. In an attempt to better understand the mechanisms by which NBFs influence the activity of the channel, we have expressed the NBF domains with C-terminally added epitopes (FLAG to NBF1 and His(6) to NBF2) in Escherichia coli and the rabbit reticulocyte lysate system and examined the ability of these domains to interact with each other and with Kir6.2. Both NBFs could be expressed to high levels in E. coli and purified to homogeneity from inclusion bodies. Re-folding of the proteins proved to be unsuccessful. However, we were able to obtain small amounts of radio-labelled NBFs in a soluble state. Using co-immunoprecipitation, we demonstrate that the radio-labelled NBF1 and NBF2 interact with each other. Neither of the NBFs bound to Kir6.2 expressed in the presence of canine microsomes.
