RESUMEN
The G protein-coupled inward rectifier K(+) channel (GIRK) is activated by direct interaction with the heterotrimeric GTP-binding protein betagamma subunits (Gbetagamma). However, the precise role of Gbeta and Ggamma in GIRK activation remains to be elucidated. Using transient expression of GIRK1, GIRK2, Gbeta1, and Ggamma2 in human embryonic kidney 293 cells, we show that C-terminal mutants of Gbeta1, which do not bind to Ggamma2, are still able to associate with GIRK, but these mutants are unable to induce activation of GIRK channels. In contrast, other C-terminal mutants of Gbeta1 that bind to Ggamma2, are capable of activating the GIRK channel. These results suggest that Ggamma plays a more important role than that of an anchoring device for the Gbetagamma-induced GIRK activation.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP Heterotriméricas , Canales de Potasio de Rectificación Interna , Canales de Potasio/química , Línea Celular , ADN Complementario/aislamiento & purificación , Activación Enzimática , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Proteínas de Unión al GTP/genética , Expresión Génica , Humanos , Immunoblotting , Mutación , Reacción en Cadena de la Polimerasa , Canales de Potasio/genética , TransfecciónRESUMEN
Muscarine and somatostatin enhance an inward rectifier K+ conductance in the AtT-20 pituitary cell line. Both effects are abolished by pertussis toxin (PTX). To determine which PTX-sensitive G protein mediates these agonist effects, we made cDNAs encoding mutant PTX-insensitive Gi alpha subtypes, in which the cysteine residue fourth from the C terminus was replaced with serine. The mutated cDNA was transfected into AtT-20 cells, resulting in stable cell lines overexpressing a Gi alpha subtype. As controls, wild-type Gi alpha cDNA was transfected into AtT-20 cells. The agonist-induced increase of the inward rectifier K+ conductance in the transfectants was examined with the whole-cell clamp method. Only in the cell lines into which the mutated (PTX-insensitive) Gi2 alpha cDNA was transfected, did the muscarine response become PTX-insensitive, suggesting that Gi2 couples to the muscarinic receptor and enhances the activity of the inward rectifier K+ channel. However, PTX-insensitive somatostatin responses were not obtained in any of the cell lines transfected with a mutated Gi alpha cDNA, suggesting either that none of the Gi subtypes is a transducer for the somatostatin effect or that the mutation prevents the coupling of the Gi alpha to the somatostatin receptor.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Canales de Potasio/fisiología , Animales , Línea Celular , ADN Complementario , Proteínas de Unión al GTP/genética , Ratones , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Toxina del Pertussis , Hipófisis/efectos de los fármacos , Hipófisis/fisiología , Somatostatina/farmacología , Transfección , Factores de Virulencia de BordetellaRESUMEN
Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.