G protein specificity of the muscarine-induced increase in an inward rectifier potassium current in AtT-20 cells.
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.
Kozasa
T
T
Department of Anatomy and Cell Biology, University of Illinois, College of Medicine, Chicago 60612, USA.
Kaziro
Y
Y
Ohtsuka
T
T
Grigg
J J
JJ
Nakajima
S
S
Nakajima
Y
Y
D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D009116:Muscarine / Q000494:pharmacology*; D018721:Muscarinic Agonists / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin; D010902:Pituitary Gland / Q000187:drug effects / Q000502:physiology; D015221:Potassium Channels / Q000502:physiology*; D013004:Somatostatin / Q000494:pharmacology; D014162:Transfection; D010566:Virulence Factors, Bordetella
D018076:DNA, Complementary; D018721:Muscarinic Agonists; D015221:Potassium Channels; D010566:Virulence Factors, Bordetella; D013004:Somatostatin; D009116:Muscarine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins