Modulation of the inward rectifier potassium channel IRK1 by the Ras signaling pathway.
In this study, we investigated the role of Ras and the mitogen-activated protein kinase (MAPK) pathway in the modulation of the inward rectifier potassium channel IRK1. We show that although expression of IRK1 in HEK 293 cells leads to the appearance of a potassium current with strong inward rectifying properties, coexpression of the constitutively active form of Ras (Ras-L61) results in a significant reduction of the mean current density without altering the biophysical properties of the channel. The inhibitory effect of Ras-L61 is not due to a decreased expression of IRK1 since Northern analysis indicates that IRK1 mRNA level is not affected by Ras-L61 co-expression. Moreover, the inhibition can be relieved by treatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD98059. Confocal microscopy analysis of cells transfected with the fusion construct green fluorescent protein-IRK1 shows that the channel is mainly localized at the plasma membrane. Coexpression of Ras-L61 delocalizes fluorescence to the cytoplasm, whereas treatment with PD98059 partially restores the membrane localization. In conclusion, our data indicate that the Ras-MAPK pathway modulates IRK1 current by affecting the subcellular localization of the channel. This suggests a role for Ras signaling in regulating the intracellular trafficking of this channel.
Giovannardi
Stefano
S
Department of Structural and Functional Biology, Università dell'Insubria, Via J. H. Dunant 3, 21100 Varese, Italy.
Forlani
Greta
G
Balestrini
Monica
M
Bossi
Elena
E
Tonini
Raffaella
R
Sturani
Emmapaola
E
Peres
Antonio
A
Zippel
Renata
R
D016475:3T3 Cells; D000818:Animals; D019921:Biotinylation; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D018613:Microscopy, Confocal; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D018631:ras Proteins / Q000502:physiology*
D004791:Enzyme Inhibitors; D005419:Flavonoids; D008164:Luminescent Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D018631:ras Proteins; D011188:Potassium; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one