Up-regulation of hERG K⁺ channels by B-RAF.
Human ether-a-go-go related-gene K⁺ channels (hERG) participate in the regulation of tumor cell proliferation and apoptosis. HERG channel activity is up-regulated by growth factors. Kinases sensitive to growth factor signaling include the serine/threonine protein kinase B-RAF. The present study thus explored whether B-RAF influences hERG channel expression and activity. To this end, hERG channels were expressed in Xenopus oocytes with or without wild-type B-RAF, hERG channel activity was determined utilizing dual-electrode voltage clamp and hERG protein abundance in the cell membrane was analyzed utilizing confocal microscopy as well as chemiluminescence. Moreover, in rhabdomyosarcoma RD cells the effect of B-RAF inhibitor PLX-4720 on hERG-mediated current was quantified by whole-cell patch clamp and hERG cell surface protein abundance by utilizing biotinylation of cell surface proteins as well as flow cytometry. As a result, co-expression of wild-type B-RAF in hERG-expressing Xenopus oocytes significantly increased hERG channel activity and hERG channel protein abundance in the cell membrane. Treatment for 24 hours of B-RAF and hERG-expressing Xenopus oocytes with B-RAF inhibitor PLX-4720 (10 µM) significantly decreased hERG-mediated current and hERG cell surface expression. Similarly, in rhabdomyosarcoma RD cells, treatment for 24 hours with B-RAF inhibitor PLX-4720 significantly decreased hERG cell membrane protein abundance and hERG-mediated current. In conclusion, B-RAF is a powerful regulator of hERG channel activity and cell surface hERG protein abundance.
Department of Physiology, University of Tübingen, Tübingen, Germany.
Tatsiana
T
Pakladok
Department of Physiology, University of Tübingen, Tübingen, Germany.
Zohreh
Z
Hosseinzadeh
Department of Physiology, University of Tübingen, Tübingen, Germany.
Ahmad
A
Almilaji
Department of Physiology, University of Tübingen, Tübingen, Germany ; Department of Immunology, Institute of Experimental Medicine, St. Petersburg, Russia.
Aleksandra
A
Lebedeva
Department of Physiology, University of Tübingen, Tübingen, Germany.
Ekaterina
E
Shumilina
Department of Physiology, University of Tübingen, Tübingen, Germany.
Ioana
I
Alesutan
Department of Physiology, University of Tübingen, Tübingen, Germany.
Florian
F
Lang
D000818:Animals; D051638:Ether-A-Go-Go Potassium Channels / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008163:Luminescent Measurements; D018613:Microscopy, Confocal; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D015854:Up-Regulation / Q000502:physiology*; D014981:Xenopus
D051638:Ether-A-Go-Go Potassium Channels; D007211:Indoles; C528407:PLX 4720; D013449:Sulfonamides; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf