Pakladok Tatsiana, Hosseinzadeh Zohreh, Almilaji Ahmad, Lebedeva Aleksandra, Shumilina Ekaterina, Alesutan Ioana, Lang Florian
Department of Physiology, University of Tübingen, Tübingen, Germany.
Department of Physiology, University of Tübingen, Tübingen, Germany ; Department of Immunology, Institute of Experimental Medicine, St. Petersburg, Russia.
PLoS One. 2014 Jan 27;9(1):e87457. doi: 10.1371/journal.pone.0087457. eCollection 2014.
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.
人类醚-去极化相关基因钾通道（hERG）参与肿瘤细胞增殖和凋亡的调控。hERG通道活性受生长因子上调。对生长因子信号敏感的激酶包括丝氨酸/苏氨酸蛋白激酶B-RAF。因此，本研究探讨了B-RAF是否影响hERG通道的表达和活性。为此，在有或没有野生型B-RAF的非洲爪蟾卵母细胞中表达hERG通道，利用双电极电压钳测定hERG通道活性，并利用共聚焦显微镜和化学发光分析细胞膜中hERG蛋白丰度。此外，在横纹肌肉瘤RD细胞中，通过全细胞膜片钳定量分析B-RAF抑制剂PLX-4720对hERG介导电流的影响，并利用细胞表面蛋白生物素化和流式细胞术分析hERG细胞表面蛋白丰度。结果显示，在表达hERG的非洲爪蟾卵母细胞中共表达野生型B-RAF可显著增加hERG通道活性和细胞膜中hERG通道蛋白丰度。用B-RAF抑制剂PLX-4720（10 µM）处理表达B-RAF和hERG的非洲爪蟾卵母细胞24小时，可显著降低hERG介导的电流和hERG细胞表面表达。同样，在横纹肌肉瘤RD细胞中，用B-RAF抑制剂PLX-4720处理24小时可显著降低hERG细胞膜蛋白丰度和hERG介导的电流。总之，B-RAF是hERG通道活性和细胞表面hERG蛋白丰度的有力调节因子。
