Up-Regulation of hERG K+ Channels by B-RAF

Abstract

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.

Biophysical Characterization and Expression Analysis of Kv1.3 Potassium Channel in Primary Human Leukemic B Cells

“…compound [25] which is able to prevent B-RAF-induced stimulation of hERG by decreasing PM-located channel protein and its activity in rhabdomyosarcoma cells [26]. In addition, a ligand of the Sigma Receptor 1, a known ion channel modulator [27,28], was tested.…”

“…Several kinases have been shown to be implicated in regulation of potassium channel protein abundance at the level of the PM. For example Janus kinase in the case of calcium-dependent potassium channels [35], mTOR kinase in the case of Kv1.3 and Kv1.5 [36] and the serine/threonine protein kinase B-RAF in the case of HERG [26] impact channel expression. Here we show that B-RAF signalling contributes to the observed increase of Kv1.3 expression in the pathologic B cells.…”

The Impact of Pazopanib on the Cardiovascular System

“…78,79 Inhibition of these kinases likely contributes to cardiovascular toxicity, as cardiac-specific C-RAF knockout mice have increased cardiomyocyte apoptosis accompanied by left ventricular systolic dysfunction and dilation of the heart. 80 B-RAF activity is a strong mediator of human ether-a-go-go-related gene (hERG) channel expression, 81 and selective inhibition of B-RAF by vemurafenib has been associated with prolonged QT interval in humans. 82 Mechanistically, it has been proposed that BRAF inhibition may lead to a compensatory increase in cyclic adenosine monophosphate (AMP) leading to overactivation of protein kinase A, an enzyme that phosphorylates hERG channels and reduces their ability to open during action potential voltages–prolonging repolarization period and contributing to prolonged QT interval.…”

Systems Medicine: The Application of Systems Biology Approaches for Modern Medical Research and Drug Development

“…Another area of drug development in which systems approaches are of significance is in the prediction of drug/target interactions. The study carried out by Babcock et al investigated drug-induced gene expression profiles for predicting novel inhibitor molecules against the human ether-a-go-go related (hERG) potassium channel, which plays an important part in the regulation of tumour cell proliferation and apoptosis [ 50 , 51 ]. The study utilized the Connectivity Map (CMap) to select candidate hERG inhibitors with similar gene signature expression profile induction, together with analytical methodologies from databases of experimental datasets for annotated hERG inhibitor activities [ 50 ].…”