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Modulation of hERG potassium currents in HEK-293 cells by protein kinase C. Evidence for direct phosphorylation of pore forming subunits
The human ether-a-go-go related gene (hERG) potassium channel is expressed in a variety of tissues including the heart, neurons and some cancer cells. hERG channels are modulated by several intracellular signalling pathways and these provide important mechanisms for regulating cellular excitability....
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Published in: | The Journal of physiology 2007-06, Vol.581 (2), p.479-493 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | The human ether-a-go-go related gene (hERG) potassium channel is expressed in a variety of tissues including the heart, neurons and some cancer cells.
hERG channels are modulated by several intracellular signalling pathways and these provide important mechanisms for regulating
cellular excitability. In this study, we investigated muscarinic modulation of hERG currents and direct phosphorylation of
channel subunits expressed in HEK-293 cells at physiologically relevant temperatures by protein kinase C (PKC). Activation
of G αq/11 -coupled M 3 -muscarinic receptors with methacholine, reduced current amplitudes at all potentials with minor effects on the voltage dependence
of activation and inactivation. The response to methacholine was insensitive to intracellular BAPTA, but was attenuated by
either acute inhibition of PKC with 300 n m bisindolylmaleimide-1 (bis-1) or chronic down-regulation of PKC isoforms by 24 h pretreatment of cells with phorbol 12-myristate
13-acetate (PMA). Stimulation of PKC with 1-oleoyl 2-acetylglycerol (OAG), an analogue of diacylglycerol (DAG), mimicked the
actions of muscarinic receptor stimulation. Direct phosphorylation of hERG was measured by [ 32 P]orthophosphate labelling of immunoprecipitated protein with an anti-hERG antibody. Basal phosphorylation was high in unstimulated
cells and further increased by OAG. The OAG dependent increase was abolished by bis-1 and down-regulation of PKC, but basal
levels of phosphorylation were unchanged. Deletion of the amino-terminus of hERG prevented both the modulation of channel
activity and the increase of phosphorylation by OAG. Our results are consistent with calcium and/or DAG sensitive isotypes
of PKC modulating hERG currents through a mechanism that involves direct phosphorylation of sites on the amino terminus of
hERG. |
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ISSN: | 0022-3751 1469-7793 |
DOI: | 10.1113/jphysiol.2006.123414 |