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This Article Full Text Full Text (PDF) All Versions of this Article: 581/2/479    most recent jphysiol.2006.123414v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cockerill, S. L. Articles by Mitcheson, J. S. Search for Related Content PubMed PubMed Citation Articles by Cockerill, S. L. Articles by Mitcheson, J. S. Related Collections Cellular

¹ Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, UK

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_q/11-coupled M₃-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 nM 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 [³²P]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.

(Received 23 October 2006; accepted after revision 13 March 2007; first published online 15 March 2007)
Corresponding author J. S. Mitcheson: University of Leicester, Department of Cell Physiology and Pharmacology, Maurice Shock Medical Sciences Building, University Road, Leicester LE1 9HN, UK. Email: jm109{at}leicester.ac.uk

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