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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 Gαq/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.

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(alpha q/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 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 [(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.

The human ether-a-go-go-related gene (hERG) potassium channel is expressed in a variety of cell types, including neurons, tumor cells, and cardiac myocytes. In the heart, it is important for repolarization of the cardiac action potential. Attenuation of hERG current can cause long QT syndrome and cardiac arrhythmias such as torsades de pointes. Caffeine is frequently used as a pharmacological tool to study calcium-dependent transduction pathways in cellular preparations. It raises cytosolic calcium by opening ryanodine receptors and may also inhibit phosphodiesterases to increase cytosolic cAMP. In this study, we show 5 mM caffeine rapidly and reversibly attenuates hERG currents expressed in human embryonic kidney 293 cells to 61.1 +/- 2.2% of control. Caffeine-dependent inhibition of hERG current is not altered by raising cAMP with forskolin, buffering cytosolic calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or inhibition of protein kinase C. Thus, the effects of caffeine are unlikely to be mediated by cAMP or intracellular calcium-dependent mechanisms. Further experiments showed caffeine directly blocks hERG in an open state-dependent manner. Furthermore, caffeine inhibition is greatly reduced by the pore mutants Y562A and F656A hERG, which disrupt block of most previously tested hERG antagonists. Thus, caffeine attenuates hERG currents by binding to a drug receptor located within the inner cavity of the channel. Dietary intake of caffeine is unlikely to cause long QT syndrome because plasma concentrations do not reach sufficiently high levels to significantly inhibit hERG currents. However, the effects of caffeine have implications for its use in examining calcium-dependent pathways in cellular preparations expressing hERG.