The human ether-a-go-go related gene (HERG) encodes a voltage gated potassium (K⁺) channel widely expressed in the nervous system and heart. Attenuation of the cardiac HERG channel current, either resulting from inhibition by drugs or inherited mutations in HERG causes abnormal action potential repolarisation and cardiac arrhythmias. Cyclic AMP and several kinases modify HERG channel activity, but little is known about its modulation by Ca²⁺-dependent pathways. Here, we have investigated the mechanisms for the decrease of HERG currents in response to elevation of intracellular [Ca ²⁺] ([Ca²⁺]_i).

HEK 293 cells either stably or transiently expressing HERG were superfused with a NaCl-based extracellular solution containing 4 mM K⁺ and 2 mM Ca²⁺, at 37 °C. Currents were recorded using whole cell patch clamp. Cells were dialysed with KCl-based, Ca²⁺-free intracellular solution and currents elicited by stepping from -80 mV to 0 mV for 5 s. Data are expressed as means ± S.E.M. from more than five cells and were compared with Student’s unpaired t test, with the level of significance taken as P < 0.05.

Elevating [Ca²⁺]_i by application of the Ca²⁺ ionophore ionomycin (5 µM) or methacholine (1 mM) to stimulate Gα_q/11-coupled muscarinic M₃ receptors resulted in a sustained decrease of HERG current of 38 ± 1 % and 30 ± 5 %, respectively. In both cases, the decrease of HERG current could be partially blocked by prior application of 300 nM bisindolylmaleimide-1 (bis-1), a selective protein kinase C (PKC) inhibitor. Higher concentrations of bis-1 had an inhibitory effect on HERG currents. As an alternative approach to reduce PKC activity, cells were treated with 1 µM PMA for 24 h to down-regulate PKC. PMA resulted in a marked decrease in the levels of PKCα, β and δ in Western blots of PKC isoforms and the inhibition of HERG currents by ionomycin and M₃ receptor stimulation was abolished. However, HERG currents from cells pre-treated with an inactive analogue of PMA (4αPMA) remained sensitive to ionomycin and methacholine. To further characterise the effect of PKC, HERG currents were recorded before and after application of 10 µM 1-oleoyl-2-acetylglycerol (OAG). OAG reduced peak currents at 0 mV by 37 ± 3 %, shifted the voltage dependence of activation by +5.8 mV, but had no significant effect on the voltage dependence of inactivation.

Taken together these results suggest there is no direct or calmodulin-mediated effect of Ca²⁺ on HERG channels under our recording conditions. However, Ca²⁺ can modulate HERG channel activity through stimulation of PKC. Ca²⁺ or diacylglycerol stimulated PKC activity causes a reduction of HERG current and positive shift of activation.

This work was supported by the British Heart Foundation