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Fig. 1. The effect of ceramide on I_HERG density in stably expressing HEK293 cells. (A) Whole-cell current recordings of I_HERG in the absence and presence of 10 µM ceramide or dihydro-ceramide. From a holding potential of –80 mV cells were stepped to potentials between –70 mV and +40 mV for 2 seconds with tail currents observed on repolarisation to –60mV for 4.5 seconds. Inset, Identification of I_HERG by the use of the inhibitor cisapride (1 µM) with the current evoked by a depolarising step to 0 mV. (B,C) Voltage-dependence of I_HERG density with I_HERG measured at the end of the depolarising steps (B) and as peak tail current (C). , control, n=12; , 10 µM ceramide, n=9. Significant differences in current density were observed (^*P<0.05, ^** P<0.001) compared to levels in the control. (D) Instantaneous I_HERG density-voltage plot. Insets show the effect of 10 µM ceramide on the instantaneous I_HERG density at 0 mV and the voltage protocol used, which consisted of a 2 second activating step to +40 mV, a 13 msecond step to –100 mV to relieve inactivation and then a step to potentials between +50 mV and –20 mV. I_HERG was measured immediately following the end of the brief hyperpolarising step (n=7 for both control () and 10 µM ceramide (); ^*P<0.05). (E) The effect of ceramide concentration on the instantaneous I_HERG density at 0 mV (voltage protocol as in D). The number of cells tested are given in parentheses (^*P<0.05 compared to control current).

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