Concentration dependence of CsA-induced inhibition of I_HERG. (A) Superimposed I_HERG traces were elicited with 4-s depolarization to +20 mV from a holding potential of –80 mV, and the tail current was recorded at –60 mV for 6 s in the absence and presence of 0.3, 1, 3, and 10μM CsA, as indicated. The protocol was applied every 15 s. The dotted line represents zero current. (B) Concentration-dependent curve of inhibition by CsA for steady-state currents (open circle) measured at the end of a depolarizing pulse to +20 mV or peak tail currents (closed circle). The respective percentage inhibitions were plotted against various concentrations of CsA. The solid lines are fitted to the data points by the Hill equation. Data are expressed as mean±SEM.

The effects of a calcineurin inhibitor on the inhibition of I_HERG by CsA. Representative superimposed currents were elicited with 4-s depolarization to +20 mV from a holding potential of –80 mV, and the tail current was recorded at –60 mV for 6 s every 15 s. (A) Control current, the current recorded after 5-min exposure to 10μM cypermethrine, and the current measured after a further 3-min treatment with 3μM CsA are shown. The dotted line represents zero current. (B) Peak tail currents under a set of experimental conditions (A) were normalized to those of the control, and are displayed as a percentage of inhibition to show the effects of 10μM cypermethrine (n=5), 10μM cypermethrine with 3μM CsA (n=5), and 3μM CsA (n=7, Fig. 1). Data are expressed as mean±SEM.

Effect of CsA on the I_HERG-voltage (I-V) relationship. (A) Representative superimposed current traces recorded under control conditions and 3-min exposure to 3μM CsA. I_HERG was produced by applying 4-s depolarizing pulses between –70 and +60 mV in 10-mV increments every 15 s from a holding potential of –80 mV. Tail current was recorded at –60 mV for 6 s. The dotted lines represent zero current. (B) The I-V relationships for I_HERG were measured at the end of the depolarizing pulses and for peak tail current. (C) Normalized activation curves were recorded under control conditions and 3 min after exposure to 3μM CsA. The solid lines represent the activation curves obtained by normalization to the tail peak amplitude from (A) and by fitting those data to the Boltzmann equation (see METHODS, equation 2).

Voltage dependence of CsA-induced inhibition of I_HERG. (A) Representative superimposed current traces under control conditions and in the presence 3μM CsA selected at three different potentials (–20, 0 and +60 mV) recorded using the pulse protocols. The dotted lines represent zero current. (B) Percentage of current inhibition (closed triangle) at different membrane potentials. The tail currents in the presence of 3μM CsA were normalized to the tail current obtained under control conditions. The dashed line represents the activation curve of the HERG channel under control conditions, which was calculated by measuring tail current amplitudes and by fitting those data to the Boltzmann equation (see METHODS, equation 2). For potentials positive to 0 mV, the solid line was drawn from a linear curve fitting (n=5; ^∗p<0.05 versus data at –20 mV). Data are expressed as mean±SEM.

State-dependent inhibition of I_HERG by CsA. (A) Representative superimposed current traces under control conditions and after application of 3μM CsA for 3 min. Cells were held at a holding potential of –80 mV to the channels in the closed state before a single depolarizing step to 0 mV for 8 s resumed in the presence of CsA. (B) The % inhibition was obtained by division and plotted as a function of time. A strong time-dependent development of I_HERG inhibition by CsA was detected, suggesting an open channel block by CsA. (C) Superimposed typical current traces under control conditions and after application of 3μM CsA for 3 min with a holding potential of –80 mV, which were obtained by a first 4-s depolarizing pulse of +80 mV followed by a second 4-s depolarizing pulse of 0 mV. (D) The normalized fractional inhibition. The % inhibition upon channel opening during the second voltage step (0 mV) was obtained by division and plotted against time. Maximum inhibition was detected in the inactivated state during the first step pulse, and no further time-dependent inhibition occurred upon channel opening during the second voltage step. The dotted lines represent zero current.