Fig. 1 Inhibition of hERG channels by bisindolylmaleimide I Fig. 1 Inhibition of hERG channels by bisindolylmaleimide I. Representative current traces recorded from the same cell are displayed in panel A. (B) Concentration–response relationship for the effect of BIM I on hERG peak tail currents (n=3 to 5 oocytes). (C) Time course of hERG tail current inhibition by 30 μM BIM I (n=4). For simplicity, not all current measurements are displayed. (D) mean relative tail current amplitudes after application of 100 μM BIM I (20 min) for hERG wild type (n=5), hERG Y652A (n=5), and hERG F656A currents (n=5), respectively (see text for voltage protocol). From: Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X) Cardiovasc Res. 2004;64(3):467-476. doi:10.1016/j.cardiores.2004.07.023 Cardiovasc Res | Copyright © 2004, European Society of Cardiology

Fig. 2 Effects of BIM I on the voltage dependence of hERG activation Fig. 2 Effects of BIM I on the voltage dependence of hERG activation. Control measurement (A) and the effects of 30 μM BIM I (20 min; B) are shown in one representative oocyte. (C) Resulting mean current amplitudes at the end of the test pulse as function of the preceding test pulse potential under control conditions and after incubation with BIM I (n=6). Panels D and E display activation curves (D, original current amplitudes; E, values normalized to peak tail currents; n=5) (see text for voltage protocol). From: Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X) Cardiovasc Res. 2004;64(3):467-476. doi:10.1016/j.cardiores.2004.07.023 Cardiovasc Res | Copyright © 2004, European Society of Cardiology

Fig. 3 Effects of BIM I on hERG current inactivation Fig. 3 Effects of BIM I on hERG current inactivation. Panels A and B show enlargements from representative single measurements of the steady-state inactivation after various potentials from −110 to +30 mV (increment 10 mV). Note that, for clarity, not all original current traces are displayed. The normalized mean inactivating current amplitudes at +20 mV are shown in panel C, giving steady-state inactivation curves (see text for voltage protocol). From: Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X) Cardiovasc Res. 2004;64(3):467-476. doi:10.1016/j.cardiores.2004.07.023 Cardiovasc Res | Copyright © 2004, European Society of Cardiology

Fig. 4 The biophysical mechanism of hERG current block by bisindolylmaleimide I. The control recording and the first pulse measured immediately after the incubation period are displayed (A). Panel B shows the degree of inhibition in %. Inhibition of activated channels increased time-dependently to 72.3% at 1000 ms in this representative experiment. (C) hERG channels were inactivated by a first voltage step to +80 mV, followed by a second pulse to 0 mV. The corresponding relative block during the 0 mV-step is displayed in panel D. Maximum inhibition was already achieved during the first pulse to +80 mV (see text for voltage protocols). From: Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X) Cardiovasc Res. 2004;64(3):467-476. doi:10.1016/j.cardiores.2004.07.023 Cardiovasc Res | Copyright © 2004, European Society of Cardiology

Fig. 5 (A) BIM I block of hERG currents is voltage-dependent Fig. 5 (A) BIM I block of hERG currents is voltage-dependent. (B) Application of BIM I did not cause significant changes in deactivation time constants at different potentials (n=5–6). (C) Lack of frequency-dependence of hERG channel block by BIM I. For the purpose of clear presentation, not all measurements are displayed (see text for voltage protocols). From: Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X) Cardiovasc Res. 2004;64(3):467-476. doi:10.1016/j.cardiores.2004.07.023 Cardiovasc Res | Copyright © 2004, European Society of Cardiology

Fig. 6 BIM I blockade of hERG in human HEK 293 cells Fig. 6 BIM I blockade of hERG in human HEK 293 cells. (A) Typical whole cell patch clamp recordings from one HEK/hERG cell. Tail currents were blocked by 49.3% and 97.5% in this representative cell. (B) Concentration–response curve for inhibition of hERG peak tail currents in HEK 293 cells (n=3–5 cells) (see text for voltage protocol). From: Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X) Cardiovasc Res. 2004;64(3):467-476. doi:10.1016/j.cardiores.2004.07.023 Cardiovasc Res | Copyright © 2004, European Society of Cardiology

Fig. 7 BIM I prolongs action potentials and reduces native I_Kr currents in guinea pig ventricular myocytes. (A) Current clamp recordings of action potentials under control conditions and after application of 1 μM BIM I at room temperature. (B) Quantitative analysis of APD₉₀ in the absence and in the presence of the drug (n=4 cells). (C) 1 μM BIM I blocked I_Kr (i.e. E4031-sensitive) tail currents recorded from this representative cardiomyocyte by 72.9%. (D) BIM I-sensitive current, isolated by subtraction of outward tail current recorded in the presence of BIM I from control current (same cell as in panel C) (see text for voltage protocol). From: Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X) Cardiovasc Res. 2004;64(3):467-476. doi:10.1016/j.cardiores.2004.07.023 Cardiovasc Res | Copyright © 2004, European Society of Cardiology

Fig. 8 BIM I does not affect KvLQT1/minK currents in Xenopus oocytes Fig. 8 BIM I does not affect KvLQT1/minK currents in Xenopus oocytes. (A, B) Representative current traces recorded from a cell expressing KvLQT1 and minK proteins before and after application of 30 μM BIM I (20 min). (C) I–V relationship for mean current amplitudes measured at the of the depolarizing test pulse. (D) Normalized I–V relationship for mean peak tail currents (see text for voltage protocol). From: Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X) Cardiovasc Res. 2004;64(3):467-476. doi:10.1016/j.cardiores.2004.07.023 Cardiovasc Res | Copyright © 2004, European Society of Cardiology