Intracellular Binding of Terfenadine Competes with Its Access to Pancreatic ß-cell ATP-Sensitive K+ Channels and Human ether-à-go-go-Related Gene Channels

Figures

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  Fig. 1. Effects of application of terfenadine via either the bath or pipette solution on hERG currents using the whole-cell configuration of the patch-clamp technique. A the voltage-clamp protocol used to study hERG currents (upper trace). The lower traces show the effects of terfenadine applied via the bath solution on hERG currents. The arrow indicates the zero current level. The extent of block of hERG peak tail currents at terfenadine concentrations of 3, 30, and 300 nM was 17.7, 57.3, and 94.9%, respectively. B hERG current traces at times 0 min (control) and 10 min after breaking into the cell in the presence of 3 µM terfenadine in the pipette solution; the extent of hERG peak tail current block was 47.1%. C concentration–response relationships for the inhibition of hERG peak tail currents by terfenadine applied via either the bath or pipette solution. The ordinate represents current amplitudes in the presence of different concentrations of terfenadine applied via either the bath or pipette solution (I) in percent of the current amplitudes obtained either before extracellular application of terfenadine or at time 0 min after breaking into the cells with intracellular application of…

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  Fig. 2. Effects of terfenadine applied via the bath solution on KATP currents from RINm5F cells using the whole-cell configuration of the patch-clamp technique. The holding potential was –70 mV and current responses to ± 10 mV pulses are shown. A in the example shown, current amplitudes increased during the first minutes of recording due to the washout of ATP from the cytoplasm. Tolbutamide (100 µM) was added to the bath solution during the period indicated by the first horizontal bar and reversibly blocked current amplitudes by 89.1%. Terfenadine (3 µM) was added to the bath solution during the period indicated by the second horizontal bar and blocked current amplitudes by 38.9%. After washout of terfenadine, channel activity did not recover. B concentration–response relationship for the inhibition of whole-cell KATP currents from RINm5F cells by terfenadine applied via the bath solution. The ordinate represents current amplitudes in the presence of different concentrations of terfenadine applied via the bath solution (I) in percent of the current amplitudes in the absence of terfenadine (control; Ic). The abscissa indicates the concentrations of terfenadine (logarithmic scale). Points…

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  Fig. 3. Effects of terfenadine and tolbutamide applied via the pipette solution on KATP currents from RINm5F cells using the whole-cell configuration of the patch-clamp technique. The holding potential was –70 mV and current responses to ± 10 mV pulses are shown. A time courses of KATP currents during 12–13 min after breaking into the cell in the absence (upper trace) and the presence of 10 µM terfenadine (middle trace) or 100 µM tolbutamide (lower trace) in the pipette solution. Maximum currents in response to 10 mV depolarizing pulses were divided by the cell capacitance and were 19.8 pA/pF under control conditions, 16.8 pA/pF in the presence of 10 µM terfenadine in the pipette solution and 6.5 pA/pF in the presence of 100 µM tolbutamide in the pipette solution. B concentration–response relationship for the inhibition of whole-cell KATP currents from RINm5F cells by tolbutamide applied via the pipette solution. The ordinate represents current densities in the absence of test substances in the pipette solution (control, Ic; mean value 18.6 pA/pF) and in the presence of different concentrations of tolbutamide applied via the pipette solution (I). The abscissa indicates the concentrations…

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  Fig. 4. Effects of terfenadine applied via the pipette solution for 10 min on hERG currents in the presence of the P-glycoprotein inhibitor cyclosporine A in the bath solution and in the absence of MgATP in the pipette solution. A the voltage-clamp protocol used to study hERG currents (upper trace). The lower traces show the effects of 3 µM terfenadine applied via the pipette solution in the presence of 10 µM cyclosporine A in the bath solution on hERG currents. The extent of block induced by 3 µM terfenadine applied via the pipette solution was 77.3% in the presence of 10 µM cyclosporine A. B hERG current traces at times 0 min (control) and 10 min after breaking into the cell in the presence of 3 µM terfenadine in the pipette solution in the absence of MgATP. The extent of block induced by 3 µM terfenadine was 76.4% in the absence of MgATP in the pipette solution (B)

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  Fig. 5. Decrease of the EGFP-induced fluorescence intensity in hERG/EGFP fusion protein-expressing HEK 293 cells after obtaining the whole-cell configuration evaluated using a laser-scanning microscope. A EGFP-induced fluorescence intensity in a hERG/EGFP fusion protein-expressing HEK 293 cell (upper left), transmission image of the same cell with an attached patch pipette (upper right), and fusion image of both (lower left). B time course of the EGFP-induced fluorescence intensity in hERG/EGFP fusion protein-expressing HEK 293 cells in the cell-attached configuration (left) and 10 min (middle) or 30 min (right), respectively, after membrane rupture. Fluorescence intensity values determined from a region of interest (ROI) located over the entire cell were 32 (left), 27 (middle), and 20 (right). C mean values ± S.E.M. of EGFP-induced fluorescence intensity in hERG/EGFP fusion protein-expressing HEK 293 cells after obtaining the whole-cell configuration as a function of time obtained from 11 experiments. Ordinate: EGFP-induced fluorescence intensity normalized to the mean value of the three time points before rupture of the membrane patch (F/Fmax; c.-a. cell attached); abscissa: time.…

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  Fig. 6. FCS experiments of hERG/EGFP channels transiently expressed in HEK 293 cells. A confocal image of fluorescence intensity from HEK 293 cells expressing hERG/EGFP channels. B fluorescence intensity fluctuations (count rate) obtained from the cytosol of the cell in the middle of the image. C fluorescence autocorrelation function (G(t)) of hERG/EGFP channels expressed in HEK 293 cells fitted with a two-component model. hERG/EGFP channels had a count rate of 25.5 kHz/molecule, and the diffusion time constants in the cytosol of this example were 0.46 and 29.4 ms, respectively. In addition, a triplet state can be seen at short correlation times with a lifetime of 24 µs

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  Fig. 7. Increase of the Bodipy-glibenclamide-induced fluorescence intensity in RINm5F cells in the whole-cell configuration evaluated using a laser-scanning microscope. A upper left: Transmission image of RINm5F cells with an attached recording pipette in the whole-cell configuration. Lower: Confocal images of the fluorescence of the cells before (left) and 6 min after administration of 10 nM Bodipy-glibenclamide (middle) and after 5 min washout of Bodipy-glibenclamide (right). Fluorescence intensity values determined from a region of interest (ROI) located over the entire cell were 10 before, 97 after 6 min administration of Bodipy-glibenclamide and 73 after 5 min of washout of Bodipy-glibenclamide. Upper right: Mean values ± S.E.M. of fluorescence intensity from 7 RINm5F cells in the whole-cell configuration as a function of time before, during administration of 10 nM Bodipy-glibenclamide and after washout. 10 nM Bodipy-glibenclamide was present throughout the period indicated by the horizontal bar. Ordinate: fluorescence intensity normalized to the mean value of the three time points before administration of 10 nM Bodipy-glibenclamide (F/Fcont); abscissa: time. Points represent means…

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  Fig. 8. Effects of terfenadine administered either via the bath solution or via the pipette solution on hERG (Y652A) currents using the whole-cell configuration of the patch-clamp technique. A the voltage-clamp protocol used to study hERG (Y652A) currents (upper trace). Effects of terfenadine administered via the bath solution on hERG (Y652A) currents (middle traces). The extent of block was 39.6% at 30 nM and 86.0% at 3 µM terfenadine, respectively, in this example. Representative current traces at times 0 min (control) and 10 min after breaking into the cell in the presence of 10 µM terfenadine in the pipette solution (lower traces). The extent of block was 33.8% in this example. B concentration–response relationships for the inhibition of hERG (Y652A) peak tail currents by terfenadine administered either via the bath solution (●) or via the pipette solution for 10 min (▲). The ordinate represents current amplitudes in the presence of different concentrations of terfenadine (I) in percent of the current amplitudes either determined before extracellular administration of terfenadine or directly after breaking into the cells for intracellular administration of terfenadine (control; Ic).…