Block of Voltage-Gated Sodium Channels by Aripiprazole in a State-Dependent Manner

Figures

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  Figure 1. Inhibition of hNav1.5 channels by aripiprazole: Representative current traces obtained for control (Co) and in the presence of 3 µM aripiprazole (ari: 3). Channel activations were carried out by short depolarizations to −20 mV for 5 ms from a holding potential of −85 mV.

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  Figure 2. Interaction with the resting state. Relative current amplitudes versus the concentration of aripiprazole are illustrated. Channel activations in the absence (control) and presence of different concentrations of aripiprazole (test) were carried out from a holding potential of −140 mV (insert). Current amplitudes obtained for the test pulses were related to their controls and plotted against the concentration of aripiprazole. Data points were fit to Equation (1). Note, even at the highest concentration of aripiprazole (10 µM), the inhibitory effect small. Thus, the calculated affinity (Kr: 54.7 ± 13.0 µM) has to be considered as an approximate estimation.

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  Figure 3. (A) Voltage dependence of activation. The graph illustrates representative data obtained from one typical cell. Normalized peak conductances in the absence (control 1 and 2; open circles and triangles) and presence of 1 µM aripiprazole (filled circles) versus test pulse potential are shown. The potential difference in activation midpoints between control 1 and 2 was taken into account as the drug-independent shift. Altogether, mean half-maximal activation for control 1 and 2 occurred at –43.6 ± 3.0 mV and −45.2 ± 3.4 mV with a mean slope of 6.5 mV. The corresponding values in the presence of 1 µM aripiprazole were −46.5 ± 4.2 mV, slope 6.7 mV. Insert: Experimental scheme. Starting from a holding potential of −140 mV, test pulses for 5 ms to potentials ranging from −90 to +20 mV (increment 5 mV) were carried out. Interpulse interval was 5 s. (B) Overlay of original current traces (control 1) obtained for the data shown in (C) Peak current amplitudes at different test pulse potentials from the traces shown in (B). All data are from the same cell.

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  Figure 4. Voltage dependence of fast inactivation. (A) Data of normalized peak currents are plotted against the prepulse potential. Solid lines represent fits according to Equation (4). Insert: Experimental scheme. Voltage dependence of fast-inactivation was determined by measuring sodium currents elicited by 5 ms depolarizations to −20 mV after conditioning prepulses for 500 ms to different potentials (range −140 to −50 mV) in the absence (open circles) and presence (filled circles) of 1 µM aripiprazole. Holding potential was −140 mV. (B) Overlay of original current traces obtained for control. (C) Relative shift of inactivation midpoints in relation to the applied concentration of aripiprazole. Data are corrected for the drug-independent shift (1.0 mV/measuring cycle) and fitted according to Equation (6) using the mean slope obtained in the presence of aripiprazole. The fit gives an affinity of 0.5 ± 0.14 µM for the fast-inactivated state.

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  Figure 5. Voltage dependence of slow inactivation. (A) Data of normalized peak currents from a representative cell are plotted versus the prepulse potential. Solid lines are fits according to Equation (5) for control (open circles) and different concentrations of aripiprazole (filled symbols). The experimental scheme as illustrated by the insert of B consisted of channel activations to −20 mV after long-lasting (10 s) conditioning prepulses to different potentials and a short (20 ms) recovery period at −140 mV immediately before the test pulse. (B) Normalized data for 1 µM aripiprazole were obtained by dividing the values obtained in the presence of 1 µM aripiprazole by those obtained for control. It is evident that current amplitudes continuously decreased with an increase in prepulse potential. (C) Concentration response curve taken from normalized current amplitudes obtained at the prepulse potential of 0 mV. The affinity to the slow inactivated state (Ki) obtained from this experiment is given in the insert and was obtained from a fitting of data according to Equation (1). Altogether, an affinity for the slow inactivated state of 0.39 ± 0.07 µM resulted.

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  Figure 6. Time course of block development. (A) The graph illustrates relative current amplitudes in dependence on the duration of the inactivating prepulse for control (open circles) and in the presence of different concentrations of aripiprazole (filled symbols). Solid lines represent fits of single (control) or double exponential functions (in the presence of aripiprazole). (B) Experimental scheme for an individual sweep. Channels were inactivated at −20 mV for a variable duration. Immediately before the test pulse a short recovery period was inserted. The amplitude of the inactivating current was used as control. Interval between individual sweeps was 5 s. (C,D). The inverse of the time constants of the data shown in A were plotted versus the concentration of aripiprazole. Association (kon) and dissociation rate constants (koff) were estimated from the slope and y-intercept of the linear fit. Inserts give fit data for this particular experiment. Altogether affinity constants for the fast and slow time constants were 0.55 µM and 0.38 µM, respectively. This evaluation was performed under the reservation that the data from the individual terms were allowed to be handled separately, as…

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  Figure 7. Apparent affinity. (A) The graph illustrates relative current amplitudes in the presence of different concentrations of aripiprazole (black circles represent measured normalized current amplitudes, solid line is the result of the fit to Equation (1)). (B) Original current traces obtained from channel activations carried out according to the protocol illustrated in Figure 1. The holding potential was set close to half-maximal inactivation (here: −90 mV). Aripiprazole was preincubated for 10 s before the test pulse was given. Interval between individual sweeps was 10 s. The apparent affinity (Kapp) was obtained from a fit of data according to Equation (1). For calculating the affinity to the inactivated state (Ki), Equation (7) was employed, which respects the affinity to the resting state (Kr) and the amount (h) of currently available (not-inactivated) channels. Fit parameters for this cell are shown as insert. Altogether, a Ki of 2.14 ± 1.16 µM was estimated.

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  Figure 8. Recovery from inactivation. (A) Overlay of 19 original current traces obtained for control from the initial part of the conditioning inactivation pulses (inact.), which served as control and their corresponding test pulses (test). The variable recovery time is not illustrated. (B) Recovery from fast inactivation was performed using an inactivation time of 500 ms. The relative amount of available channels expressed as relative current amplitude (test/inact.) versus the recovery time at −140 mV is illustrated. The pulse protocol is illustrated by the inset. Interval between individual sweeps was 10 s and aripiprazole was applied at 1 µM. Solid lines represent fits with two exponential functions according to Equation (11). (C) Otherwise identical protocol as in (B), but with the inactivation time set to 5 s.

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  Figure 9. Analysis for use-dependence. Short activations (1 ms) were carried out at a frequency of 10 Hz in the absence and presence of 10 µM aripiprazole. Current amplitudes declined under control from the first to the last activation (50th pulse) by 1.8 ± 1.1%. In the presence of aripiprazole (10 µM), current reduction amounted to 8.2 ± 2.1%. Aripiprazole was preincubated for 20 s.

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  Figure 10. Interaction with the open state. (A) Original current traces obtained from the inactivation deficient mutant Nav1.5_CW in the absence and presence of different concentrations of aripiprazole. Individual activations were carried out from a holding potential of −140 mV by stepping to −20 mV for 50 ms. Drugs were preincubated for 60 s before individual activations were carried out. (B) For evaluation, relative plateau current amplitudes (measured at the end of the depolarization) were plotted versus the concentration of aripiprazole (the amplitude obtained for control was set to 1). Data fitting according to Equation (1) revealed fit parameters of this experiment as illustrated. Ko indicates the affinity for the open state. Altogether, an affinity for the open state of 0.94 ± 0.25 µM was estimated.

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  Figure 11. Time course of block development at the F1760K mutant. (A) Identical experiments as shown by Figure 6 were carried out with the F1760K mutant. Note: differently to the wildtype, time course of block development for the mutant can be described with one single exponential function. (B) Inverse of the time constants versus the concentration of aripiprazole. Listed fit parameters refer to this particular experiment. Altogether, a Ki of 2.39 ± 0.28 was estimated.