Effects of Mexiletine on a Race-specific Mutation in Nav1.5 Associated With Long QT Syndrome

Figures

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  FIGURE 1. Asian-specific mutation P1090L in the Nav1.5 sodium channel. (A). Topological diagram showing P1090L located in the intracellular loop between the DII-DIII domain of the Nav1.5 channel. (B). Nav1.5 sequence alignment indicates that the P1090 is highly conserved across vertebrate species. (C,D): Representative wild-type (WT) (C) and P1090L (D) sodium currents. Currents were measured from a holding potential of −120 mV to test potentials ranging between −110 and +60 mV in 5 mV steps. (E). Average peak current-voltage (I-V) relationships for WT channel and P1090L mutation (n = 8 cells in each group, data are presented as the mean ± SEM).

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  FIGURE 2. Effects of P1090L mutation on voltage-dependent gating of the Nav1.5 channel. (A). Steady-state activation and inactivation curves of Nav1.5 WT and P1090L mutation. Inactivation currents were elicited to −20 mV following prepulses between −120 and −20 mV in 5 mV steps for 500 ms. Activation currents were measured from a holding potential of −120 mV to test potentials ranging between −110 and +20 mV in 5 mV steps. Curves are Boltzmann fits of the data points (n = 8). (B). The view of the overlapping area between activation and steady-state inactivation was expanded from (A). Left inset shows sodium window current of WT Nav1.5 channel (black). Right inset shows sodium window current of P1090L mutation (red). (C). Summary of V1/2 of steady-state inactivation for WT and P1090L channel. (D). Summary of V1/2 of voltage-dependent activation for WT and P1090L channel. Data are presented as the mean ± SEM. *p < 0.05.

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  FIGURE 3. Effects of P1090L mutation on the channel inactivation. (A). Development of inactivation for WT and P1090L mutation was assessed by varying the duration of a conditioning pulse P1 (1–2000 ms) to −20 mV followed by a return to -120 mV for 20 ms followed by a 100 ms test pulse P2 to −20 mV. (B). Recovery of inactivation for WT and P1090L mutation was assessed using insert protocol with a first pulse duration of 1000 ms (P1) and a second pulse of 200 ms (P2) to −20 mV with varying rest intervals from 1 to 2000 ms at −120 mV. Curves in (A,B) are exponential function fits of the data points, n = 8 in each group. (C). Summary of time constant for development (Ʈdev) for WT and P1090L channel. (D). Summary of time constant for fast recovery (Ʈrec,f) for WT and P1090L. (E). Summary of time constant for slow recovery (Ʈrec,s) for WT and P1090L. Data are presented as the mean ± SEM. *p < 0.05.

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  FIGURE 4. P1090L mutation does not increase the late sodium currents of the Nav1.5 channel. (A,B): Representative wild-type (WT) (A) and P1090L (B) persistent sodium current traces. Currents were activated by depolarization to −20 mV from a holding potential of −120 mV, and the dash line represented 0 pA. (C). The late sodium currents were measured and normalized to peak currents, n = 6 cells in each group, data are presented as the mean ± SEM. NS, non-significant.

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  FIGURE 5. Inhibition of P1090L mutation currents by mexiletine. (A). Representative current traces of P1090L recorded in the absence (Control) and presence of 200 µM mexiletine. Currents were elicited by a test pulse of −20 mV from a holding potential of −120 mV. (B). Quantification of P1090L mutation current densities without (Control) or with the treatment of 200 µM mexiletine. n = 6 for each group, data are presented as the mean ± SEM. *p < 0.05. (C). Dose-dependent effects of mexiletine on P1090L sodium current inhibition (n = 4-6 for each concentration; data are presented as the mean ± SEM). Curves are Hill fits of the data points.

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  FIGURE 6. Effects of mexiletine on voltage-dependent gating of the P1090L mutation. (A). Steady-state inactivation and activation curves of P1090L mutation currents in the absence (Control) and presence of 200 µM mexiletine. Inactivation currents were elicited to −20 mV following prepulses between −120 and −20 mV in 5 mV steps for 500 ms. Activation currents were measured from a holding potential of −120 mV to test potentials ranging between −110 and +20 mV in 5 mV steps. Curves are Boltzmann fits of the data points (n = 8 for Control, n = 6 for Mexiletine; data are presented as the means ± SEM). (B). The view of the overlapping area between activation and steady-state inactivation was expanded from (A). Right inset shows P1090L mutation sodium window current without mexiletine (Control), left inset shows P1090L window current in presence of the drug (Mexiletine). (C). Summary of V1/2 of steady-state inactivation for control group and mexiletine group. (D). Summary of V1/2 of voltage-dependent activation for control and mexiletine. Data are presented as the mean ± SEM. *p < 0.05.

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  FIGURE 7. Effects of mexiletine on the development and recovery of inactivation of the P1090L mutation. (A). Development of inactivation for P1090L mutation in the absence (Control) or presence of 200 µM mexiletine was assessed by varying the duration of a conditioning pulse P1 (1–2000 ms) to −20 mV followed by a return to −120 mV for 20 ms followed by a 100 ms test pulse P2 to −20 mV. (B). Recovery of inactivation for P1090L mutation in the presence or absence of mexiletine was assessed using insert protocol with a first pulse duration of 1000 ms (P1) and a second pulse of 200 ms (P2) to −20 mV with varying rest intervals from 1 to 2000 ms at −120 mV. Curves in (A,B) are exponential function fits of the data points, n = 8 for Control, n = 6 for Mexiletine; data are presented as the mean ± SEM. (C). Summary of time constant for development (Ʈdev) for control group and mexiletine group. (D). Summary of time constant for fast recovery (Ʈrec,f) for control and mexiletine. (E). Summary of time constant for slow recovery (Ʈrec,s) for control and mexiletine. Data are presented as the mean ± SEM. *p < 0.05.