Fig 1.
Effects of mexiletine and lidocaine on voltage-dependent activation of Nav1.5 and Nav1.7 channels.
A, B, Representative activation traces were recorded in HEK293 cells stably expressing either Nav1.5 (A, left panel) or Nav1.7 (B, left panel) channels. In the presence of 0.3 mM mexiletine (middle panel) or lidocaine (right panel), the peak currents of both channels evoked by 50 ms pulses to -20 mV were inhibited. The protocol of voltage-dependent activation is shown in A inset. C, Normalized conductance versus voltage was plotted for Nav1.5 (left panel) and Nav1.7 (right panel) channels. Curves were fitted by Boltzmann function: G/Gmax = 1-1/ (1 + exp ((V-V1/2)/k)). The V1/2 and slope factor (k) of Nav1.5 and Nav1.7 channels are listed in Table 1. Mexiletine and lidocaine caused no significant shift of V1/2 and slope factor for both Nav1.5 and Nav1.7 channels. D, Effects of mexiletine and lidocaine on inactivation kinetics. The inactivation decay time constants were estimated with one exponential fit from the decay of current elicited by a 50 ms pulse to indicated voltages. Mexiletine or lidocaine slightly but no significantly accelerated the decay phase of Nav1.5 at -40 mV and -35 mV (left panel), whereas slightly accelerated decay of Nav1.7 inactivation was only found at -40 mV (right panel).
Table 1.
Parameters obtained from voltage-dependent activation curve fittings with Boltzmann function.
Fig 2.
A hyperpolarizing shift of steady-state fast inactivation of Nav1.5 and Nav1.7 channels by mexiletine and lidocaine.
Steady-state fast inactivation curves of Nav1.5 and Nav1.7 channels were obtained by Boltzmann fitting: I/Imax = 1/ (1 + exp ((V-V1/2)/k)). The V1/2 and slope factor (k) of Nav1.5 and Nav1.7 channels are listed in Table 2. A. The steady-state channel availability between Nav1.5 and Nav1.7 has a 5.5-mV difference (Nav1.5: V1/2 = -83.5 ± 1.4, n = 23; Nav1.7: V1/2 = 78.0 ± 1.4, n = 8; p<0.05, unpaired t-test,). The protocol is shown in right panel. B and C, the V1/2 values of both channels were shifted to the hyperpolarized direction significantly at 0.3 mM and 1 mM of mexiletine or lidocaine. The shift displayed a concentration-dependent effect. Both drugs caused the curves less steep compared to drug-free condition. See Table 2 for all the time constants.
Table 2.
Parameters obtained from steady-state fast inactivation curve fittings with Boltzmann Function.
Fig 3.
A hyperpolarizing shift of steady-state slow inactivation of Nav1.5 and Nav1.7 channels by mexiletine and lidocaine.
Steady-state slow inactivation curves of Nav1.5 and Nav1.7 channels were obtained by Boltzmann fitting: I/Imax = 1/ (1 + exp ((V-V1/2)/k)). The V1/2 and slope factor (k) of Nav1.5 and Nav1.7 channels are listed in Table 3. A. The midpoint of Nav1.5 is more hyperpolarized than that of Nav1.7 (Nav1.5: V1/2 = -65.4 ± 11.9 mV, n = 5; Nav1.7: V1/2 = -41.0 ± 3.4 mV, n = 5; p<0.001, unpaired t-test). The protocol is shown in right panel. B and C, the V1/2 values of both channels were shifted to the hyperpolarized direction significantly at 0.3 mM and 1 mM of mexiletine or lidocaine. The shift displayed a concentration-dependent effect. The steepness factor is similar as the control for Nav1.7, while for Nav1.5 the curves become steeper in the presence of mexiletine or lidocaine. See Table 3 for all the time constants.
Table 3.
Parameters obtained from steady-state slow inactivation curve fittings with Boltzmann Function.
Fig 4.
Enhanced closed-state inactivation of Nav1.5 and Nav1.7 channels by mexiletine and lidocaine.
The closed-state inactivation curves were best fitted with two-exponential function. Both mexiletine and lidocaine accelerated the development of closed-state inactivation in Nav1.5 (A) and Nav1.7 (B) channels. The fast (τ1) and slow (τ2) constants of Nav1.5 and Nav1.7 channels are in the same range in control condition. All the τ values and the corresponding fraction in each condition are summarized in Table 4. Note that both mexiletine and lidocaine caused a significant reduction of slow τ2 values in dose-dependent manner, leading to speedy inactivation of either Nav1.5 or Nav1.7.
Table 4.
Parameters obtained from fitting closed-state inactivation curves by two-exponential function.
Fig 5.
Delayed recovery from inactivation by mexiletine and lidocaine.
Recovery of Nav1.5 or Nav1.7 from inactivation was measured with two-pulse protocol (inset). After 50 ms inactivation pulse to -20 mV, channels were allowed to recover at the holding potential. Normalized peak currents were plotted versus recovery time. The curves were best fitted with two-exponential function. Mexiletine and lidocaine prolonged the recovery from inactivation in Nav1.5 (A) and Nav1.7 (B) channels. The fast and slow constants as well as their relative weights were summarized in Table 5.
Table 5.
Parameters obtained from fitting the recovery curves by two-exponential function.
Fig 6.
Differential use-dependent inhibition of Nav1.5 and Nav1.7 by mexiletine and lidocaine.
Cells were held at -120 mV and pulsed at -20 mV for three different frequencies (1, 5 and 10 Hz), with interpulse potential set at -120 mV. The peak currents elicited by each pulse were normalized to the current of first pulse and were plotted against the pulse number. Black symbols represent control condition without drugs, while red and blue symbols represent experiments in the presence of 0.3 mM mexiletine or 0.3 mM lidocaine, respectively. A and B, Nav1.5 and Nav1.7 were stimulated by a train of 60 pulses at 1 Hz and a train of 150 pulses at 5 Hz or 10 Hz in the presence and absence of 0.3 mM mexiletine or lidocaine, respectively. C, Bar graphs represent the relative amplitudes at the last sweep (60th or 150th) of use-dependent protocol for each frequency. Increasing the number of pulses resulted in current reduction for Nav1.5 in a frequency-dependent manner in the presence of mexiletine or lidocaine at 0.3 mM, whereas mexiletine showed a stronger use-dependent inhibition than lidocaine. In contrast, there is no difference in the inhibition of Nav1.7 at 5- and 10-Hz between mexiletine and lidocaine. Besides, the overall current reduction in Nav1.5 is more prominent than that of Nav1.7 due to the drug effects. Asterisk indicates the significance compared to drug-free (control) condition at each frequency by one-way ANOVA, ***p<0.001.