Mexiletine block of wild-type and inactivation-deficient human skeletal muscle hNav1.4 Na+ channels

Abstract

Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium-aggravated myotonia, long QT-3 syndrome, and neuropathic pain. This drug elicits tonic block of Na(+) channels when cells are stimulated infrequently and produces additional use-dependent block during repetitive pulses. We examined the state-dependent block by mexiletine in human skeletal muscle hNav1.4 wild-type and inactivation-deficient mutant Na(+) channels (hNav1.4-L443C/A444W) expressed in HEK293t cells with a beta1 subunit. The 50% inhibitory concentrations (IC(50)) for the inactivated-state block and the resting-state block of wild-type Na(+) channels by mexiletine were measured as 67.8 +/- 7.0 microm and 431.2 +/- 9.4 microm, respectively (n= 5). In contrast, the IC(50) for the block of open inactivation-deficient mutant channels at +30 mV by mexiletine was 3.3 +/- 0.1 microm (n= 5), which was within the therapeutic plasma concentration range (2.8-11 microm). Estimated on- and off-rates for the open-state block by mexiletine at +30 mV were 10.4 microm(-1) s(-1) and 54.4 s(-1), respectively. Use-dependent block by mexiletine was greater in inactivation-deficient mutant channels than in wild-type channels during repetitive pulses. Furthermore, the IC(50) values for the block of persistent late hNav1.4 currents in chloramine-T-pretreated cells by mexiletine was 7.5 +/- 0.8 microm (n= 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open-channel block of persistent late Na(+) currents, which may arise during various pathological conditions.

Figure 1. Activation of hNav1.4 wild-type channels in the absence and presence of 100 μm mexiletine

Currents were evoked by 5 ms test pulses from –120 to +50 mV in 10 mV increments without (A) or with (B) 100 μm mexiletine. The inward current evoked by a pulse to –50 mV and the outward current evoked by a pulse to +50 mV are labelled. C, normalized membrane conductance (g_m) was plotted against the corresponding membrane voltage. g_m was determined from the equation g_m=I_Na/(E_m–E_Na), where I_Na is the peak current, E_m is the amplitude of the voltage step, and E_Na is the estimated reversal potential of the Na⁺ current. Plots were fitted with a Boltzmann function, y= 1/{1 + exp[(V_0.5–V)/k]}. The average midpoint voltage (V_0.5) and slope (k) for hNav1.4 wild-type (○, n= 7; fitted value ±s.e.m. of the fit) were –29.4 ± 0.7 mV and 10.1 ± 0.7 mV, respectively, and –34.5 ± 0.5 mV and 7.6 ± 0.4 mV for the mexiletine-treated cell (•, n= 5; P < 0.05), respectively. Cells were cotransfected with the β1 subunit. Holding potential was set at –140 mV and the time interval between pulses was 10 s.

Figure 2. Steady-state inactivation of hNav1.4 with or without 100 μm mexiletine

Currents were evoked by a 5 ms test pulse to +30 mV in the absence (A) or presence (B) of 100 μm mexiletine. Test pulses were preceded by 100 ms conditioning pulses, increased in 5 mV increments between –160 mV and –120 mV. The interval between pulses was 10 s. C, normalized Na⁺ current availability (h_∞) of hNav1.4 was obtained from data as shown in A and B and plotted against the conditioning voltage. Data were fitted with the Boltzmann function, y= 1/{1 + exp[(V–V_0.5)/k]}. The average midpoint (V_0.5) and slope factor (k) for the wild-type (○, n= 8) were –75.9 ± 0.2 mV and 6.4 ± 0.2 mV, respectively, and –80.6 ± 0.2 mV and 5.9 ± 0.2 mV for the cells treated with mexiletine (•, n= 5). The difference between two V_0.5 values is significant (P < 0.05).

Figure 3. Voltage-dependent block of hNav1.4 by 100 μm mexiletine

A, a 10 s conditioning prepulse ranging in amplitude from –180 to –40 mV was applied. After a 100 ms interval at –140 mV, Na⁺ currents were evoked by the delivery of a 5 ms test pulse at +30 mV. Currents obtained in control solution and with 100 μm mexiletine were normalized to the current obtained with the –180 mV control conditioning pulse. Mexiletine data were then renormalized at each conditioning pulse voltage. Normalized control data (○, n= 6) and renormalized mexiletine data (•, n= 5) were plotted against the conditioning prepulse voltages. Mexiletine data were fitted with a Boltzmann function (1/[1 + exp((V_0.5–V)/k_E)]). The average V_0.5 and k_E (slope factor) values for the fitted functions were –91.8 ± 0.5 mV and 7.2 ± 0.5 mV, respectively. B, a 10 s conditioning pulse to –70 mV or –160 mV was followed by a 100 ms interval at –140 mV and a 5 ms test pulse to +30 mV to evoke Na⁺ current. Pulses were delivered at 30 s intervals. The peak amplitudes of Na⁺ current were measured at various mexiletine concentrations, normalized to the peak amplitude of the control, and plotted against drug concentration. Continuous lines represent fits to the data with the Hill equation. IC₅₀ values ±s.e.m. and Hill coefficients ±s.e.m. (in square brackets) for inactivated-state block at –70 mV (□, n= 5), and for the resting-state block at –160 mV (○, n= 5) are 67.8 ± 7.0 μm[0.85 ± 0.07], and 431.2 ± 9.4 μm[1.3 ± 0.3], respectively.

Figure 4. Development of and recovery from the inactivated-state block of hNav1.4 by 100 μm mexiletine

A, for the development of the inactivated block, the pulse protocol shown at the top was applied. The peak currents at the test pulse were measured and normalized to the initial peak amplitude (t= 0) and plotted against the prepulse duration. The data were fitted by a single-exponential function or, where appropriate, a double-exponential function. The fast time constant for mexiletine-treated cells (•, n= 5) was 0.203 ± 0.011 s. The slow time constant in the control (τ= 5.9 ± 1.3 s; ○, n= 7) and in mexiletine-treated cells (τ= 5.7 ± 1.0 s; n= 5) probably represents the slow inactivation of the Na⁺ channel. B, the recovery from the inactivated-state block was measured by the pulse protocol shown at the top. The peak currents at the test pulse were measured, normalized and plotted against the interpulse duration. The recovery time courses were fitted by the sum of two exponentials. The fast and slow time constants for the 100 ms conditioning pulse were 0.8 ± 0.1 ms and 8.0 ± 0.3 ms (control, □, n= 5), and 2.0 ± 0.2 ms and 0.70 ± 0.06 s (mexiletine, ○, n= 5). The fast and slow time constants for the 500 ms conditioning pulse were 1.0 ± 0.1 ms and 0.080 ± 0.015 s (control, ▪, n= 5), and 5.1 ± 0.7 ms and 0.74 ± 0.03 s (mexiletine, •, n= 5). The slow time constant represented the recovery from the inactivated-state block by mexiletine.

Figure 5. Activation of hNav1.4 l443C/A444W mutant channels without and with mexiletine

Superimposed current traces were evoked by 5 ms pulses from –120 to +50 mV in 10 mV increments in the absence (A) or presence (B) of 100 μm mexiletine. The inward current evoked by a pulse to –50 mV and the outward current evoked by a pulse to +50 mV are labelled. C, normalized membrane conductance (g_m) was plotted against the membrane voltage. g_m was determined as described in Fig. 1C. Plots were fitted with a Boltzmann function. The average midpoint voltage (V_0.5) and the slope factor (k) of the function in the control solution (○, n= 11) were –12.4 ± 0.9 mV and 17.0 ± 0.9 mV, respectively, and –29.4 ± 0.7 mV and 14.8 ± 0.6 mV for the mexiletine-treated cell (•, n= 6; P < 0.05).

Figure 6. Steady-state inactivation of hNav1.4 l443C/A444W mutant channels without and with mexiletien

Superimposed current traces were evoked by a 5 ms test pulse to +30 mV in the absence (A) or presence (B) of 100 μm mexiletine. Test pulses were preceded by 100 ms conditioning pulses and increased in 5 mV increments between –160 and –15 mV C, peak currents in A and B were measured and normalized with respect to the amplitude with the –160 mV conditioning pulse and plotted against the conditioning voltage. Plots were fitted with the Boltzmann function as described in Fig. 2C. The average midpoint (V_0.5) and slope factor (k) for the control solution (○, n= 11) were –83.6 ± 1.2 mV and 10.6 ± 1.0 mV, respectively, and –88.6 ± 0.7 mV and 11.1 ± 0.6 mV for the cells treated with mexiletine (•, n= 6; dashed line). A second Boltzmann function (V_0.5=–57.2 ± 2.7 mV; k= 4.5 ± 0.2 mV; continuous line) was applied to fit the mexiletine data.

Figure 7. Voltage-dependent block of hNav1.4 l443C/A444W mutant channels by mexiletine

The same pulse protocol as described in Fig. 3A was used. Peak currents obtained in control solution and with 100 μm mexiletine were normalized to the peak current obtained at the –180 mV control conditioning pulse. Mexiletine data were then renormalized at each conditioning pulse voltage. Normalized control data (○, n= 8) and renormalized mexiletine data (•, n= 5) were plotted against the conditioning voltages. Data were fitted with a Boltzmann function (1/[1 + exp((V_0.5–V)/k)]). The average V_0.5 and k (slope factor) values for the fitted functions were –108.9 ± 1.4 mV and 7.5 ± 1.3 mV, respectively, for mexiletine. Notice that the slow inactivation is more pronounced in the inactivation-deficient mutant channels (○) than in wild-type (Fig. 3A).

Figure 8. Open-channel block in inactivation-deficient mutant channels by mexiletine

A, superimposed mutant Na⁺ currents were recorded at various concentrations of mexiletine. The Na⁺ currents were evoked by a 50 ms test pulse to +30 mV every 30 s. A steady state at each concentration was established before application of the next concentrated solution. B, the decaying phase of the normalized relative Na⁺ current was fitted with a single exponential function, and the corresponding τ value (time constant) was inverted and plotted against the corresponding mexiletine concentration (0.3–30 μm). Data were fitted with a linear regression y= 10.4x+ 54.4. The on-rate (k_on) corresponded to the slope of the fitted line (10.4 μm⁻¹ s⁻¹) and the off-rate (k_off) corresponded to the y-intercept (54.4 s⁻¹). The dissociation constant determined by the equation K_D=k_off/k_on was 5.2 μmC, dose–response curves for open-channel block (relative block at the end of the 50 ms test pulse) and resting-state block (relative block at the peak current) were constructed using the data set as shown in A. All pulses were delivered at 30 s intervals. The amplitudes of Na⁺ current were measured, normalized to the amplitude of the control, and plotted against the mexiletine concentration. Continuous lines represent fits to the data with the Hill equation. IC₅₀ values ±s.e.m. [Hill coefficients ±s.e.m.] are 3.3 ± 0.1 μm[0.90 ± 0.02] for open-channel block (□, n= 5), and 336.4 ± 20.0 μm[1.2 ± 0.1] for resting-state block (○, n= 5).

Figure 9. Recovery from the open-channel block of hNav1.4 l443C/A444W mutant channels by 100 μm mexiletine

Recovery time course at –140 mV was measured by the pulse protocol shown at the top and fitted by the sum of two exponentials. The fast and slow time constants for the 100 ms conditioning pulse to +30 mV were 1.7 ± 0.5 s and 3 ± 1 ms (control, ○, n= 5), and 0.91 ± 0.05 s and 4 ± 1 ms (mexiletine, •, n= 5). Notice that most of mutant channels (70%; ○) are not inactivated by this pulse protocol. However, we might overestimate the slow recovery τ value for mexiletine block since its time course overlapped considerably with that of drug-free mutant channels (∼20%; ○).

Figure 10. Use-dependent block of hNav1.4 wild-type and inactivation-deficient mutant channels by 100 μm mexiletine

A, twenty 24 ms pulses to +30 mV were delivered to hNav1.4 wild-type channels at 5 Hz from a holding potential of –140 mV. The peak current amplitude of each data set was normalized to the first pulse of the set and plotted against the pulse number. In control solution, the pulse protocol did not elicit use-dependent decreases in current amplitude. Data were best fitted by a single exponential function for 100 μm mexiletine (•, n= 5) with a time constant of 1.16 ± 0.03 pulses and reached steady state at 70.1 ± 0.1% of the remaining current (∼30% block). B, use-dependent block of hNav1.4 l443C/A444W by 100 μm mexiletine was measured as described in A. The time constant for the mexiletine solution (•, n= 7) was too fast to be measured. Steady state was reached at 48.9 ± 0.6% of the remaining current (∼50% block). Most of the persistent currents at the end of 24 ms pulse were blocked by 100 μm mexiletine as shown in Fig. 8A.

Figure 11. Open-channel block of hNav1.5-L409C/A410W and hNav1.5-L409C/A410W/1760K at various mexiletine concentrations

Superimposed current traces of hNav1.5-L409C/A410W (A) or hNav1.5-L409C/A410W/F1760K (B) mutant channels were recorded with a test pulse at +30 mV for 50 ms before and after mexiletine at various concentrations. Notice that mutation F1760K renders the open channel relatively resistant to mexiletine block. C, dose–response curves for open-channel block (relative block at end of 50 ms test pulse to +30 mV) and resting-state block (relative block of peak currents) were constructed using the data set as shown in A and B. Data were fitted with the Hill equation. IC₅₀ values ±s.e.m. [Hill coefficients ±s.e.m.] for hNav1.5-L409C/A410W are 11.1 ± 0.3 μm[1.1 ± 0.2] for open-channel block (▪, n= 5), and 157.4 ± 11.4 μm[1.2 ± 0.1] for resting-state block (□, n= 6). IC₅₀ values ±s.e.m. [Hill coefficients ±s.e.m.] for hNav1.5-L409C/A410W/F1760K are 207.9 ± 7.0 μm[1.0 ± 0.1] for open-state block (•, n= 5), and 290.7 ± 9.6 μm[1.1 ± 0.1] for resting-state block (○, n= 5).

Figure 12. Open-channel block in chloramine-T-treated hNav1.4 Na⁺ channels by mexiletine

A, superimposed Na⁺ currents were recorded at various concentrations of mexiletine. Before these records were taken, the cell was treated with 0.5 mm chloramine-T for 2.5 min and then washed with drug-free bath solution for 5 min. The Na⁺ currents were evoked by a 400 ms test pulse to +30 mV every 30 s. A steady state at each mexiletine concentration was established before application of the next solution. TTX at 1 μm was also applied to identify hNav1.4 Na⁺ currents. Treatment with chloramine-T was limited to 2.5 min since prolonged incubation of this oxidant increased the non-linear leak currents. B, the dose–response curve for the open-channel block (relative block at the end of the 400 ms test pulse) was constructed using the data set as shown in A. All pulses were delivered at 30 s intervals. The amplitudes of Na⁺ current were measured, normalized to the amplitude of the control, and plotted against the mexiletine concentration. Leak currents after 1 μm TTX were subtracted from the current measurements. Continuous lines represent fits to the data with the Hill equation. IC₅₀ values ±s.e.m[Hill coefficients ±s.e.m.] are 7.5 ± 0.8 μm[1.0 ± 0.1] for open-channel block (n= 5). Cells were cotransfected with the β1 subunit.