Different flecainide sensitivity of hNav1.4 channels and myotonic mutants explained by state-dependent block

Abstract

Flecainide, a class IC antiarrhythmic, was shown to improve myotonia caused by sodium channel mutations in situations where the class IB antiarrhythmic drug mexiletine was less efficient. Yet little is known about molecular interactions between flecainide and human skeletal muscle sodium (hNa(v)1.4) channels. Whole-cell sodium currents (I(Na)) were recorded in tsA201 cells expressing wild-type (WT) and mutant hNa(v)1.4 channels (R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). At a holding potential (HP) of -120 mV, flecainide use-dependently blocked WT and G1306E I(Na) equally but was more potent on R1448C channels. For WT, the extent of block depended on a holding voltage more negative than the activation threshold, being greater at -90 mV as compared to -120 and -180 mV. This behaviour was exacerbated by the R1448C mutation since block at -120 mV was greater than that at -180 mV. Thus flecainide can bind to inactivated sodium channels in the absence of channel opening. Nevertheless, all the channels showed the same closed-state affinity constant (K(R) approximately 480 microM) and the same inactivated-state affinity constant (K(I) approximately 18 microM). Simulations according to the modulated receptor hypothesis mimic the voltage-dependent block of WT and mutant channels by flecainide and mexiletine. All the results suggest similar blocking mechanisms for the two drugs. Yet, since flecainide exerts use-dependent block at lower frequency than mexiletine, it may exhibit greater benefit in all myotonic syndromes. Moreover, flecainide blocks hNa(v)1.4 channel mutants with a rightward shift of availability voltage dependence more specifically than mexiletine, owing to a lower K(R)/K(I) ratio. This study offers a pharmacogenetic strategy to better address treatment in individual myotonic patients.

Figure 1. Frequency-dependent flecainide block of wild-type and mutant hNav1.4 channels at a holding potential of –120 mV

A, block of sodium currents by flecainide was assessed 3 min after drug application by measuring the reduction of I_Na elicited from –120 to –30 mV at stimulation frequencies of 0.1 Hz and 10 Hz. B, concentration–response curves for flecainide block were constructed at 0.1 Hz using the protocol described in A and fitted with eqn (1) (see Results). Each data point is the mean ± s.e.m. of at least 3 cells. The calculated IC₅₀ values ± s.e. of the fit were 83.5 ± 16.9 μm for WT, 82.8 ± 11.2 μm for G1306E, and 21.4 ± 2.2 μm for R1448C. C, concentration–response curves for flecainide block were constructed at 10 Hz using the protocol described in A and fitted with eqn (1) (see Results). Each data point is the mean ± s.e.m. of at least 3 cells. The calculated IC₅₀ values ± s.e. of the fit were 36.6 ± 6.1 μm for WT, 38.2 ± 1.8 μm for G1306E, and 8.2 ± 0.4 μm for R1448C.

Figure 2. Effect of holding potential on flecainide block of wild-type and R1448C hNav1.4 channels and flecainide affinity for closed channels

A, time course evolution of peak I_Na amplitude in a tsA201 cell expressing R1448C channels. The cell was held at the HP of –180 mV and depolarized to –30 mV at 0.1 Hz frequency, except under the open bar where the HP was maintained with no depolarization for determination of tonic block (I_TB). The filled bars indicate application of 100 μm flecainide. B, traces of R1448C I_Na measured at the times indicated by arrows in A. I_CTRL was measured just before application of the drug, while I_0.1 was measured when steady-state block was reached at 0.1 Hz stimulation frequency. C and D, tonic block is expressed as 100 × (I_CTRL–I_TB)/I_CTRL, while use-dependent block is expressed as 100 × (I_TB–I_0.1)/I_CTRL, measured as in A with HP =–180, –120 and –90 mV. Each bar corresponds to the mean ± s.e.m. of at least 4 cells. The P-values reported on bars were calculated using Student's unpaired t test versus respective block at HP =–180 mV. In addition, use-dependent block of R1448C channels was significantly greater than that of WT channels (at least P < 0.02). E, concentration–response curves were constructed for I_TB/I_CTRL measured as in A at HP =–180 mV and fitted with eqn (1). Each data point is the mean ± s.e.m. of at least 4 cells. The calculated IC₅₀ values ± s.e. of the fit were 469.0 ± 31.8 μm for WT and 481.2 ± 21.4 μm for R1448C.

Figure 3. Flecainide affinity for inactivated wild-type and R1448C hNav1.4 channels and recovery from flecainide block

A, effects of flecainide on voltage dependence of I_Na availability in a tsA201 cell expressing R1448C channels. I_Na was evoked by a 20 ms-long test pulse to –30 mV after a 50 ms-long conditioning pulses to potentials ranging from –150 to –30 mV in 10 mV increments. Pulses were delivered at 10 s or 30 s interval duration (ID) as indicated and HP was –180 mV. The peak I_Na recorded during the test pulse was plotted against the conditioning pulse potential. The relationships were fitted with eqn (2) (see Results). The values of the half-maximum inactivation potential, V_1/2, along with the s.e. of the fit were –87.0 ± 0.2 mV in control, –108.2 ± 0.4 mV in the presence of 300 μm flecainide with 30 s ID, and –164.7 ± 0.8 in the presence of 300 μm flecainide with 10 s ID. The values of the slope factor, S, were 11.0 ± 0.2 mV in control, 12.6 ± 0.3 mV in the presence of 300 μm flecainide with 30 s ID, and 17.9 ± 0.6 mV in the presence of 300 μm flecainide with 10 s ID. Availability curves were normalized with respect to their own I_Na,max. B, the affinity of flecainide for inactivated channels (K_I) was evaluated by plotting V_1/2 values, determined as in A, against flecainide concentration. Each data point is the mean ± s.e.m. from at least 4 cells. The relationships were fitted with eqn (3) (see Results). The values of K_I along with the s.e. of the fit were 17.1 ± 1.1 μm for WT (V_1/2,CTRL was –75.7 mV and S_CTRL was 5.8 mV in eqn (3)) and 17.8 ± 2.9 μm for R1448C channels (V_1/2,CTRL was –85.0 mV and S_CTRL was 11.1 mV). C and D, the recovery of WT and R1448C channels from inactivation and from flecainide block was measured at –180 mV. A recovery pulse at the HP of increasing duration was included between two test pulses at –30 mV. The peak I_Na recorded during the second test pulse was normalized with respect to the peak I_Na recorded during the first test pulse and means ± s.e.m. were calculated from at least 5 cells to be plotted against the recovery time. The relationships were fitted with eqn (4) (see Results). Fitted parameters are reported in Table 1.

Figure 4. Effects of flecainide on I_Na decay rate of WT and R1448C hNav1.4 channels

The I_Na was evoked by 25 ms-long test pulses to –30 mV applied at 0.1 Hz from the V_1/2 of –180 mV before (CTRL) and after application of 100 μm flecainide. To allow direct inspection of drug effect on current decay, I_Na measured during drug exposure was scaled with respect to peak amplitude of control I_Na (dashed line). The parameters τd₁, τd₂ and Q were calculated form the fit of current decay with eqn (5). Each bar represents the mean ± s.e.m. from 8 (WT) and 11 cells (R1448C). Statistical analysis was performed using Student's paired t test, * indicating at least P < 0.01 versus CTRL wild-type and # indicating at least P < 0.02versus relative control.

Figure 5. Effects of internal application of drugs on wild-type hNav1.4 channels

Development of use-dependent block after internal diffusion of control pipette solution (CTRL, □), or pipette solution supplemented with 1 mm mexiletine (MEX, ▪), 1 mm flecainide (FLECA, ○), or 300 μm QX-314 (QX, •). The tsA201 cells expressing WT hNav1.4 channels were held at –120 mV and received a 25 ms-long depolarizing pulse to –30 mV every 0.1 s (10 Hz) to elicit I_Na. This protocol was applied about 5 min after achieving the whole-cell configuration to allow pipette solution to diffuse well within the cell. Peak I_Na measured at each test pulse was normalized with respect to the first pulse I_Na. Each data point is the mean from at least 3 cells.

Figure 6. Simulation of flecainide and mexiletine effects on hNav1.4 channels using the modulated receptor model

A and B, experimental concentration–response curves for flecainide and mexiletine effect on wild-type, R1448C, and G1306E hNav1.4 channels were constructed at a holding potential (HP) of –120 mV in absence of depolarization as described in Fig. 2. Each data point is the mean ± s.e.m. of at least 3 cells. The relationships were fitted with eqn (1) (see Results) and the IC₅₀ values are reported in Table 2. C and D, the theoretical curves according to the modulated receptor hypothesis were built using eqn (1) (see Results) with the K_APP values calculated for WT, G1306E, and R1448C channels using eqn (6) at a HP of –120 mV and reported in Table 2. The lines labelled with K_R and K_I were obtained using K_R and K_I values reported in Table 2 for each drug and describe the theoretical relationships for a hypothetical pure block of closed channels (K_R) and a hypothetical pure block of inactivated channels (K_I).