State- and use-dependent block of muscle Nav1.4 and neuronal Nav1.7 voltage-gated Na+ channel isoforms by ranolazine

Abstract

Ranolazine is an antianginal agent that targets a number of ion channels in the heart, including cardiac voltage-gated Na(+) channels. However, ranolazine block of muscle and neuronal Na(+) channel isoforms has not been examined. We compared the state- and use-dependent ranolazine block of Na(+) currents carried by muscle Nav1.4, cardiac Nav1.5, and neuronal Nav1.7 isoforms expressed in human embryonic kidney 293T cells. Resting and inactivated block of Na(+) channels by ranolazine were generally weak, with a 50% inhibitory concentration (IC(50)) >/= 60 microM. Use-dependent block of Na(+) channel isoforms by ranolazine during repetitive pulses (+50 mV/10 ms at 5 Hz) was strong at 100 microM, up to 77% peak current reduction for Nav1.4, 67% for Nav1.5, and 83% for Nav1.7. In addition, we found conspicuous time-dependent block of inactivation-deficient Nav1.4, Nav1.5, and Nav1.7 Na(+) currents by ranolazine with estimated IC(50) values of 2.4, 6.2, and 1.7 microM, respectively. On- and off-rates of ranolazine were 8.2 microM(-1) s(-1) and 22 s(-1), respectively, for Nav1.4 open channels and 7.1 microM(-1) s(-1) and 14 s(-1), respectively, for Nav1.7 counterparts. A F1579K mutation at the local anesthetic receptor of inactivation-deficient Nav1.4 Na(+) channels reduced the potency of ranolazine approximately 17-fold. We conclude that: 1) both muscle and neuronal Na(+) channels are as sensitive to ranolazine block as their cardiac counterparts; 2) at its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na(+) channels; and 3) ranolazine block of open Na(+) channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.

Fig. 1

Chemical structures of ranolazine and lidocaine. Notice that ranolazine is larger and contains a lidocaine moiety.

Fig. 2

Block of resting and inactivated rNav1.4 Na⁺ channels at various ranolazine concentrations. A, representative current traces are superimposed before and after application of 100 μM ranolazine. Cells were held at −140 mV and received 5-ms test pulses of +30 mV at 30-s intervals. B, representative current traces are superimposed for ranolazine concentrations of 0, 30, and 100 μM. After a −70-mV conditioning pulse for 10 s, Na⁺ currents were evoked by the 5-ms test pulse at 30 mV, administered every 30 s. An interpulse of 95 ms at the holding potential was inserted to allow the recovery of the drug-free inactivated Na⁺ channels. C, a dose-response curve was constructed using data described in Fig. 2A (resting block: ■, n = 5) and 2B (inactivated block: ▲, n = 6). The peak current was measured, normalized to the control (0 μM), and plotted against the ranolazine concentration. The curve was fitted with the Hill equation (solid line). The IC₅₀ value for resting block was >300 μM and could not be experimentally attained. The IC₅₀ value for inactivated block was estimated at 75.0 ± 3.5 μM (Hill coefficient, 1.41 ± 0.10, n = 6).

Fig. 3

Use-dependent block of rNav1.4 wild-type Na⁺ currents by 100 μM ranolazine during repetitive pulses. A, repetitive pulses at + 50 mV for 10 ms were applied at 5 Hz for a total of 60 pulses. Representative traces of Na⁺ currents were recorded in the presence of 100 μM ranolazine; the number near the trace indicates the corresponding pulse number applied. B, peak currents were measured, normalized with respect to the peak amplitude at 1P, and plotted against the corresponding pulse. The curve (solid line) was best fitted by an exponential function with a time constant of τ = 3.43 ± 0.30 pulse (n = 6).

Fig. 4

Duration dependence of the use-dependent block of rNav1.4 wild-type Na⁺ currents induced by 100 μM ranolazine. A, repetitive pulses were applied as described in Fig. 3A, except that the pulse duration was shortened to 1 ms. Representative traces of Na⁺ currents were recorded and labeled with the corresponding pulse number. B, repetitive pulses with durations of 0.5, 2, 4, 10, and 20 ms were applied, and Na⁺ currents were recorded as shown in Fig. 4A. Peak currents were measured and plotted against the pulse number. Notice that the block reaches the same level with pulse duration as short as 2 ms. Data in A and B were obtained from the same cell. Comparable results were recorded in 5 cells.

Fig. 5

Use-dependent block of wild-type hNav1.7 and hNav1.5 Na⁺ channels by 100 μM ranolazine. Repetitive pulses of + 50 mV for 10-ms at 5 Hz were applied in the presence of 100 μM ranolazine. Representative current traces of wild-type hNav1.7 (A) and hNav1.5 (B) were recorded and superimposed, with the label indicating the corresponding pulse number. The degree of the steady-state block of each isoform by ranolazine after repetitive pulses is given under Results.

Fig. 6

Block of inactivation-deficient rNav1.4-WCW and rNav1.4-WCW/F1579K Na⁺ channels. A, representative current traces of inactivation-deficient rNav1.4-WCW Na⁺ channel block were superimposed at various ranolazine concentrations. Cells were held at −140 mV and received 50-ms test pulses of +30 mV at 30-s intervals. B, a dose-response curve was constructed from the data presented in Fig. 6A. Both peak (■) and late (□) currents were measured, normalized to the control (0 μM), and plotted against ranolazine concentration. The curve was fitted with the Hill equation (solid lines). The IC₅₀ value for the peak current block was estimated 225.4 ± 16.3 μM (Hill coefficient, 0.92 ± 0.05) (n = 5) and the IC₅₀ value for rNav1.4-WCW of the late current was 2.4 ± 0.2 μM (1.15 ± 0.09) (n = 5). C, representative current traces of rNav1.4-F1579K inactivation-deficient Na⁺ channel block were superimposed at various ranolazine concentrations. Currents were evoked using the pulse-protocol represented in A. Peak and late Na⁺ currents were measured, normalized, and plotted against concentration as shown in B (triangles). The estimated IC₅₀ value for rNav1.4-F1579K inactivation-deficient channel block of the late current was 40.8 ± 1.3 μM (Hill coefficient, 1.12 ± 0.04) (n = 5).

Fig. 7

Time-dependent block of inactivation-deficient rNav1.4-WCW Na⁺ currents at various ranolazine concentrations. The decaying phase of Na⁺ currents in the presence of ranolazine shown in Fig. 6A were each normalized with respect to the control current trace without drug and fitted by a single exponential function. Normalization was applied to remove the slow-decaying component found in the control. The inverse of the time constant, τ, was plotted against ranolazine concentration (y-intercept: 22.1 ± 2.3, n = 5; slope: 8.2 ± 0.2; linear correlation coefficient r = 0.997).

Fig. 8

Recovery time course from ranolazine open-channel block at 100 μM. A, a conditioning pulse of +50 mV for 50 ms was applied to induce the time-dependent block of rNav1.4-WCW Na⁺ currents at 100 μM ranolazine. Representative traces of rNav1.4-WCW Na⁺ currents were evoked by a +30-mV test pulse after an interpulse at −140 mV with an increasing duration and superimposed. The pulse protocol is shown in the inset. B, normalized peak current after drug treatment (100 μM ranolazine; squares) was plotted against the interpulse duration and fitted by an exponential function with a τ value of 558.2 ± 40.7 ms (n = 5). The control data before drug treatment (circles; n = 5) was shown for comparison; most currents (>90%) reappeared within 3 ms.

Fig. 9

Time-dependent block of inactivation-deficient hNav1.7-WCW Na⁺ currents at various ranolazine concentrations. A, representative traces of inactivation-deficient hNav1.7-WCW Na⁺ currents were superimposed before and after application of ranolazine at various concentrations. Cells were held at −140 mV and received 5-ms test pulses of +30 mV at 30-s intervals. B, the decaying phase of Na⁺ currents at various ranolazine concentrations were each normalized with the control current without drug and fitted with a single exponential function as described in Fig. 7. Normalization was necessary because of the presence of a slow-decaying component in the control trace. The inverse of the time constant, τ, was plotted against ranolazine concentration (y-intercept, 14.1 ± 1.1 (n = 5); slope, 7.1 ± 0.5; r = 0.986).