Gating properties of Na(v)1.7 and Na(v)1.8 peripheral nerve sodium channels

Abstract

Several distinct components of voltage-gated sodium current have been recorded from native dorsal root ganglion (DRG) neurons that display differences in gating and pharmacology. This study compares the electrophysiological properties of two peripheral nerve sodium channels that are expressed selectively in DRG neurons (Na(v)1.7 and Na(v)1.8). Recombinant Na(v)1.7 and Na(v)1.8 sodium channels were coexpressed with the auxiliary beta(1) subunit in Xenopus oocytes. In this system coexpression of the beta(1) subunit with Na(v)1.7 and Na(v)1.8 channels results in more rapid inactivation, a shift in midpoints of steady-state activation and inactivation to more hyperpolarizing potentials, and an acceleration of recovery from inactivation. The coinjection of beta(1) subunit also significantly increases the expression of Na(v)1.8 by sixfold but has no effect on the expression of Na(v)1.7. In addition, a great percentage of Na(v)1.8+beta(1) channels is observed to enter rapidly into the slow inactivated states, in contrast to Nav1.7+beta(1) channels. Consequently, the rapid entry into slow inactivation is believed to cause a frequency-dependent reduction of Na(v)1.8+beta(1) channel amplitudes, seen during repetitive pulsing between 1 and 2 Hz. However, at higher frequencies (>20 Hz) Na(v)1.8+beta(1) channels reach a steady state to approximately 42% of total current. The presence of this steady-state sodium channel activity, coupled with the high activation threshold (V(0.5) = -3.3 mV) of Na(v)1.8+beta(1), could enable the nociceptive fibers to fire spontaneously after nerve injury.

Fig. 1.

Effects of the β₁ subunit on Na_v1.7 and Na_v1.8 sodium channels heterologously expressed in Xenopus oocytes. The data show the whole-cell sodium currents of oocytes expressing either the Na_v1.7 or Na_v1.8 sodium channel with and without the β₁ subunit. Currents were elicited by depolarizing steps between −50 and +65 mV in 5 mV increments from a holding potential of −100 mV (see inset).A, Whole-cell Na_v1.7 currents measured in the absence (left) and presence (right) of the β₁ subunit. B, Na_v1.8 sodium currents expressed without (left) and with (right) the β₁ subunit. Dashed lines are the zero current levels.

Fig. 2.

Effects of the β₁ subunit on the expression of Na_v1.7 and Na_v1.8 sodium channels. Shown are the whole-cell sodium currents of paired groups of oocytes expressing either the Na_v1.7 or Na_v1.8 channels with or without the β₁ subunit. Na_v1.7 peak currents at −20 mV were measured from oocytes expressing Na_v1.7 or Na_v1.7+β₁after 24 hr of incubation. There was no significant difference in the peak amplitude of current between Na_v1.7 and Na_v1.7+β₁ even at 3 d after injection (data not shown). For Na_v1.8 channels the peak currents measured at +20 mV were compared 6 d after cRNA injection. Peak amplitude recorded 3 d after injection for Nav1.8 channels was small (38.1 ± 2.8 nA; n = 3), whereas the coexpression increased expression by 17-fold (695 ± 117 nA;n = 5; data not shown). The β₁subunit significantly increased (p < 0.05) the currents of Na_v1.8 (n = 7), but not the Na_v1.7 (n = 6), sodium channels (p < 0.05). The holding potential was −100 mV.

Fig. 3.

Effects of the β₁ subunit on the kinetics of Na_v1.7 and Na_v1.8 inactivation. Whole-cell sodium currents of Na_v1.7 and Na_v1.7+β₁ sodium channels were elicited by a depolarizing step from −100 to −20 or +20 mV. Currents were normalized to facilitate comparison of the kinetics. A, At −20 mV the time constants of current decay were 19.8 ± 3.6 msec (n = 6) and 1.8 ± 0.2 msec (n = 6) for Na_v1.7 and Na_v1.7+β₁, respectively.B, Na_v1.8 and Na_v1.8+β₁ currents were elicited by a step depolarization to +20 mV and had decay time constants of 4.3 ± 0.2 msec (n = 6) and 2.6 ± 0.1 msec (n = 6), respectively. Dashed linesare the zero current levels.

Fig. 4.

The β₁ subunit accelerates the inactivation of Na_v1.7 and Na_v1.8 channels. The decay of the Na_v1.7 and Na_v1.8 sodium currents (Fig. 1) was fit to an exponential function, and the time constants were plotted versus the test voltage: I =A_I · exp (−t/τi) + C, where I is the current,A_i is the percentage of channels inactivating with time constant τ_i,t is time, and C is the steady-state asymptote. The data are the means ± SEM of n= 6 for Na_v1.7 and Na_v1.7+β₁ andn = 6 for Na_v1.8 and Na_v1.8+β₁ channels. A, The inactivation time constants of Na_v1.7 (filled squares) and Na_v1.7+β₁ (open squares) plotted versus voltage. B, The time constants of Na_v1.8 (filled circles) and Na_v1.8+β₁ (open circles) plotted versus voltage.

Fig. 5.

Effects of the β₁ subunit on the activation, inactivation, and recovery of Na_v1.7 and Na_v1.8 channels. Activation was measured by applying a series of depolarizing voltage pulses between −80 and +60 mV from a holding potential of −100 mV. The peak currents were measured, and the relative conductance was calculated by using the standard procedures (see Materials and Methods). Also plotted is the steady-state availability curve that was determined by using 500 msec conditioning pulses to voltages between −110 and +30 mV and a standard test pulse to either −20 mV (Na_v1.7 and Na_v1.7+β₁) or +20 mV (Na_v1.8 and Na_v1.8+β₁). Test currents were normalized and plotted versus conditioning voltage.A, The normalized conductance versus voltage and steady-state inactivation plots of Na_v1.7 (filled squares) and Na_v1.7+β₁ (open squares) channels. The smooth curves are Boltzmann fits:G = 1/(1 + exp ((V −V_0.5)/−k)), with midpoints (V_0.5) and slope factors (k) of activation of −22 ± 2.7 and 5.4 ± 0.4 mV for Na_v1.7 (n = 10) and −27.7 ± 1.3 and 3.7 ± 0.2 mV for Na_v1.7+β1 (n = 11). For inactivation the V_0.5 and k values are −68.2 ± 0.43 and 6.4 ± 0.45 mV for Na_v1.7 (n = 4) and −69.8 ± 0.3 and 3.9 ± 0.2 mV for Na_v1.7+β₁ (n = 4).B, Steady-state activation and inactivation of Na_v1.8 channels. The smooth curves haveV_0.5 and k values for activation of 4.7 ± 0.7 and 6.8 ± 0.1 mV for Na_v1.8 (filled circles;n = 8) and −3.3 ± 1.0 and 5.5 ± 0.1 mV for Na_v1.8+β₁ (open circles;n = 9). The V_0.5 andk values for inactivation are −54.8 ± 1.7 and 8.4 ± 0.2 mV for Na_v1.8 (n = 3) and −62.6 ± 2.3 and 6.3 ± 0.7 mV for Na_v1.8+β₁ (n = 6).C, D, The time course of recovery from inactivation of Na_v1.7 and Na_v1.8 channels. Inactivation was induced by depolarizing to −20 mV (Na_v1.7 and Na_v1.7+β₁) or +20 mV (Na_v1.8 and Na_v1.8+β₁) for 50 msec before returning to −100 mV for intervals between 1 msec and 5 sec. A standard test pulse was used to monitor recovery, and the normalized test currents were plotted versus the recovery interval.C, Recovery from inactivation of Na_v1.7 (filled squares) and Na_v1.7+β₁ (open squares) channels. The smooth curves are fits to the sum of two exponentials:I/I_O =A_F · (1 − exp(−t/τ_F)) +A_S · (1 − exp(−t/τ_S)), with time constants (τ) and weighting factors (A) of 19.6 ± 0.8 msec (τ_F;A_F = 0.46 ± 0.02) and 933.4 ± 54.6 msec (τ_S;A_S = 0.54 ± 0.02) for Na_v1.7 (n = 7) and 6.6 ± 0.6 msec (τ_F; A_F = 0.89 ± 0.02) and 53.2 ± 12.7 msec (τ_S;A_S = 0.11 ± 0.02) for Na_v1.7+β₁ (n = 7).D, The recovery of Na_v1.8 (filled circles) and Na_v1.8+β₁ (open circles) channels is described best by the sum of three exponentials:I/I_O =A_F · (1 − exp(−t/τ_F)) +A_I · (1 − exp(−t/τ_I)) +A_S · (1 − exp(−t/τ_S)), where τ_F, τ_I, and τ_Sare the fast, intermediate, and slow recovery time constants, andA_F,A_I, and A_Sare the relative weighting factors. t is the interpulse duration, and I/I_o is the normalized current amplitude. Data are the means ± SEM. The time constants of Na_v1.8 are 9.9 ± 1.8 msec (τ_F; A_F = 0.41 ± 0.04), 168.6 ± 52.2 msec (τ_I;A_I = 0.28 ± 0.04), and 787.6 ± 112.6 msec (τ_S;A_S = 0.28 ± 0.04) (filled circles; n = 5). Recovery time constants of Na_v1.8+β₁ are 2.0 ± 0.3 msec (τ_F;A_F = 0.32 ± 0.05), 243.8 ± 85.4 msec (τ_I; A_I= 0.34 ± 0.05), and 1070.1 ± 59.0 msec (τ_S; A_S = 0.34 ± 0.02) (open circles; n= 4).

Fig. 6.

Development of slow inactivation of Na_v1.7+β₁ and Na_v1.8+β₁ sodium channels. Time course of entry into the slow inactivated state was measured by using a triple-pulse protocol consisting of a variable duration conditioning pulse (2 msec to 10 sec) to −20 mV (Na_v1.7+β₁; open squares) or to +20 mV (Na_v1.8+β₁; open circles) to inactivate the channels. A 20 msec pulse to −100 mV was applied to promote the rapid recovery of inactivated channels, and a standard test pulse was used to assay availability. The test currents were normalized and plotted versus the conditioning pulse interval. The decay of the currents is fit best by the sum of three exponentials: I/I_O= A_F · (1 − exp(−t/τ_F)) +A_I · (1 − exp(−t/τ_I)) +A_S · (1 − exp(−t/τ_S)), where τ_F, τ_I, and τ_Sare the time constants, and A_F,A_I, and A_Sare the relative weighting factors. t is the conditioning pulse duration, andI/I_o is the normalized current. The data are the means ± SEM. The time constants of Na_v1.7+β₁ (open squares) are τ_F = 32.6 ± 1.5 msec (A_F = 0.25 ± 0.03), τ_I = 556.5 ± 104.4 msec (A_I = 0.33 ± 0.02), and τ_S = 4071.9 ± 155.1 msec (A_S = 0.42 ± 0.03) (n = 6). For Na_v1.8+β₁(open circles) the time constants are τ_F = 8.4 ± 1.5 msec (A_F = 0.57 ± 0.07), τ_I = 200.0 ± 30.0 msec (A_I = 0.16 ± 0.07), and τ_S = 8880.0 ± 1150.0 msec (A_S = 0.27 ± 0.04) (n = 5).

Fig. 7.

Frequency-dependent inhibition of Na_v1.7+β₁ and Na_v1.8+β₁ sodium currents. A,B, A train of 50 pulses was applied to −20 mV (Na_v1.7+β₁; n = 11) or +20 mV (Na_v1.8+β₁;n = 8) at frequencies between 0.5 and 100 Hz. The peak currents elicited by each test pulse were normalized to the current of the first pulse (P_n/P₁, where n = 1–50) and were plotted versus pulse number. The pulse duration was 8 msec for frequencies between 0.5 and 50 Hz but was reduced to 5 msec for the 100 Hz experiments. The holding and interpulse potential was −100 mV. C, Plotted is the ratio of the currents elicited by the 50th and first pulses (P₅₀/P₁) versus the pulsing frequency. Insets are representative raw current traces of Na_v1.7+β₁ (open squares) and Na_v1.8+β₁ (open circles) stimulated at 20 Hz.