Differential modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by the local anesthetic lidocaine

Abstract

1 Voltage-gated Na+ channels are transmembrane proteins that are essential for the propagation of action potentials in excitable cells. Nav1.7 and Nav1.8 dorsal root ganglion Na+ channels exhibit different kinetics and sensitivities to tetrodotoxin (TTX). We investigated the properties of both channels in the presence of lidocaine, a local anesthetic (LA) and class I anti-arrhythmic drug. 2 Nav1.7 and Nav1.8 Na+ channels were coexpressed with the beta1-subunit in Xenopus oocytes. Na+ currents were recorded using the two-microelectrode voltage-clamp technique. 3 Dose-response curves for both channels had different EC50 (dose producing 50% maximum current inhibition) (450 microm for Nav1.7 and 104 microm for Nav1.8). Lidocaine enhanced current decrease in a frequency-dependent manner. Steady-state inactivation of both channels was also affected by lidocaine, Nav1.7 being the most sensitive. Only the steady-state activation of Nav1.8 was affected while the entry of both channels into slow inactivation was affected by lidocaine, Nav1.8 being affected to a larger degree. 4 Although the channels share homology at DIV S6, the LA binding site, they differ in their sensitivity to lidocaine. Recent studies suggest that other residues on DI and DII known to influence lidocaine binding may explain the differences in affinities between Nav1.7 and Nav1.8 Na+ channels. 5 Understanding the properties of these channels and their pharmacology is of critical importance to developing drugs and finding effective therapies to treat chronic pain.

Figure 1

Dose-dependent block of Na_v1.7 and Na_v1.8 currents by different lidocaine concentrations. Whole-cell Na⁺ currents were evoked every 5 s by 40 ms pulses to −15 mV from a holding potential of −100 mV until current stabilized (Na_v1.7). Solid lines represent control currents while dotted lines represent the current amplitude after superfusion with lidocaine to produce a steady-state lidocaine effect. For Na_v1.8, whole-cell Na⁺ currents were evoked every 20 s by 40 ms pulses to +20 mV from a holding potential of −100 mV until current stability was obtained. Solid lines represent currents under control conditions while dotted lines represent currents after lidocaine superfusion to produce a steady-state lidocaine effect. (a) 10 μM , 100 μM (b) and 300 μM (c). Na_v1.7 (A) and Na_v 1.8 (B). (C) Dose–response curves for both Na_v1.7 and Na_v1.8 channels (n=5) were obtained from fits of a four parameters Hill's equation described in the Methods section. The values of the Hill coefficients for Na_v1.7 were: a=100.79, b=1.31, c=477.10 and y₀=1.52. For Na_v1.8, the values were: a=96.98, b=1.06, c=118.31 and y₀=4.78. Filled circles represent Na_v1.7 and filled triangles represent Na_v1.8. Na_v1.8 exhibits greater sensitivity to lidocaine than Na_v1.7 (∼4.4-fold), with EC₅₀ values of 104 and 450 μM, respectively.

Figure 2

Effect of lidocaine on Na_v1.7 Na⁺ channels heterologously expressed in Xenopus oocytes. Whole-cell Na⁺ current traces of oocytes expressing Na_v1.7 before (a) and after (b) superfusion with 300 μM of lidocaine. Also shown in (c) are the effects of lidocaine (300 μM) on the current–voltage relationship (I/V curves) in control conditions (open circles) and in the presence of the anesthetic (filled circles). Currents were elicited by depolarizing steps between −80 and +20 mV in 5 mV increments from a holding potential of −100 mV (see figure inset for protocol). Dashed lines are zero current. I/V curves were obtained by plotting the current amplitude versus the voltage for the currents shown in (a) and (b).

Figure 3

Effect of lidocaine on Na_v1.8 Na⁺ channels heterologously expressed in Xenopus oocytes. Whole-cell Na⁺ current traces of oocytes expressing Na_v1.8 before (a) and after (b) superfusion with 300 μM of lidocaine. Also shown in (c) are the effects of lidocaine (300 μM) on the current–voltage relationship (I/V curves) in control conditions (open circles) and in the presence of the anesthetic (filled circles). Currents were elicited by depolarizing steps between −80 and +40 mV in 5 mV increments from a holding potential of −100 mV (see figure inset for protocol). Dashed lines are zero current. I/V curves were obtained by plotting the current amplitude versus the voltage for the currents shown in (a) and (b).

Figure 4

Effect of lidocaine on the steady-state inactivation and steady-state activation curves of Na_v1.7 (a) and Na_v1.8 (b). Steady-state activation curves were derived from the same family of currents used for the I/V curves (Figures 2c and 3c) using the standard procedure (see Methods). Steady-state inactivation were determined using 500 ms conditioning pulses to voltages between −110 and +30 mV and a standard test pulse to −20 mV for Na_v1.7 or +15 mV for Na_v1.8. Test currents were normalized and plotted against the conditioning voltage. The steady-state properties for Na_v1.7 (a, open circles and open triangles, respectively) and Na_v1.8 (b, open squares and open reversed triangles, respectively) in the absence of lidocaine are shown on the same graph as in the presence of lidocaine 100 μM (filled circles and filled triangles (a) and filled squares and filled reversed triangles (b)). The smooth curves are Boltzmann fits (the equations are shown in Methods). See Table 1 for V_1/2 and k_v values for both activation and inactivation.

Figure 5

Frequency-dependent inhibition of Na_v1.7 (a) and Na_v1.8 (b) Na⁺ currents in the presence and absence of lidocaine 300 μM. Oocytes were held at −100 mV and a train of fifty 8 ms pulses was applied to −10 mV (Na_v1.7) or +15 mV (Na_v1.8) at three different frequencies (0.5, 2 and 5 Hz), with the interpulse potential also set at −100 mV. The peak currents elicited by each pulse were normalized to the current of the first pulse (P_n−P₁, where n=1–50) and were then plotted versus pulse number. Different open symbols represent control conditions while filled symbols represent the protocol in the presence of 300 μM lidocaine for the different frequencies (circles represent 0.5 Hz, squares represent 2 Hz and triangles represent 5 Hz). Examples of current traces at the 1st and 50th pulse of the protocol for each channel in the presence and absence of lidocaine are shown in the right panel. The central panel shows a schematic representation of the electrical protocol used.

Figure 6

Bar plot representation of the relative amplitudes at the 50th sweep of the frequency dependence protocol used in Figure 5 for each frequency. The amplitudes of the last step were normalized versus the first step of the protocol. White columns are control currents, gray columns are currents' amplitudes after perfusion with 100 μM lidocaine and black columns are currents' amplitudes in the presence of 300 μM lidocaine. (^*=P<0.05).

Figure 7

Development of slow inactivation by both Na_v1.7 (a) and Na_v1.8 (b) channels with and without lidocaine. Control conditions are open circles (Na_v1.7) and open squares (Na_v1.8) while experiments with the drug are represented by filled circles and squares. The entry into slow inactivation was measured using a double-pulse protocol consisting of a conditioning pulse of variable duration (1 ms to 10 s) to −10 mV (Na_v1.7) or 15 mV (Na_v1.8) to inactivate the channels. A 150 ms pulse to −100 mV was then applied to allow rapid recovery and a standard test pulse was used to measure the amount of available channels (see inset). The measured currents were then normalized and plotted against the duration of the conditioning pulse. The decrease in currents was best fitted in all cases with the sum of three exponentials (solid lines). See Table 1 for the time-constant values.