Modulation of human dorsal root ganglion neuron firing by the Nav1.8 inhibitor suzetrigine

Abstract

Nav1.8 voltage-gated sodium channels are strongly expressed in human primary pain-sensing neurons (nociceptors) and a selective Nav1.8 inhibitor VX-548 (suzetrigine) has shown efficacy for treating acute pain in clinical trials. Nociceptors also express other sodium channels, notably Nav1.7, raising the question of how effectively excitability of the neurons is reduced by inhibition of Nav1.8 channels alone. We used VX-548 to explore this question, recording from dissociated human dorsal root ganglion neurons at 37 °C. Applying VX-548 at 10 nM (about 25 times the IC₅₀ determined using cloned human Nav1.8 channels at 37 °C) had only small effects on action potential threshold and upstroke velocity but substantially reduced the peak and shoulder. Counterintuitively, VX-548 shortened the refractory period-likely reflecting reduced potassium channel activation by the smaller, narrower action potential-sometimes resulting in faster firing. Generally, repetitive firing during depolarizations was diminished but not eliminated by VX-548. Voltage clamp analysis suggested two reasons that repetitive firing often remains in 10 to 100 nM VX-548. First, many neurons had such large Nav1.8 currents that even 99% inhibition leaves nA-level Nav1.8 current that could help drive repetitive firing. Second, Nav1.7 current dominated during initial spikes and could also contribute to repetitive firing. The ability of human neurons to fire repetitively even with >99% inhibition of Nav1.8 channels may help explain the incomplete analgesia produced by even the largest concentrations of VX-548 in clinical studies.

Keywords: VX-548; action potential; nociceptor; refractory period; sodium channel.

Fig. 1.

VX-548 is less potent on human Nav1.8 channels at 37 °C than 21 °C. (A) Dose–response for inhibition of human Nav1.8 currents at 21 °C, mean ± SD. Measurements from 49 cells, 40 exposed to a single concentration and 9 to first a low (0.03, 0.3, or 1 nM) then a high (3, 10, or 30 nM) concentration; n = 6 for 0 (relative current measured after 10 min of exposure to control drug-free solution containing 0.1% DMSO), 4 for 0.003 nM, 7 for 0.01 nM, 8 for 0.03 nM, 9 for 0.1 nM, 5 for 0.3 nM, 5 for 1 nM, 5 for 3 nM, 5 for 10 nM, 4 for 30 nM. (B) Dose–response at 37 °C, mean ± SD. Measurements from 43 cells, 24 exposed to a single concentration and 19 to first a lower (0.03 or 1 nM) then a higher (3, 10, or 30 nM) concentration; n = 6 for 0 (relative current measured after 10 min of exposure to control drug-free solution containing 0.1% DMSO), 6 for 0.03 nM, 7 for 0.1 nM, 15 for 0.3 nM, 8 for 1 nM, 8 for 3 nM, 9 for 10 nM, 3 for 30 nM. Fitted curves: I_max/[1 + (Drug)/K_d], where both I_max and K_d were allowed to vary and the fit included weighting of data points by SD. 21 °C: I_max = 1.00, K_d = 0.072 nM. 37 °C: I_max = 0.99, K_d = 0.38 nM.

Fig. 2.

Effect of VX-548 on action potentials in human DRG neurons. (A) Action potential evoked by a short (0.5-ms) current injection before and after application of 10 nM VX-548. (B) Phase-plane plot of dV/dt versus V showing modest reduction of maximum upstroke velocity and substantial reduction of peak and shoulder. Upper dashed lines: maximum upstroke velocity in control (black) and with 10 nM VX-548 (red). (C) Collected results for effect of 10 nM VX-548 on action potential threshold determined as in SI Appendix, Fig. S3. (D) Collected results for effect of 10 nM VX-548 on peak of the action potential evoked by a short current injection. (E) Collected results for effect of 10 nM VX-548 on the maximal upstroke velocity of the action potential. (F) Collected results for effect of 10 nM VX-548 on the width of the action potential measured at half-maximum amplitude.

Fig. 3.

Effect of 10 nM VX-548 on action potentials during repetitive firing. (A) Firing evoked by a 1-s 300-pA current injection before and after application of 10 nM VX-548. (B) First 100 ms on an expanded time scale. (C) Phase-plane plot of dV/dt versus V for the first action potential. (D) Collected results for effect of 10 nM VX-548 on peak of the first action potential (Left) and 4th action potential (Right). Peak of first action potential was +54 ± 2 mV in control and +43 ± 4 mV in VX-548 (mean ± SEM, n = 21; P = 0.0001, two-tailed Wilcoxon signed rank test) with an average decrease of 11 ± 2 mV. Peak of 4th action potential was +47 ± 4 mV in control and +20 ± 4 mV in VX-548 (mean ± SEM, n = 11; P = 0.0036, two-tailed Wilcoxon signed rank test) with an average decrease of 28 ± 3 mV. (E) Collected results for effect of 10 nM VX-548 on width of first action potential, measured at half-maximal amplitude. In 16 of 21 cells, the width decreased with VX-548; in the 5 cells where width at half-maximal amplitude increased, peak was greatly reduced so that half-maximal amplitude occurred much more negative than in control. Average widths were 2.92 ± 0.33 ms in control and 2.66 ± 0.48 ms in VX-548 (mean ± SEM, n = 21; P = 0.197, two-tailed Wilcoxon signed rank test).

Fig. 4.

Effect of 10 nM VX-548 on maximal repetitive firing. (A) Firing evoked by 1-s injections of current of increasing magnitude in control and after application of 10 nM VX-548. (B) Number of action potentials as a function of the injected current. (C) Collected results for the effect of 10 nM VX-548 on the maximal number of action potentials during 1-s current injections over a range of magnitudes. Dashed line drawn at 1 action potential.

Fig. 5.

Reduction of refractory period by VX-548. (A) Action potentials were evoked by a pair of 0.5-ms current injections with a variable time between them, with magnitude of both at 1.5-times the threshold current determined in control. The time between the two current injections was varied from longer to shorter to determine the refractory period with this stimulus. The figure shows superimposed sweeps from 8 different sets of times in control (black) and with 10 nM VX-548 (red). In control, the second stimulus evoked an action potential with a spacing (start to start) of 42 ms but not with 35 ms, while after VX-548, a spacing of 21 ms evoked an action potential using the same stimuli. Note faster decay of afterhyperpolarization in VX-548. (B) Collected results in 14 neurons, plotting the maximum spacing between the stimuli (with magnitude set at 1.5-times the threshold in control) in which the second stimulus failed to evoke a spike.

Fig. 6.

Effect of 30 nM and 100 nM VX-548 on action potential firing. (A) Example of a neuron testing successive application of 10 nM, 30 nM, and 100 nM VX-548 on firing evoked by a 1-s current pulse. (B) The beginning of the record on a faster time scale, showing the earlier firing of the second spike in 10 nM, 30 nM, and 100 nM VX-548. (C) Number of action potentials evoked by 1-s current injections of different magnitudes in this neuron. Note increased firing for current injections below 500 pA. (D) Collected data for the maximum number of action potentials evoked by a series of 1-s current injections in 8 neurons in which concentrations of 10 nM, 30 nM, and 100 nM VX-548 were applied successively. Dashed line drawn at 1 action potential.

Fig. 7.

Action potential clamp determination of Nav1.8 and Nav1.7 components of current during repetitive firing. (A) The record of repetitive firing evoked by 1-s current injection was used as command waveform after switching the amplifier to voltage clamp mode and currents evoked by the waveform were recorded in control (black) and after application of 30 nM VX-548 (red). (B) Top, current during 1st, 2nd, and 5th (last) action potentials before and after application of VX-548. Middle, subtraction yielding Nav1.8 (VX-548-sensitive) current. Bottom, subtraction yielding Nav1.7 current by application of 30 nM GsAF-1 in the continuing presence of VX-548. (C) Collected results of peak current sensitive to 30 nM VX-548 during the 1st, 2nd, 3rd, 4th, and last action potentials in 10 neurons. Each cell used a command voltage from its own repetitive firing. (D) Collected results of peak current sensitive to 30 nM GsAF-1 (in the presence of VX-548) in these neurons.

Fig. 8.

Additional sodium current component with both Nav1.7 and Nav1.8 inhibited. Action potential clamp experiments were performed as in Fig. 7. (A) Example of a neuron in which a large sodium current remained in the first action potential in the presence of 30 nM VX-548 and 30 nM GsAF-1 and was inhibited by application of 1 µM TTX. (B) Collected results for the different components of sodium current evoked during the 1st, 2nd, and last action potentials in 10 neurons. Asterisks indicate neurons in which only VX-548 and GsAF-1 were applied.