Concentration-dependence for inhibition of cloned human Nav1.7 channels by AM-2099 at 22 °C (A) and 37 °C (B).

Recordings were made on a Qube 384 automated patch clamp instrument (Sophion Bioscience) using a stable cell line with human Nav1.7 channels expressed in HEK293 cells (Liu et al., 2012) using 10-hole recording wells. Sodium channel current was evoked by a step from-80 mV to +5 mV delivered every 3 seconds. Each concentration of AM-2099 was applied in 16 wells and data from each well were accepted if the leak-corrected current at the holding voltage of-80 mV was less than 0.5 nA and the peak sodium current in control was > 10 nA. Symbols show mean ± SD for wells meeting these criteria with current measured after 16 minutes of exposure to compound. A:12 wells for control solution containing 0.1% DMSO (0 nM), 8 for 1 nM, 8 for 3 nM, 8 for 10 nM, 8 for 30 nM, 8 for 100 nM, 12 for 300 nM, 15 for 1000 nM. B: 23 wells for control drug-free solution containing 0.1% DMSO, 12 for 1 nM, 13 for 3 nM, 15 for 10 nM, 9 for 30 nM, 11 for 100 nM, 11 for 300 nM, 15 for 1000 nM. Fitted curves: Imax/(1+[Drug]/Kd), where Imax was fixed at the average value in the DMSO control wells (concentration of 0 nM) and the fit included weighting of data points by SD. Fit to data in A ignored the values from the column corresponding to 3 nM AM-2099 as it was obvious that there was an error in preparing this dilution. Internal solution (mM):140 CsF, 10 NaCl, 1 EGTA, 10 HEPES, 25 sucrose pH 7.3 w CsOH. External Solution (mM):145 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 Glucose, pH 7.4 w NaOH. All solutions contained 1mg/mL Pluronic-F68.

Effect of AM-2099 on voltage-clamped sodium current in human DRG neurons.

A-B, Effect of cumulative addition of 600 nM AM-2099 and 600 nM TTX on sodium current evoked after the inhibition of Nav1.8 channels by 30 nM VX-548 (in the continuing presence of VX-548). C, Collected results. In 7 neurons, the cell was exposed first to 100 nM AM-2099 and then to 600 nM AM-2099 and then to 600 nM AM-2099 plus 600 nM TTX, all on a background of 10 nM VX-548. Light red bars show the fraction of current inhibited by 100 nM AM-2099, dark red bars show the additional effect of 600 nM AM-2099, and green bars show the additional effect of 600 nM TTX. In three neurons, cells were exposed to 600 nM AM-2099 and then 600 nM AM-2099 plus 600 nM TTX in the presence of 30 nM VX-548. D, Mean ± SD for the effect of 600 nM AM-2099 on the sodium current remaining in VX-548 in these ten neurons.

Effect of AM-2099 on action potentials in human DRG neurons.

A, Action potentials in an example capsaicin-sensitive human DRG neuron evoked by a short (0.5-ms) current injection before and after application of 600 nM AM-2099. Stimulating current was adjusted to be slightly larger in AM-2099 so that the voltage immediately after cessation of the stimulating current was the same as in control (because control has an active component in the last 0.1 ms of the stimulating pulse that is inhibited with AM-2099). B, Phase-plane plot of dV/dt versus V for the action potentials in A showing reduction of maximum upstroke velocity and peak. Upper dashed lines: maximum upstroke velocity in control (black) and with 600 nM AM-2099 (red). C, Collected results for effect of 600 nM AM-2099 on the maximal upstroke velocity of the action potential in 40 neurons. D, Tukey box-plot of collected results for upstroke in 600 nM AM-2099 relative to the control. Middle bar shows median, with notch indicating 95% confidence interval for median, lower bar indicates 25% percentile, and upper bar indicates 75% percentile. E, Collected results for effect of 600 nM AM-2099 on peak of the action potential evoked by a short current injection (N=40). F, Tukey box-plot of collected results for change in action potential peak by 600 nM AM-2099. G, Collected results for effect of 600 nM AM-2099 on the width of the action potential measured at 0 mV. N=39 for cells with control peak >0 mV; values only in control shown for 5 cells in which the action potential in AM-2099 had a peak < 0 mV.

Effect of AM-2099 on action potential threshold.

Short (0.5-ms) current injections of different size were delivered to find the level of current injection that first evoked an action potential. The voltage immediately after the smallest current injection that evoked a spike was considered as the threshold voltage. A, Just sub-threshold (red) and supra-threshold (blue) current injections in control (left) and after application of 600 nM AM-2099 (right). B, Collected data for threshold voltage before and after 600 AM-2099 (n=38). C, Tukey box-plot of collected results for change in voltage threshold in 600 nM AM-2099 relative to the control. Middle bar shows median, with notch indicating 95% confidence interval for median, lower bar indicates 25% percentile, and upper bar indicates 75% percentile. D, Collected data for minimum current (0.5-ms current injection) that evoked an action potential (n=38). E, Tukey box-plot of collected results of ratio of minimum current to evoke an action potential after application of 600 nM AM-2099 relative to control.

Effect of 600 nM AM-2099 on rheobase and repetitive firing.

A, Firing evoked in a capsaicin-sensitive human DRG neurons by 1-s injections of current of increasing magnitude in control and after application of 600 nM AM-2099. B, Number of action potentials as a function of the injected current before and after 600 nM AM-2099 in this neuron. Asterisks indicate rheobase current in control (black) and in AM-2099 (red). C, Collected data for the effect of 600 nM AM-2099 on rheobase current. N=44 neurons. D, Tukey box-plot of collected results for rheobase in 600 nM AM-2099 relative to control rheobase. Middle bar shows median, with notch indicating 95% confidence interval for median, lower bar indicates 25% percentile, and upper bar indicates 75% percentile. E, Collected results for the effect of 600 nM AM-2099 on the maximal number of action potentials during 1-s current injections over a range of magnitudes for neurons that fired more than one action potential in control. N=31 neurons. Dashed line drawn at 1 action potential. F, Tukey box-plot of collected results for maximum number of action potentials in 600 nM AM-2099 relative to maximum number in control.

Prolongation of refractory period by AM-2099.

A, Action potentials were evoked by a pair of 0.5-ms current injections with a variable time between them, with magnitude of both set 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 11 different sets of times in control (black) and with 600 nm AM-2099 (red). In control, the second stimulus evoked an action potential with a spacing (start to start) of 20.6 ms, while after AM-2099, a spacing of 35.2 ms was required to evoke an action potential using the same stimuli. B, Collected results in 15 neurons. In three other neurons, after application of 600 nM AM-2099 the neuron did not fire with injection of 1.5 times the threshold current in control and data are not included.

Comparison of Nav1.7 and Nav1.8 inhibition on action potential firing in human DRG neurons.

A, Change in action potential threshold by inhibiting Nav1.7 channels (600 nM AM-2099, n=41 neurons) or Nav1.8 channels (10 nM VX-548, n=18 neurons). B, Inhibition of maximum upstroke of action potential by inhibiting Nav1.7 channels (600 nM AM-2099, n=41 neurons) or Nav1.8 channels (10 nM VX-548, n=18 neurons). C, Reduction of action potential peak by inhibiting Nav1.7 channels (600 nM AM-2099, n=41 neurons) or Nav1.8 channels (10 nM VX-548, n=18 neurons). D, Effect of inhibiting Nav1.7 channels (600 nM AM-2099, n=31 neurons) or Nav1.8 channels (100 nM VX-548, n=15 neurons) on maximum number of action potentials evoked by 1-s depolarizations of different magnitudes, confining analysis to cells that fired more than one action potential in control. Data for effects of VX-548 from Stewart et al., (2025).

Donor demographics and distribution of data from cells used from each donor.

Distribution of cell sizes, input resistance, resting potential, and action potential upstroke velocity of neurons used for experiments (all capsaicin-sensitive).

A, Histogram of cell capacitances (n=72). B, Histogram of approximate cell diameters calculated from cell capacitance assuming a spherical cell and specific membrane capacitance of 1 μF/cm2 (n=72). C, Tukey box plot of collected data for input resistance, calculated from step from-90 to-85 mV immediately after establishing whole cell recording (22°C, before moving cell to heated solution, n=58). Middle bar shows median, with notch indicating 95% confidence interval for median, lower bar indicates 25% percentile, and upper bar indicates 75% percentile. D, Tukey box plot of collected data for resting potential (37°C, n=57). E, Tukey box plot of collected data for maximal upstroke velocity of the action potential (37°C, n=57).

Effects of 200 nM AM-2099 on action potential parameters compared in cells from individual donors.

Effects of 200 nM PF-04856264 compared to 600 nM AM-2099.

A, Effect of 200 nM PF-04856264 on the action potential in a human capsaicin-sensitive DRG neuron. B, Phase-plane plot of dV/dt versus V for the action potentials in A showing reduction of maximum upstroke velocity and peak. C-E, Collected effects of 200 nM PF-04856264 on action potential parameters (green symbols, n=11) compared to those of 600 nM AM-2099 (red symbols, replotted from Figures 3 and 4). Middle bar shows median, with notch indicating 95% confidence interval for median, lower bar indicates 25% percentile, and upper bar indicates 75% percentile. F, Time-course of inhibition of sodium current from cloned human Nav1.7 channels by 200 nM PF-04856264. Manual patch clamp, 37°C. Mean ± SD. N=7 to 150 s, 6 to 160 s, 5 to 220 s. G, Same for inhibition by 600 nM AM-2099. N=8 to 70 s, 7 to 90 s, 5 to 120 s, 4 to 160 s. H, Effect of 200 nM PF-04856264 on sodium current remaining in the presence of 10 nM VX-548 in human capsaicin-sensitive DRG neurons (green, mean ± SD, n=9) compared to 600 nM AM-2099 (red, re-plotted from Fig 2).