Contributions of NaV1.8 and NaV1.9 to excitability in human induced pluripotent stem-cell derived somatosensory neurons

Abstract

The inhibition of voltage-gated sodium (Na_V) channels in somatosensory neurons presents a promising novel modality for the treatment of pain. However, the precise contribution of these channels to neuronal excitability, the cellular correlate of pain, is unknown; previous studies using genetic knockout models or pharmacologic block of Na_V channels have identified general roles for distinct sodium channel isoforms, but have never quantified their exact contributions to these processes. To address this deficit, we have utilized dynamic clamp electrophysiology to precisely tune in varying levels of Na_V1.8 and Na_V1.9 currents into induced pluripotent stem cell-derived sensory neurons (iPSC-SNs), allowing us to quantify how graded changes in these currents affect different parameters of neuronal excitability and electrogenesis. We quantify and report direct relationships between Na_V1.8 current density and action potential half-width, overshoot, and repetitive firing. We additionally quantify the effect varying Na_V1.9 current densities have on neuronal membrane potential and rheobase. Furthermore, we examined the simultaneous interplay between Na_V1.8 and Na_V1.9 on neuronal excitability. Finally, we show that minor biophysical changes in the gating of Na_V1.8 can render human iPSC-SNs hyperexcitable, in a first-of-its-kind investigation of a gain-of-function Na_V1.8 mutation in a human neuronal background.

Figure 1

Generation and characterization of iPSC-SNs. (A) iPSC-SNs express canonical sensory neuronal markers. 50 µm scale bar for reference. (B) iPSC-SNs express very high levels of Na_V1.7 mRNA, but virtually no levels of Na_V1.8 and Na_V1.9 mRNA, as determined by droplet digital PCR. (C) Representative current traces recorded from iPSC-SNs confirm large total sodium currents (top). However, application of 1 µM tetrodotoxin reveals very little tetrodotoxin-resistant current, which behaves like Na_V1.5 and not Na_V1.8 or Na_V1.9 (middle). Consistent with the lack of Na_V1.8 expression by PCR analysis, there is also no Na_V1.8 current when cells are clamped at a holding potential of − 50 mV to inactivate all other sodium channels besides Na_V1.8 (bottom). (D) The V_1/2 of activation (open circles) and fast-inactivation (open diamonds) of total sodium current in iPSC-SNs was − 29.83 ± 2.43 mV and − 74.08 ± 2.55 mV, respectively. The V_1/2 of activation (orange circles) and fast-inactivation (orange diamonds) of TTX-R sodium currents in iPSC-SNs was − 38.17 ± 1.80 mV and − 87.79 ± 2.93 mV, respectively.

Figure 2

Na_V1.8 contributes to action potential overshoot, half-width, and repetitive firing. (A) Representative traces from the same iPSC-SN illustrating the action potential waveform in the setting of varying levels of Na_V1.8 current density. (B) Increasing Na_V1.8 current density increases the overshoot of iPSC-SNs, although the effect is more robust at lower initial overshoot amplitudes. For neurons with an initial overshoot amplitude between 40 and 45 mV (far left), the change in overshoot is best fit with a linear model with slope 0.1706 and an r² of 0.5093. For neurons with an initial overshoot between 45 and 50 mV (center-left), 50–55 mV (center-right), and 55–60 mV (far right), the change in overshoot amplitudes are best fit with exponential association equations. (C) Increasing Na_V1.8 current density directly increases the action potential half-width of iPSC-SNs linearly (% change in half-width = 0.4254*current density) with an r² of 0.65. An equivalent transformation of the data into base-10 logarithmic form illustrates a similarly robust relationship (% change in half-width = 0.4816*e^{2.253(log[current density])}) with an r² of 0.6502. (D) Increasing Na_V1.8 current density enhances iPSC-SN repetitive firing following (Δ action potential count = 524.9*(1 − e^{−0.002266(current density)}) with an r² of 0.4164. (E) Representative traces depicting the response of the same iPSC-SN to a 1 s duration 500 pA suprathreshold stimulus with no Na_V1.8 currents injected via dynamic clamp (left), approximately 50 pA/pF Na_V1.8 current density (middle), and 100 pA/pF current density (right).

Figure 3

Na_V1.9 is responsible for setting the neuronal resting membrane potential and plays a role in setting the threshold to action potential firing. (A) Increasing Na_V1.9 current density depolarizes iPSC-SN resting membrane potentials (% depolarization = 1.629*e^{0.007674(current density)}) with an r² of 0.5764. (B) Adding Na_V1.9 currents to iPSC-SNs results in a statistically significant reduction in threshold to action potential firing (left, paired t-test p = 0.0043). However, there does not appear to be a strong correlation between the levels of Na_V1.9 current density and the change in threshold (right).

Figure 4

While the resting membrane potential is primarily set by Na_V1.9, both Na_V1.9 and Na_V1.8 play important roles in repetitive firing. (A) The relationship between Na_V1.8 and Na_V1.9 current densities with the change in iPSC-SN resting membrane potential can be approximated with a 3-dimensional polynomial curve with two degrees of freedom for the x- and y-axes (right, adjusted r² = 0.4147). A contour plot (left) of this fit illustrates that Na_V1.9 current density has a stronger effect on changing the neuronal resting membrane potential. (B) The relationship between Na_V1.8 and Na_V1.9 current densities with the change in iPSC-SN repetitive firing can be approximated with a 3-dimensional polynomial curve with two degrees of freedom for the x- and y-axes (right, adjusted r² = 0.3514). A contour plot of this fit illustrates that both Na_V1.8 and Na_V1.9 contribute to enhanced repetitive firing.

Figure 5

Small biophysical shifts in Na_V1.8 gating can substantially alter neuronal excitability. (A) Conductance-voltage relationship between the wild-type Na_V1.8 dynamic clamp model and the model with a 4.5 mV hyperpolarized voltage-dependence of activation, approximating the gain-of-function mutation Na_V1.8-I1706V. (B) Representative traces of Na_V1.8 current recorded from human autopsy-derived DRG neurons. (C) Box-and-whisker plot showing the Na_V1.8 current density recorded from autopsy-derived human DRG neurons in 70 mM NaCl (− 143.69 ± 24.54 pA/pF, n = 24). (D) Injection of 50% wild-type Na_V1.8 current density and 50% “Na_V1.8-I1706V” current density resulted in a statistically significant (paired t-test p = 0.0023, n = 13) reduction in current threshold to action potential firing. (E) Injection of 50% wild-type Na_V1.8 current density and 50% “Na_V1.8-I1706V” current density resulted in a statistically significant (paired t-test p = 0.0367, n = 7) enhancement of repetitive action potential firing.