The role of Nav1.7 in human nociceptors: insights from human induced pluripotent stem cell-derived sensory neurons of erythromelalgia patients

Abstract

The chronic pain syndrome inherited erythromelalgia (IEM) is attributed to mutations in the voltage-gated sodium channel (NaV) 1.7. Still, recent studies targeting NaV1.7 in clinical trials have provided conflicting results. Here, we differentiated induced pluripotent stem cells from IEM patients with the NaV1.7/I848T mutation into sensory nociceptors. Action potentials in these IEM nociceptors displayed a decreased firing threshold, an enhanced upstroke, and afterhyperpolarization, all of which may explain the increased pain experienced by patients. Subsequently, we investigated the voltage dependence of the tetrodotoxin-sensitive NaV activation in these human sensory neurons using a specific prepulse voltage protocol. The IEM mutation induced a hyperpolarizing shift of NaV activation, which leads to activation of NaV1.7 at more negative potentials. Our results indicate that NaV1.7 is not active during subthreshold depolarizations, but that its activity defines the action potential threshold and contributes significantly to the action potential upstroke. Thus, our model system with induced pluripotent stem cell-derived sensory neurons provides a new rationale for NaV1.7 function and promises to be valuable as a translational tool to profile and develop more efficacious clinical analgesics.

Figure 1.

Na_V1.7/I848T mutation in IEM patients. (A) Segregation of Na_V1.7/I848T mutation in IEM study subjects. IEM 1, mother; IEM 2, daughter; both previously investigated in Ref. . (B) Location of the I848T mutation in the Na_V1.7 channel protein. IEM, inherited erythromelalgia.

Figure 2.

Functional sensory neurons are generated from iPS cells. (A) Differentiation scheme of iPS cells into sensory neurons with dual-SMAD inhibition (LDN193189 and SB431542), VEGF/FGF/PDGF inhibition (SU5402), Notch inhibition (DAPT), and WNT activation (CHIR99021) for 10 days (d0-d10), followed by growth factor (NGF, BDNF, and GDNF)-driven neuron maturation for 8 weeks. On maturation day M35 and M55, neurons were used for analysis. (B) Representative phase-contrast and immunofluorescence images of iPS cell–derived neurons expressing peripherin (green) and TUJ-1 (red) of IEM 1. Scale bar 100 µm. (C and D) Representative immunofluorescence images of neurons from IEM 1 stained positive for Na_V1.8 (C) (see also Supplementary Fig. S2, available at http://links.lww.com/PAIN/A749) and TRPV1 (D) (both red) and TUJ-1 (green). Nuclei were counterstained with DAPI (blue). Scale bar 100 µm. (E) Representative calcium imaging recording performed on iPS cell–derived neurons from clone IEM 1. Neurons were stimulated with capsaicin (1 µM), AITC (100 µM), menthol (100 µM), pH 6.0, and KCl (60 mM) for 30 seconds each as indicated. Response profiles of 18 cells are overlaid. For quantification, see Supplementary Figure S3 (available at http://links.lww.com/PAIN/A749). (F) Representative recording of an IEM 1 neuron. Traces display the neuron's total (left) and TTX-r (right) currents, showing functional expression of TTX-r Na_V channels. See also Table 1. (G) Representative recording of an IEM 1 neuron, showing functional expression of Na_V1.7. Total Na_V current is shown before (black) and after (red) 20 minutes of application of the Na_V1.7-specific blocker ProTx-II (5 nM). Data shown in (B–E) were gathered on maturation day M35. IEM, inherited erythromelalgia; iPS cell, induced pluripotent stem cell.

Figure 3.

The IEM mutation changes action potential characteristics. (A) Representative action potentials recorded in IEM 2 (left) and Ctrl 2 (right) nociceptors. Action potential thresholds for each particular neuron are indicated. Dotted line represents 0 mV. (B) The RMP was −47.7 ± 2.4 mV for IEM and −39.3 ± 3.3 for Ctrl nociceptors (n = 24 and 22; P = 0.05 unpaired t test: t = 2.07, df = 44). (C) The action potential threshold was significantly lowered in IEM nociceptors (−45.9 ± 0.6 mV in IEM and −40.1 ± 2.8 mV in Ctrl; n = 23 and 21; P = 0.02, Mann–Whitney test). (D) IEM nociceptors display a stronger hyperpolarization after each action potential (−61.2 ± 0.8 mV for IEM and −45.9 ± 3.7 mV for Ctrl; n = 23 and 21; P < 0.0001, Mann–Whitney test). (E) IEM action potentials had a mean amplitude of 120.9 ± 1.7 mV, compared with 103.1 ± 4.3 mV in Ctrl nociceptors (n = 23 and 21; P = 0.004, Mann–Whitney test). (F) Action potentials in IEM nociceptors had a mean half-width of 2.3 ± 0.6 ms, compared with 8.5 ± 3.8 ms in Ctrl nociceptors (n = 23 and 21; P = 0.02, Mann–Whitney test). (G) The time from pulse onset to the action potential peak is significantly shorter in IEM nociceptors (28.6 ± 2.4 ms, compared with 56.8 ± 11.8 ms in Ctrl; n = 23 and 21; P = 0.019, unpaired t test: t = 2.44, df = 42). (H) Nociceptors for IEM patients have a steeper maximum slope of the action potential upstroke (223.7 ± 13.2 V/s, compared with 136.3 ± 29 V/s in Ctrl; n = 23 and 21; P = 0.007, unpaired t test: t = 2.83, df = 42). (I) The slope of the subthreshold depolarization is not different between IEM and Ctrl nociceptors (1.5 ± 0.1 V/s for IEM vs 1.8 ± 0.3 V/s for Ctrl; n = 23 and 21; P = 0.8, Mann–Whitney test). (J) Action potential (AP) frequency induced by increasing stepwise current injections (n = 14 for IEM and n = 5 for Ctrl; note that some traces are overlaid). None of the measured Ctrl nociceptors fired more than 2 action potentials. The current injection protocol is identical to the one in Supplementary Fig. S5b (available at http://links.lww.com/PAIN/A749). Neurons were held at an RMP around −70 mV and depolarized by square current injections in steps of 0.5 × rheobase to evoke action potential firing. IEM, inherited erythromelalgia; RMP, resting membrane potential.

Figure 4.

Voltage-clamp protocol to measure TTX-s currents of iPS cell–derived nociceptors. (A–F) Example recordings from a Ctrl 2 nociceptor explain the protocols used in voltage-clamp recordings on iPS cell–derived cells. (A) Shown is the prepulse protocol, used to eliminate space clamp artifacts in this particular Ctrl 2 neuron. The protocol consisted of a short prepulse (generally 4-6 ms, −50 to −15 mV), followed by a repolarizing interpulse (generally 1 ms, −120 to −70 mV), followed by the regular testpulse (40 ms, −80 to +40 mV, 10 mV increments). Prepulse and interpulse voltage and duration were adjusted to each cell individually to eliminate artefacts (F) and obtain optimal voltage-clamp conditions. When tested in Na_V1.7-expressing HEK293 cells, such prepulse protocols did not affect voltage dependence of activation of Na_V1.7 and provided measurable current amplitudes during the testpulse (Supplementary Fig. S6, available at http://links.lww.com/PAIN/A749). (B) Total current measured in this particular neuron using the protocol shown in (A) before application of TTX. (C) TTX-resistant (TTX-r) current obtained from the same neuron after application of 500 nM TTX. (D) The TTX-sensitive (TTX-s) current component was calculated by subtracting the TTX-r current (C) from the total current (B). (E) Current–voltage (IV) relationships of the same neuron, obtained from the total current (black) shown in (B), the TTX-r current (blue) shown in (C), and the TTX-s current (red) shown in (D). The IV relationships confirm accurate voltage-clamp conditions, achieved by the prepulse protocol shown in (A). Incomplete inactivation of Na_Vs during the interpulse (here −70 mV) prevents current from reaching zero before the onset of the test pulse (see also B). (F) A regular voltage protocol without prepulse (40 ms, −80 to 40 mV, 10 mV increments) was run on the same neuron at the end of the recording after TTX had already been applied. The recorded TTX-r current shows bad voltage clamp and components from multiple cells, visible as delayed inward current peaks (red and blue with arrows). This confounds current–voltage analysis. iPS cell, induced pluripotent stem cell.

Figure 5.

The IEM mutation hyperpolarizes the voltage dependence of activation. (A–E) Analysis of the voltage dependence of the TTX-s current component in iPS cell–derived nociceptors. (A) Example traces of TTX-s currents measured in nociceptors from the indicated clones. (B) Current–voltage relationship of TTX-s currents show a left shift in both IEM clones. Current was normalized to the maximum inward current for each group. Due to the applied prepulse protocol, current does not start at 0.0 (Fig. 4). (C) Voltage dependence of activation of TTX-s currents obtained from data in (B). The left shift of the activation curves in both IEM clones is clearly visible. (D) Values for half-maximal voltage-dependent activation (V_1/2) of TTX-s currents obtained from data in (C): −24.4 ± 1.8 mV for IEM 1 (P = 0.034 vs Ctrl 1 and P = 0.018 vs Ctrl 2), −24.7 ± 1.1 mV for IEM 2 (P = 0.021 vs Ctrl 1 and P = 0.0099 vs Ctrl 2), −17.6 ± 1.6 mV for Ctrl 1, and -18.7 ± 1.1 mV for Ctrl 2. (E) TTX-s current density showed no significant differences between different clones: 173 ± 15 pA/pF for IEM 1, 151 ± 25 pA/pF for IEM 2, 88 ± 24 pA/pF for Ctrl 1, and 116 ± 14 pA/pF for Ctrl 2 (all P ≥ 0.07). (F) Voltage dependence of activation of total currents, measured before application of TTX. The Na_V1.7-mediated left shift is discernible between IEM and Ctrl clones. (G) V_1/2 values of total currents obtained from data in (F): −26.5 ± 1.8 mV for IEM 1 (P = 0.006 vs Ctrl 1 and P = 0.002 vs Ctrl 2), −24.4 ± 1.2 mV for IEM 2 (P = 0.073 vs Ctrl 1 and P = 0.092 vs Ctrl 2), −17 ± 1.8 mV for Ctrl 1, and −18.9 ± 1.1 mV for Ctrl 2. (H) Voltage dependence of activation of TTX-r currents. See Table 1 for all V_1/2, slope, current density, and n values. All 1-way ANOVA with Bonferroni multiple comparisons test. ANOVA, analysis of variance; IEM, inherited erythromelalgia; iPS cell, induced pluripotent stem cell.

Figure 6.

Block of Na_V1.7 using ProTx-II reveals a hyperpolarized voltage dependence. (A) Representative traces displaying tonic block by 10 nM ProTx-II or vehicle in HEK293 cells, expressing Na_V1.7 (upper and middle) or Na_V1.6 (lower). Voltage protocol (above) was applied every 2 seconds for 10 minutes. (B) ProTx-II- or vehicle-mediated block in Na_V1.7 and 1.6 expressing HEK293 cells. For Na_V1.7, vehicle and ProTx-II for 10 minutes reduced peak current by 14.2 ± 4.7% and 56.8 ± 6.4% (P = 0.0001; n = 7 and 8), respectively. For Na_V1.6, block was 2.7 ± 4% for vehicle and 20.4 ± 9.8% for ProTx-II (P = 0.45 vs Na_V1.6 + vehicle and P = 0.004 vs Na_V1.7 + ProTx-II; n = 7 and 4). (C and D) Analysis of the TTX-s current component in iPS cell–derived nociceptors (IEM 2 and Ctrl 2) after incubation in 5 nM ProTx-II for ≥ 10 minutes. (C) Left: TTX-s currents with ProTx-II. Right: TTX-s voltage dependence of activation with ProTx-II (solid lines). For comparison, TTX-s curves without ProTx-II are presented with dashed lines (same as in Fig. 5C). Block of Na_V1.7 induces a left shift of activation in control but not in IEM nociceptors. (D) V_1/2 values of TTX-s currents with or without ProTx-II: −26.8 ± 1.1 mV (IEM 2) and −25.3 ± 1.9 mV (Ctrl 2) with ProTx-II (P = 0.99). V_1/2 values without ProTx-II are presented for comparison (same as in Fig. 5D) (IEM 2: −24.7 ± 1.1 mV, P = 0.99 vs IEM 2 + ProTx-II; Ctrl 2: −18.7 ± 1.1 mV, P = 0.002 vs IEM 2 w/o ProTx-II, P = 0.004 vs Ctrl 2 + ProTx-II, and P = 0.0002 vs IEM 2 + ProTx-II). (E) The total current also shows a left shift of activation in control neurons. For Ctrl 2, V_1/2 was −25.9 ± 1.7 mV with ProTx-II, but −18.9 ± 1.1 mV without ProTx-II (P = 0.003). For IEM 2, V_1/2 was −26.8 ± 1.1 mV with ProTx-II and −24.4 ± 1.2 mV without ProTx-II (P = 0.99 vs IEM 2 + ProTx-II, P = 0.015 vs Ctrl 2 w/o ProTx-II). Values without ProTx-II are the same as in Fig. 5G. All 1-way ANOVA with Bonferroni multiple comparisons test. ANOVA, analysis of variance; IEM, inherited erythromelalgia; iPS cell, induced pluripotent stem cell.

Figure 7.

Proposed distribution of Na_V channels during action potential generation. Top: schematic representations of action potentials from wild-type (WT, left) or Na_V1.7/I848T (right) nociceptors. The individual phases of the action potential are shaded in different colors to match the assumed contribution of different Na_V isoforms to each particular phase. Bottom: schematic activation curves for the different Na_V channel categories, based on the data presented in this study. We propose that in the wild-type (left panel) Na_V1.1, 1.2, 1.3, 1.6, and 1.9 (purple) contribute mainly to subthreshold depolarizations because their voltage dependence is very hyperpolarized. Na_V1.7 (red) has a more depolarized activation curve and defines the action potential threshold but also contributes to the upstroke. Na_V1.8 (blue) with its very depolarized activation contributes mainly to the action potential upstroke. In patients carrying the IEM mutation Na_V1.7/I848T (right panel), activation of Na_V1.7 is crucially shifted towards negative potentials. This leads to more synchronized activation of Na_V1.7 and other TTX-s Na_Vs (note the vicinity of the red and purple activation curves). This synchronized activity leads to a lowering of the action potential threshold and to a speeding up of the upstroke. It is possible that Na_V1.2 and 1.3 now also contribute to the action potential upstroke in addition to Na_V1.7 and 1.8. This further enhances slope and amplitude of the action potential upstroke. Taken together, action potentials in IEM (I848T) patients are faster and larger and display particular changes, summarized on the right. Comparing the 2 graphs of activation curves (bottom panel), please note the shorter X-axis in the bottom right graph. We do not propose a shift of either the purple or the blue (Na_V1.8) activation curve. IEM, inherited erythromelalgia.