a Structure of HwTxIV-Nvoc with K³² and Nvoc group reported in blue and orange sticks respectively. HwTxIV structure has been isolated from PDB 7K48. The sequence chosen for caging is shown upon sequence alignment with nHwTxIV. b Illustration of steric van der Walls clashes (red cylinders) resulting from the addition of the Nvoc protecting group onto K³² upon binding of HwTxIV onto the chimeric Na_v1.7-Na_vAb in the position as published³⁰ (PDB code 7K48). c RP-HPLC elution profiles of crude, crude folded/oxidized, and purified folded/oxidized HwTxIV-Nvoc. RP-HPLC elution profile of purified folded/oxidized non-caged HwTxIV has been superimposed for comparison. d LC-ESI QTOF analysis of HwTxIV-Nvoc denotes an exact mass of 722.1785 [M + 6H]⁶⁺. e Superimposition of TOCSY spectra (sidechains area only for clarity) of the non-caged HwTxIV (in black), and of HwTxIV-Nvoc before illumination (in orange). f Residues showing major chemical shifts modifications in the presence of Nvoc are reported in orange on the surface of the molecule (180°views). K³² is highlighted in blue. The Nvoc group is reported in sticks. Source data are provided as a Source Data File.

a i) Representative recording of hNa_V1.6 currents elicited at 10 mV from a holding potential of -100 mV illustrating the extent of current block by different concentrations of noncaged HwTxIV (black) and HwTxIV-Nvoc (orange). ii) Average dose-response curves for hNa_V1.6 current inhibition by non-caged HwTxIV (black) and HwTxIV-Nvoc (orange). The data were fitted according to a Hill equation (non-caged HwTxIV IC₅₀ = 22.4 nM (n = 5) and HwTxIV-Nvoc IC₅₀ = 6539 nM (n = 7) (292-fold reduction in IC₅₀)). Scales: 2 ms, 1 nA. Note that on hNa_v1.6, high concentrations of HwTxIV-Nvoc produce a slowing of channel inactivation due to binding on a low-affinity site absent on hNa_v1.2 (Supplementary Figure ³). b i) Representative recording of hNa_V1.1 current elicited at 10 mV from a holding potential of -100 mV illustrating the extent of current block by different concentrations of non-caged HwTxIV (black) and HwTxIV-Nvoc (orange). ii) Average dose-response curves for hNa_v1.1 current inhibition by noncaged HwTxIV (black) and HwTxIV-Nvoc (orange). The data were fitted according to a Hill equation (noncaged HwTxIV IC₅₀ = 3.60 nM (n = 12); HwTxIV-Nvoc IC₅₀ = 2794 nM (n = 8) (776-fold reduction in IC₅₀). Scales: 2 ms, 1 nA. c i) Representative recording of hNa_V1.2 current elicited at 10 mV from a holding potential of -100 mV illustrating the extent of current block by different concentrations of noncaged HwTxIV (black) and HwTxIV-Nvoc (orange). Scales: 2 ms, 1 nA. ii) Average dose-response curves for hNa_v1.2 current inhibitions by non-caged HwTxIV (black) and HwTxIV-Nvoc (orange). The data were fitted according to a Hill equation (noncaged HwTxIV IC₅₀ = 3.23 nM (n = 11); HwTxIV-Nvoc IC₅₀ = 5141 nM (n = 6) (1592-fold reduction in IC₅₀)). d i) Representative recording of hNa_V1.7 current elicited at 10 mV from a holding potential of -100 mV illustrating the extent of current block by different concentrations of non-caged HwTxIV (black) and HwTxIV-Nvoc (orange). Scales: 2 ms, 0.5 nA. ii) Average dose-response curves for hNa_v1.7 current inhibitions by non-caged HwTxIV (black) and HwTxIV-Nvoc (orange). The data were fitted according to a Hill equation (non-caged HwTxIV IC₅₀ = 15.23 nM (n = 92 cells); HwTxIV-Nvoc IC₅₀ = 4467 nM (n = 83 cells) (293-fold reduction in IC₅₀)). All data are presented as mean ± SEM. Source data are provided as a Source Data File.

a Top: Analytical RP-HPLC profiles of HwTxIV-Nvoc after different durations of illuminations (365 nm, 45 mW/cm²) demonstrating time-dependent control of uncaging. Bottom: Uncaged/Caged ratio of HPLC chromatogram peaks area versus irradiation time at 365 nm (45 mW/cm²). b Top: Analytical RP-HPLC profiles of HwTxIV-Nvoc after different intensities of illuminations (365 nm, 5 min) demonstrating intensity-dependent control of uncaging. Bottom: Uncaged/Caged ratio of HPLC chromatogram peaks area versus irradiation power at 365 nm (3 min). c Analytical RP-HPLC profiles of non-caged HwTxIV (top), 50:50 ratio of noncaged HwTxIV after partial uncaging of HwTxIV-Nvoc (middle) and of HwTxIV-Nvoc (bottom). d Mass analyses of noncaged HwTxIV and uncaged HwTxIV by LC-ESI QTOF ([M + 6H]⁶⁺ values). e Superimposition of TOCSY spectra (same area as Fig. 1e) of the noncaged HwTxIV (in black), and of the uncaged HwTxIV after illumination of HwTxIV-Nvoc (in purple). f Representative recording of hNa_V1.6 currents elicited at 10 mV from a holding potential of -100 mV illustrating the extent of current block by different concentrations of non-caged HwTxIV (black) and uncaged HwTxIV (purple). Scales: 2 ms, 1 nA. g Average dose-response curves for hNa_v1.6 current inhibitions by non-caged HwTxIV (black) and uncaged HwTxIV (purple). The data were fitted according to a Hill equation (noncaged HwTxIV IC₅₀ = 22.4 nM (n = 5) and uncaged HwTxIV IC₅₀ = 15.20 nM (n = 6)). Data are presented as mean ± SEM. Source data are provided as a Source Data File.

a–c Light-induced inhibition of hNa_V1.6 current by photolysis of 100 nM HwTxIV-Nvoc. Note that not all of the HwTxIV-Nvoc is uncaged by photolysis during this exposure time. a Representative recordings of hNa_V1.6 current with caged HwTxIV (orange) and after various illumination times (purple) and (b) average normalized time courses of hNa_V1.6 current inhibition during and following light application (n = 11, mean ± SEM). Scale: 2 ms, 1 nA. c Average normalized current at steady-state in control (dark), caged (orange) and uncaged (purple) conditions (n = 11, ***p < 0.001, two-sided repeated-measures 1-way ANOVA followed by Bonferroni’s post-test.). d Representative recordings of hNa_V1.2 current with caged HwTxIV (orange) and after illumination (purple). Scale: 2 ms, 1 nA. e Average normalized current at steady-state in control (dark), caged (orange), and uncaged (purple) conditions (n = 12, mean ± SEM ***p < 0.001, two-sided repeated-measures 1-way ANOVA followed by Bonferroni’s post-test). f–g Controllable uncaging of HwTxIV-Nvoc via UV irradiation. f Left: Superimposition of normalized hNa_V1.6 currents at steady-state recorded after varying durations of illumination. Middle: Average normalized time courses of hNa_V1.6 current inhibition (n = 40 cells, mean ± SEM). Arrow indicates steady-state. Right: Plot of current amplitude at the end of 800 s recording versus duration of illumination at 365 nm. Scale: 2 ms, 20% of amplitude. g Left: Superimposition of normalized hNa_v1.6 currents at steady-state recorded after varying power of illumination (4 min). Middle: Average normalized time courses of hNa_v1.6 current inhibition (n = 43 cells, mean ± SEM). Arrow indicates steady-state. Right: Plot of current amplitude at the end of 800 s recordings versus duration of illumination at 365 nm. Scale: 2 ms, 20% of amplitude. h Representative examples of photocontrol (365 nm, 45 mW/cm²) of 1 nM AaHII-R⁶²K-Nvoc activity on hNa_V1.2 current (left, AaHII PDB code 6NT4⁸), 100 nM BeKm1-Nvoc activity on hERG current (middle, BeKm-1 PDB code 1J5J⁴⁰) and 100 nM charybdotoxin-Nvoc activity on K_V1.2 current (right, ChTx PDB code 4JTA⁴¹). AaHII-R⁶²K toxin induces slowing of inactivation of hNa_V1.2, while BeKm1 and charybdotoxin induce block of hERG and K_V1.2 channels, respectively. For AaHII-R⁶²K-Nvoc and BeKm1-Nvoc, scale is 2 ms and 1 nA. For charybdotoxin-Nvoc, scale is 400 ms and 200 pA. Source data are provided as a Source Data File.

a Inhibition of AP by 500 nM non-caged HwTxIV (n = 6). b AP in control condition, in presence of 2.5 µM HwTxIV-Nvoc and 1 min after uncaging. c Left: mean ± SEM (n = 8 cells) of normalized AP peak after addition of HwTxIV-Nvoc and 1 min after photolysis (*p = 0.0078, two-sided paired t test). Right: mean ± SEM (n = 5 cells) of normalized AP width after addition of HwTxIV-Nvoc. d APs 1 min after HwTxIV-Nvoc uncaging over a spot centered ~100 µm from the cell body or over the soma. e Mean ± SEM (n = 4 cells) of the normalized AP peak after uncaging 100 µm away from the soma or directly on the soma. (*p = 0.0005, two-sided paired t test). f Image of the ~40 µm diameter UV spot. Purple curve represents the light intensity profile on cell schematic. g Top: images of a L5 pyramidal neuron relative to UV (405 nm) illumination spot position. Bottom: AP in the presence of HwTxIV-Nvoc (orange traces) and 1 min after uncaging (purple traces) with cell positioned at 100, 80, 60, 40, 20, and 0 µm distance from spot. h Mean ± SEM (n = 5 cells) of normalized AP peak 1 min after uncaging with the cell positioned at decreasing distances from spot center (100 µm; 80 µm, p = 0.2302; 60 µm, *p = 0.0434; 40 µm, *p = 0.0420; 20 µm, *p = 0.0133; 0 µm, ***p < 0.0001). Two-sided paired t-test. i Left, L5 pyramidal neuron filled with 500 µM of ING-2. AIS area of Δ[Na⁺] measurement in red cylinder. Right, images before and during UV uncaging pulse illustrating photolysis area. j Left, somatic AP (top) and associated Δ[Na⁺] signal in the presence of HwTxIV-Nvoc. Right, after toxin uncaging, the cell was depolarized to +20 mV to correspond to control AP peak and measure Δ[Na⁺] signal in the same condition. k mean ± SEM (n = 4 cells) of the Δ[Na⁺] signal maximum (peak) before and after uncaging the toxin. (*p < 0.0174, two-sided paired t-test). Source data are provided as a Source Data File.

a Representative trajectory plots of mice injected with vehicle (left), non-caged HwTxIV (center) or HwTxIV-Nvoc (right). b–c Quantification of total movements (b) and rearing (c) in mice injected with vehicle (white circles), HwTxIV (black circles) or HwTxIV-Nvoc (orange circles). (n = 5 in each group, mean ± SEM *p < 0.05; **p < 0.01; ***p < 0.001 versus vehicle, two-sided repeated-measures 2-way ANOVA test followed by Tukey’s post test). d Representative twitches obtained from mice injected with vehicle or HwTxIV-Nvoc at t = 0; t = 5; t = 10; t = 15; t = 20 min after injection. e EDL twitch force normalized to muscle mass (g/mg of EDL) in vehicle or HwTxIV-Nvoc mice before and after illumination (365 nm, 50 mW/cm²). (n = 5 in each group, mean ± SEM **p = 0.0041; ***p = 0.0002 versus t0, two-sided repeated-measures Friedman test followed by Dunn’s post-test). Source data are provided as a Source Data File.