(a) Pedigree of the family with episodic pain. Genotype of the p.V1184A mutation is indicated. V/A=heterozygous mutation carrier, V/V=wild type. Whole-exome sequencing was performed in individuals III.2 and III.4 (marked with ‘E') (b) Confirmation of the SCN11A mutation by Sanger sequencing. (c) Membrane topology of the α subunit of Na_V1.9, encoded by SCN11A. The four homologous domains (DI-DIV) are indicated; position of mutations p.L811P (blue) causing pain insensitivity and p.V1184A (red) causing cold-aggravated peripheral pain is highlighted. (d) Reduced epidermal nerve fiber density in the father of the patient. Arrow: PGP9.5-immunoreactive epidermal nerve fiber. (Scale bar, 20 μm). (e) Stacks of Schwann cell processes containing only few unmyelinated axons. (Scale bar, 1 μm). (f) Autophagic vacuole in an unmyelinated axon. (Scale bar, 500 nm). (g) Small dermal nerve fascicle containing myelinated and unmyelinated axons. Arrow: abnormal vacuoles in an endoneurial Schwann cell. (Scale bar, 2 μm). (h) At higher magnification, autophagic material is found in one of the vacuoles (arrow). Another vacuole (arrowheads) is lined by ribosomes and connected to the nuclear envelope. (Scale bar, 1 μm). (i) Prominent rough endoplasmic reticulum, multivesicular bodies (white arrowheads) and prominent osmiophilic structures, probably lysosomes (black arrowheads) in an endoneurial Schwann cell. (Scale bar, 1 μm).

(a) Representative whole-cell current traces recorded at 20 °C from a ND7/23 cell transiently expressing Na_V1.9 (black) or Na_V1.9-V1184A (red) channels in response to depolarizing voltages ranging from –107 to –17 mV in steps of 10 mV. (b,c) Mean current densities of ND7/23 cells expressing either Na_V1.9 (black) or Na_V1.9-V1184A (red) measured at 30 °C (b) or 20 °C (c) as a function of voltage. Superimposed fits describe the voltage dependence of channel activation according to . (d) Representative whole-cell tail currents recorded at 20 °C from a ND7/23 cell transiently expressing Na_V1.9 (black) or Na_V1.9-V1184A (red) channels in response to a hyperpolarizing voltage step from –47 to –87 mV. (e,f) Single-exponential time constants of channel activation (τ_m, squares) and deactivation (τ_d, circles) obtained at 30 °C (e) or 20 °C (f) from experiments as shown in a and d, plotted as function of voltage. Superimposed curves are data fits according to characterizing the voltage dependence of activation and deactivation kinetics of Na_V1.9 and Na_V1.9-V1184A. Data points represent mean values with numbers of experimental replicates indicated in parentheses. Error bars in all plots represent s.e.m.

(a,b) Single-exponential time constants of fast inactivation of Na_V1.9 (black) and Na_V1.9-V1184A (red) channels obtained from experiments as shown in at 30 °C (a) or 20 °C (b) plotted as a function of voltage. Straight lines connect data points for clarity. (c,d) Voltage dependence of steady-state fast inactivation of Na_V1.9 (black) and Na_V1.9-V1184A (red) channels at 30 °C (c) or 20 °C (d), analysed according to . Data points represent mean values with numbers of experimental replicates indicated in parentheses. Error bars in all plots represent s.e.m.

Representative single action potentials at 30 °C (a) or 20 °C (b), elicited in murine DRG neurons transfected with wild-type Na_V1.9 (black) or mutant Na_V1.9-V1184A (red) in response to current injections of 100–200 pA for 5 ms (a) or 10 ms (b). Dotted lines indicate 0 mV; dashed lines mark levels of the RMP. (c) Parameters characterizing action potential properties. V_th: action potential voltage threshold, V_max: maximum action potential voltage, V_min: minimum voltage during after-hyperpolarization, width: action potential width at 0 mV, τ_AHP: single-exponential time constant characterizing the relaxation of the membrane voltage from the action potential after-hyperpolarization (V_min) back to the resting level. Data points represent mean values with numbers of experimental replicates from four (30 °C) or five (20 °C) animals indicated in parentheses. Error bars in all plots represent SEM. Significance between pairs of data was tested with a two-sided Student's t-test. ***P<0.001, *P<0.05.

(a) Representative trains of action potentials at 30 °C, recorded from murine DRG neurons transfected with wild-type Na_V1.9 (black) or mutant Na_V1.9-V1184A (red), in response to 2 s current injections of 60 pA. Dotted lines mark 0 mV; dashed lines indicate the RMP. (b) Action potential frequencies obtained from experiments as shown in a as a function of the injected current. (c,d) Equivalent set of experiments as shown in a and b, performed at 20 °C. Data points represent mean values obtained from four (30 °C) or five (20 °C) animals with numbers of experimental replicates indicated in parentheses. Significance between pairs of data was tested with a two-sided Student's t-test. ***P<0.001, **P<0.01, *P<0.05.

(a) Representative trains of action potentials at 30 °C, recorded from murine DRG neurons transfected with Na_V1.9 (black), Na_V1.9-V1184A (red) or Na_V1.9-L811P (blue), in response to 30 s current injections of 40 pA. Dotted lines mark 0 mV; dashed lines indicate the RMP. (b) Trains of action potentials as shown in a, obtained from repetitively firing neurons, were analysed to determine the fraction of neurons that maintained stable action potential amplitudes over 30 s. The criterion for ‘persistent firing' was defined as a reduction of peak action potential amplitude (V_max) by <10 mV throughout the train. Numbers of experimental replicates are given in parentheses. Significance was tested with a Z-test. **P<0.01.

(a) Representative whole-cell current traces recorded at −37 mV from ND7/23 cells transiently expressing Na_V1.9 (black), Na_V1.9-V1184A (red) or Na_V1.9-L811P (blue). Fast inactivation of Na_V1.9 is not affected by mutation p.V1184A while mutation p.L811P slows down channel inactivation, thus prolonging channel opening. (b) Boltzmann functions describing the voltage dependences of opening (Γ/Γ_max) and inactivation (I₅₀₀/I₀) of wild-type Na_V1.9 (black) and mutants L811P (blue) and V1184A (red). Coloured areas indicate overlap between activation and inactivation curves, which is expected to result in a pro-excitatory window current; this window current is largest for mutation p.L811P followed by p.V1184A and wild-type Na_V1.9. (c) Current traces as shown in a were analysed to determine the fraction of channels not inactivated 80 ms after triggering channel activation with various depolarizing voltage steps. Notably, fast inactivation of mutant p.L811P (n=11) is impaired in the entire voltage range while mutation p.V1184A (n=9) does not affect fast inactivation (n=10 for Na_V1.9). Data points represent mean values with error bars indicating s.e.m. Data from Na_V1.9-L811P as in Leipold et al. (2013).