## Results: 5

Figure 2. From: Temperature dependence of erythromelalgia mutation L858F in sodium channel Nav1.7.

**Activation midpoint shifts to more depolarized potentials upon cooling for L858F, but not for Nav1.7**.

**A**. Voltage dependences of conductance for Nav1.7 at 16°C (filled squares, n = 12), 25°C (filled triangles, n = 13) and 35°C (filled circles, n = 12). Conductance curves were derived from current-voltage families, normalized, and fitted with a Boltzmann equation, as described in methods.

**B**. Voltage dependences of conductance for L858F at 16°C (open squares, n = 8), 25°C (open triangles, n = 15) and 35°C (open circles, n = 14).

**C**. Midpoint of activation for Nav1.7 (black bars) and L858F (white bars) plotted versus temperature.

**D**. Slope factor of activation for Nav1.7 (black bars) and L858F (white bars) plotted versus temperature. * indicate significant differences between values with p < 0.05, tested with ANOVA and Tukey HSD post hoc analysis.

Figure 5. From: Temperature dependence of erythromelalgia mutation L858F in sodium channel Nav1.7.

**Cooling increases deactivation time constants for Nav1.7 and L858F**.

**A**. Nav1.7 deactivates more slowly at -50 mV, -45 mV and -40 mV when temperatures are lowered from 35°C (filled circles, n = 8) to 25°C (filled triangles, n = 8) and 16°C (filled squares, n = 6). Deactivation time constants were obtained by a single exponential fit of tail currents elicited by repolarization to the indicated potentials from a brief depolarization of 0.5 ms to -20 mV.

**B**. L858F deactivates more slowly at potentials ranging from -55 mV to -40 mV when temperatures are lowered from 35°C (open circles, n = 7) to 25°C (open triangles, n = 7) and 16°C (open squares, n = 6). * indicate significant differences to the values at 35°C with p < 0.05, tested with ANOVA and Tukey HSD post hoc analysis.

Figure 4. From: Temperature dependence of erythromelalgia mutation L858F in sodium channel Nav1.7.

**Cooling increases currents elicited by slow ramp depolarizations, and diminishes the difference between Nav1.7 and L858F**. Representative current traces of Nav1.7 (

**A.**) and L858F (

**B.**) ramp currents at 16°C, 25°C and 35°C. Cells were held at -120 mV and stimulated with a depolarizing voltage ramp that increased to 20 mV within 600 ms.

**C**. The bar graph shows the mean peak currents recorded during the voltage ramps expressed as percent of transient peak current obtained during initial I-V families, 3 min after breaking into the cell; black bars represent Nav1.7 at 16°C (n = 8), 25°C (n = 9) and 35°C (n = 7); white bars represent L858F at 16°C (n = 8), 25°C (n = 9) and 35°C (n = 8). * indicate significant differences between values with p < 0.05, tested with ANOVA and Tukey HSD post hoc analysis.

Figure 1. From: Temperature dependence of erythromelalgia mutation L858F in sodium channel Nav1.7.

**Cooling decreases current density for Nav1.7 and L858F**.

**A.**Representative current-voltage (I-V) families recorded from HEK293 cells stably expressing Nav1.7 (left column) or the mutation L858F (right column) at 16°C, 25°C or 35°C. Cells were held at -120 mV and depolarizing steps were applied to membrane potentials ranging from -80 mV to 40 mV in 5 mV steps.

**B**. Temperature dependence of the current density for Nav1.7 (black bars, n = 17, 15, 25) and L858F (white bars, n = 15, 16, 28) at the indicated temperatures. Current density was measured as peak current divided by cell capacitance. * indicate significant differences between values with p < 0.05, tested with ANOVA and Tukey HSD post hoc analysis.

Figure 3. From: Temperature dependence of erythromelalgia mutation L858F in sodium channel Nav1.7.

**Steady-state fast inactivation changes in a similar way for Nav1.7 and L858F**.

**A**. Voltage dependences of steady-state fast inactivation for Nav1.7 at 16°C (filled squares, n = 12), 25°C (filled triangles, n = 11) and 35°C (filled circles, n = 9). Availability was assessed using a 500 ms prepulse ranging from -150 mV to 0 mV followed by a 40 ms test pulse to -20 mV. The current was normalized to the largest current response evoked by the test pulse. Steady-state inactivation curves were fitted with a Boltzmann equation, as described in methods.

**B**. Voltage dependences of steady-state fast inactivation for L858F at 16°C (open squares, n = 9), 25°C (open triangles, n = 11) and 35°C (open circles, n = 10).

**C**. Temperature dependence of the midpoint of steady-state inactivation for Nav1.7 (black bars) and L858F (white bars) at the indicated temperatures.

**D**. The slope factor of steady-state inactivation for Nav1.7 (black bars) and L858F (white bars) plotted versus temperature. * indicate significant differences between values with p < 0.05, tested with ANOVA and Tukey HSD post hoc analysis.