Temperature dependence of erythromelalgia mutation L858F in sodium channel Nav1.7

Abstract

Background: The disabling chronic pain syndrome erythromelalgia (also termed erythermalgia) is characterized by attacks of burning pain in the extremities induced by warmth. Pharmacological treatment is often ineffective, but the pain can be alleviated by cooling of the limbs. Inherited erythromelalgia has recently been linked to mutations in the gene SCN9A, which encodes the voltage-gated sodium channel Nav1.7. Nav1.7 is preferentially expressed in most nociceptive DRG neurons and in sympathetic ganglion neurons. It has recently been shown that several disease-causing erythromelalgia mutations alter channel-gating behavior in a manner that increases DRG neuron excitability.

Results: Here we tested the effects of temperature on gating properties of wild type Nav1.7 and mutant L858F channels. Whole-cell voltage-clamp measurements on wild type or L858F channels expressed in HEK293 cells revealed that cooling decreases current density, slows deactivation and increases ramp currents for both mutant and wild type channels. However, cooling differentially shifts the midpoint of steady-state activation in a depolarizing direction for L858F but not for wild type channels.

Conclusion: The cooling-dependent shift of the activation midpoint of L858F to more positive potentials brings the threshold of activation of the mutant channels closer to that of wild type Nav1.7 at lower temperatures, and is likely to contribute to the alleviation of painful symptoms upon cooling in affected limbs in patients with this erythromelalgia mutation.

Figure 1

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 2

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 3

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.

Figure 4

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 5

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.