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. 2014 Sep 4;9(9):e105895. doi: 10.1371/journal.pone.0105895

Figure 7. Nav1.7 KOs show minimal pain behaviors upon injection of veratridine or grayanotoxin III.

Figure 7

A. hNav1.7 currents recorded from HEK 293 cells in control (left) and after addition of 30 µM veratridine (right). Currents were evoked by a family (traces overlaid) of depolarizing voltage pulses incremented by +10 mV from a holding voltage of −100 mV, with repolarization to −80 mV followed by return to the holding voltage of −100 mV. Inward currents were evoked by depolarizations to −50 mV and more positive. Note the prolonged opening following repolarization in veratridine. Scale bars, 500 pA and 10 ms. B. Nav1.7 currents recorded with 300 µM grayanotoxin III in the internal (pipette) solution. Currents shown at left are in response to a family (traces overlaid) of step depolarizations in +5 mV increments from −120 mV to −50 mV, followed by repolarization to −120 mV. Depolarizations to −95 mV and more positive evoked inward currents, and these currents showed little or no inactivation during the test pulse. Dotted line marks the zero current level (I = 0). Currents shown at right are records from the same cell, after switching the holding voltage to −80 mV. Note that the holding current increased despite the decreased driving force (compare current at −80 mV to the dashed line marking the holding current at −120 mV), presumably reflecting steady influx of sodium ions through grayanotoxin-modified Nav1.7. Step depolarizations from −80 mV to −40 mV at +10 mV increments evoked additional currents with slow inactivation and deactivation kinetics. Scale bars, 500 pA and 20 ms. C. Licking and lifting in male CD-1 mice in response to increasing intraplantar doses of veratridine. At doses of 1 µg and 10 µg, veratridine caused a statistically significant increase in paw licking and lifting behaviors compared to saline or vehicle (1% ethanol/99% PBS) controls. The licking and lifting caused by 1 µg veratridine was completely prevented by pre-dosing the animals with mexiletine (MEX; 30 mg/kg, i.p. or p.o.). A separate animal cohort was used for each dose. D. Licking and lifting in male CD-1 mice in response to increasing intraplantar doses of grayanotoxin III. At doses of 0.1 µg and 1 µg, grayanotoxin III caused a statistically significant increase in paw licking and lifting behaviors compared to saline or vehicle (1% ethanol/99% PBS) controls. A separate animal cohort was used for each dose. E. In a separate experiment, the licking and lifting induced by grayanotoxin III was prevented by pre-dosing the animals with mexiletine (MEX; 30 mg/kg, i.p. or p.o.). F. Total paw licking and lifting behavior time in Nav1.7 KO (n = 5) and WT/HET littermates (n = 7) mice in the 20 minutes following i.pl. injection of 1 µg veratridine. Responses from KO (44.4 sec ±26.6 sec) were smaller than from WT/HET (292.1 sec ±34.7 sec) (p = 0.0073) (mean ± S.E.M., homogeneous ANOVA model with Tukey-Kramer adjusted t-test). G. Total painful paw lifting and licking behavior time in Nav1.7 KO (n = 3) and WT/HET littermates (n = 7) in the 15 minutes following i.pl. injection of 0.1 µg grayanotoxin III. Responses from KO (7.33 sec ±3.5 sec) were smaller than from WT/HET (170.9 sec ±23.1 sec) (p = 0.0003) (mean ± S.E.M., heterogeneous ANOVA model with Welch’s test).