PMC

(a) Linear schematic of the human Na_V1.7 channel topology showing the mutations S241T, V400M and F1449V. (b) Intra-membrane view of structural model of Na_V1.7 channel transmembrane domains. Domain I, lightblue; Domain II, salmon; Domain III, cyan; Domain IV, lime. (c) Cyotsolic view of the structural model of Na_V1.7 channel transmembrane domains. Boxed area containing S241, V400 and F1449 residues was enlarged in (e). (d) Close-up intra-membrane view of the area containing V400, S241 and F1449 residues. (e) Close-up cytosolic view of the boxed area of (c). V400, S241 and F1449 were shown as stick and colored red, black, and yellow, respectively.
Waxman

(a–d) Representative traces of current families recorded using the activation protocol from HEK293 cells expressing S241T mutant channel treated with DMSO (a), or with CBZ (b); F1449V mutant channel treated with DMSO (c), or with CBZ (d). (e) The averaged voltage-dependence of activation of S241T mutant channel treated with DMSO or CBZ (30 μM) was plotted and fitted with Boltzmann equation. A depolarizing shift of activation of 7.0 mV was observed when S241T mutant channel was treated with CBZ. (f) The averaged voltage-dependence of activation of F1449V mutant channel treated with DMSO or CBZ was plotted and fitted with Boltzmann equation. No notable shift in activation curve of F1449V mutant channel was observed. (g–h) The voltage dependence of fast inactivation of S241T (g) or F1449V (h) mutant channel treated with DMSO or CBZ was plotted and fitted with Bolzmann equation. No notable shift was observed.
Waxman

(a–b) The sub- and supra-threshold responses of representative DRG neurons expressing S241T mutant channel treated with DMSO (a) or 30 μM CBZ (b) are shown. (c) Comparison of current threshold for DRG neurons expressing S241T mutant channel treated with DMSO or 30 μM CBZ. The CBZ treatment increased the current threshold significantly (P<0.01). Current threshold for DMSO treated DRG neurons: 90.4±13.2 pA (n=27); for CBZ treated DRG neurons: 162.7±24.4 pA (n=28). (d–e) The sub- and supra-threshold of DRG neurons expressing F1449V mutant channel treated with DMSO (d) or 30 μM CBZ (e) are shown. (f) Comparison of current threshold for DRG neurons expressing F1449V mutant channel with the treatment of DMSO (153.5±17.9 pA, n=29) or 30 μM CBZ (165.5±19.7 pA, n=28). No significant difference was found (P>0.05).
Waxman

(a) Voltage-dependence of activation curves of Na_V1.7 WT, V400M, S241T, and VM/ST double mutant channels. Curves were Boltzmann fits of the data. (b) Voltage-dependence of activation curves of Na_V1.7 WT, V400M, F1449V, and VM/FV double mutant channels. (c–h) Representative traces of current families recorded from HEK293 cells expressing WT (c), V400M (d), S241T (e), F1449V (f), VM/ST double mutant (g), and VM/FV double mutant (h) channels. (i) Data summary for the mutant cycle analysis and interpretation (n=4–5). ΣΔG° was used to determine the level of additivity and was calculated based on the equation described in methods.
Waxman

(a–c) Responses of a representative DRG neuron expressing S241T mutant channel treated with DMSO to 1sec long depolarization current steps at 100 (a), 200 (b) and 400 (c) pA current injection. (d–f) Similar recording from a representative DRG neuron expressing S241T mutant channel treated with 30 μM CBZ at 100 (d), 300 (e), 400 (f) pA current injection. (g) The averaged response for DRG neurons expressing S241T mutant channel treated with DMSO or CBZ were summarized. CBZ statistically reduced firing frequency starting from 100 pA current injection (P<0.05). (h) Averaged resting membrane potential between DRG neurons expressing S241T mutant channel treated with DMSO or CBZ were not statistically different (DMSO, −55.3±1.4 mV, n=27; CBZ, −54.9±1.3 mV, n=28, P>0.05).
Waxman

(a–c) Responses of a representative DRG neurons expressing F1449V mutant channel treated with DMSO to 1s long depolarization current steps at 200 (a), 350 (b) and 425 (c) pA current injection. (d–f) Similar recording from a representative DRG neuron expressing F1449V mutant channel treated with CBZ at 200 (d), 350 (e), and 425 (f) pA current injection. (g) The averaged response of the firing frequency for DRG neurons expressing F1449V mutant channel treated with DMSO or CBZ were compared and no statistical difference was found across the entire range (P>0.05). (h) Averaged resting membrane potentials between DRG neurons expressing F1449V mutant channel treated with DMSO or CBZ were not statistically different (DMSO, −54.8±1.5 mV, n=29; CBZ, −54.1±1.0 mV, n=28, P>0.05).
Waxman

(a) Representative DRG neuron expressing Na_V1.7 WT channel showed sub-threshold response to 225 pA current injection and subsequent action potential evoked by injection of 230 pA, which was the current threshold for this neuron. (b) Representative DRG neuron expressing Na_V1.7-S241T mutant channel showed sub-threshold response to 55 pA current injection and subsequent action potential evoked by injection of 60 pA. (c) Comparison of current threshold for DRG neurons expressing WT and S241T mutant channels. Expression of S241T channel reduced current threshold significantly (P<0.01). Current threshold for WT: (227.6±36.7 pA, n= 19); for S241T: (83.5±18.2 pA, n=20). (d–f) responses of a representative DRG neuron expressing WT channel to 1s long depolarization current steps at 100 (d), 300 (e) and 400 (f) pA current injection. (g–i) Responses of a representative DRG neuron expressing S241T mutant channel to 1s long depolarization current steps at 100 (g), 300 (h) and 400 (i) pA current injection. The difference in responses is apparent across this range. (j) The averaged number of action potentials between DRG neurons expressing WT and S241T mutant channel was compared. The response of DRG neurons expressing WT channel to current injection was significantly different compared with DRG neurons expressing S241T mutant channel across a range (125–500 pA) of step current injections (P<0.05). (k) Averaged membrane potentials between DRG neurons expressing WT or S241T mutant channel were not statistically different.
Waxman