L858H renders DRG neurons hyperexcitable. (A and B) Action potentials were evoked from small (≤25 μm in diameter) DRG neurons by using depolarizing current injections from the RMP. V_m, membrane potential. (A) Representative traces from a cell expressing WT Na_v1.7 show subthreshold responses to 50- to 130-pA current injections and subsequent all-or-none action potentials evoked by injections of 135 pA (current threshold for this neuron) and 155 pA. (B) In contrast, in a cell expressing L858H, action potentials were evoked by a 60-pA current injection. The voltage for take-off of the all-or-none action potential (approximately −14.5 mV, dashed line) was similar for the neurons in A and B. (C) L858H causes a depolarizing shift in the RMP of DRG neurons. DRG neurons expressing WT Na_v1.7 had an average RMP of −50.1 ± 0.9 mV (n = 20), whereas those expressing L858H mutant channels had a significantly (∗, P < 0.001) depolarized RMP of −44.9 ± 1.1 (n = 25). (D) The average current threshold for action potential firing of DRG neurons expressing WT Na_v1.7 channels was 120.6 ± 23.9 pA (n = 20), whereas that of neurons expressing L858H mutant channels was significantly (∗, P < 0.01) reduced to 69.2 ± 9.8 pA (n = 25). (E) Action potential overshoot in cells expressing WT Na_v1.7 channels (67.8 ± 3.0 mV, n = 20) was not significantly different from that in cells expressing L858H mutant channels (64.4 ± 2.6 mV, n = 20; P > 0.05). The voltage of action potential take-off was unchanged (WT, −14.5 ± 1.2 mV, n = 20; L858H, −14.5 ± 1.3 mV, n = 25; P > 0.05). n.s., not significant.

L858H renders SCG neurons hypoexcitable. (A and B) Action potentials were evoked by using depolarizing current injections from resting potential. (A) Representative traces from a cell expressing the WT channel show subthreshold responses to 15- to 20-pA current injections and subsequent all-or-none action potentials evoked by injections of ≥25 pA. (B) In contrast, in a cell expressing the L858H channel, action potentials required a ≥70-pA current injection. The voltage for take-off (dashed line) of the all-or-none action potential was unchanged. (C) L858H channels caused a depolarizing shift in the RMP of SCG neurons. SCG neurons expressing WT channels had an average RMP of −46.3 ± 0.8 mV (n = 15), whereas those expressing L858H had a significantly (P < 0.001) depolarized RMP of −41.6 ± 0.8 (n = 17). (D) The average current threshold for action potential firing of SCG neurons expressing WT channels was 22.7 ± 3.6 pA (n = 15), whereas that of neurons expressing L858H channels was significantly (∗, P < 0.01) increased to 42.9 ± 6.3 pA (n = 17). (E) Action potential overshoot in cells expressing WT channels (47.8 ± 3.4 mV, n = 15) was significantly larger (∗, P < 0.001) than that in cells expressing L858H (23.8 ± 4.7 mV, n = 20). The voltage of action potential take-off was unchanged (WT, −23.1 ± 1.2 mV, n = 15; L858H, −19.8 ± 1.3 mV, n = 17; ∗, P > 0.05).

The L858H mutation increases firing frequency in DRG and decreases firing frequency in SCG neurons. (A) Representative DRG neuron expressing WT Na_v1.7 fires a single action potential in response to a 950-ms input of 100 pA from the RMP of this neuron (approximately −50 mV). (Inset) The same neuron fires multiple action potentials in response to a 250-pA stimulus. (B) Representative DRG neuron expressing L858H fires five action potentials in response to a 100-pA current injection from the RMP of this neuron (approximately −42 mV). (C) For the entire population of DRG neurons studied, the firing frequency evoked by 50-pA current stimuli was 0.32 ± 0.13 Hz after transfection with WT channels (n = 20) and 2.06 ± 0.79 Hz after transfection with L858H (n = 24; ∗, P < 0.05), and the firing frequency evoked by 100-pA stimuli was 0.89 ± 0.28 Hz after transfection with WT and 3.37 ± 1.13 Hz after transfection with L858H (∗, P < 0.05). (D) Representative SCG neuron expressing WT Na_v1.7 fires six action potentials in response to a 950-ms input of 40 pA from the RMP (approximately −45 mV). (E) Representative SCG neuron expressing L858H fires only two action potentials in response to a 100-pA current injection from the RMP (approximately −40 mV). (Inset) When the cell was held at −60 mV to overcome the depolarization of the RMP caused by L858H, it produced four action potentials with an identical stimulus. (F) For the entire population of SCG neurons studied, the firing frequency evoked by 30-pA stimuli was 5.33 ± 1.5 Hz after transfection with WT channels (n = 14) and 0.63 ± 0.01 Hz after transfection with L858H channels (n = 15; P < 0.05). The firing frequency evoked by 40-pA stimuli was 7.05 ± 1.86 Hz after transfection with WT and 1.96 ± 1.0 Hz after transfection with L858H channels (∗, P < 0.05).

DRG neurons express Na_v1.7 and Na_v1.8; SCG neurons express Na_v1.7 but not Na_v1.8. (A) Restriction analysis of multiplex PCR amplification products from sodium channel domain 1 from adult DRG (lanes 1–9) and SCG (lanes 10–18). M, 100-bp ladder marker (Promega). Lanes 1 and 10 contain amplification products from DRG and SCG, respectively. Lanes 2–9 and 11–18 show results of cutting this DNA with EcoRV, EcoNI, AvaI, AccI, SphI, BamHI, AflII, and EcoRI, which are specific to subunits Na_v1.1, Na_v1.2, Na_v1.3, Na_v1.5/1.9, Na_v1.6, Na_v1.7/1.8, Na_v1.8, and Na_v1.9 (details can be found in Table 1, which is published as supporting information on the PNAS web site). Restriction products in lanes 2 and 5–9 show the presence of Na_v1.1, Na_v1.6, Na_v1.7, Na_v1.8, and Na_v1.9 in DRG, in agreement with previous results (19). Restriction products in lanes 13, 15, and 16 show the presence of Na_v1.3, Na_v1.6, and Na_v1.7 in SCG. (B and C) Immunostaining of Na_v1.7 and Na_v1.8 channels in DRG and SCG neurons in vivo and in cultured neurons. (B) Na_v1.7 (a) and Na_v1.8 (b) proteins are present in adult DRG neurons in vivo; Na_v1.7 (c) and Na_v1.8 (d) proteins are present in cultured DRG neurons from postnatal day 2 (P2) rat pups. (C) Na_v1.7 (a), but not Na_v1.8 (b), protein is present in adult SCG neurons in vivo; Na_v1.7 (c), but not Na_v1.8 (d), protein is present in cultured SCG neurons from P2 rat pups. (Scale bars, 50 μm.)

Coexpression of L858H and Na_v1.8 channels rescues electrogenic properties in SCG neurons. When Na_v1.8 was coexpressed with L858H, current threshold and action potential overshoot were restored, although the depolarization of the RMP induced by L858H persisted. (A) Suprathreshold action potentials recorded from representative SCG neurons transfected with WT (blue), L858H (red), and L858H plus Na_v1.8 (green) channels. (B) Depolarized RMP in cells with L858H channels (−41.6 ± 0.76 mV, n = 17) was maintained with coexpression of Na_v1.8 (−40.5 ± 1.01 mV, n = 17; P > 0.05). n.s., not significant. (C) Current threshold for action potential firing was reduced from 42.9 ± 6.3 pA (n = 17) for L858H to 26.8 ± 4.3 pA (n = 17) for L858H coexpressed with Na_v1.8 (∗, P < 0.05). (D) Action potential overshoot in SCG neurons with L858H channel (23.8 ± 4.7 mV, n = 17) was increased when Na_v1.8 was coexpressed with L858H (41.5 ± 4.6 mV, n = 17; ∗, P < 0.05).