Novel SCN9A Mutations Underlying Extreme Pain Phenotypes: Unexpected Electrophysiological and Clinical Phenotype Correlations

Electrophysiological characterization of L245V in HEK293 cells. A, Left, Representative raw trace of L245V channel currents in response to the pulse protocol shown (activation). Right, Current–voltage relationship of the peak activation currents normalized to cell size (pA/pF) for L245V (red). Averaged WT (black dash) and β₁/β₂ only (blue dash) traces are overlaid. All traces were fitted with a Boltzmann equation (see Materials and Methods), where V_0.5 = −25.2 ± 1.3 mV, k = −4.9 ± 0.4, V_Rev = 74.0 ± 3.1 mV, and I_Max = −398 ± 44.7 pA/pF (n = 16). Comparisons to WT values are shown in Table 1. B, Left, Representative raw trace of L245V currents in response to a test pulse to −10 mV following a series of prepulses (protocol shown; fast inactivation). Right, Peak fast inactivation currents for L245V (red) were normalized to the maximum current and plotted against the prepulse potential used. Averaged WT (black dash) traces are overlaid. L245V traces were fitted with a double Boltzmann equation (see Materials and Methods), where V_0.5#1 = −71.2 ± 0.6 mV and V_0.5#2 = −31.4 ± 1.8 mV (n = 14). Comparisons to WT values are shown in Table 1. C, Left, Representative tail current traces measured at −40 mV from wt, L245V, and A1632E expressing cells without (black) and with (red) addition of β4-peptide in the intracellular pipette solution. The voltage-stimulus protocol is shown below. Right, Compared with WT, the mutations L245V (top) and A1632E (bottom) cause a significant increase (p < 0.05) in tail current, measured as a fraction of the peak activation current at voltages between −80 and −20 mV; there was no significant difference between L245V and A1632E or between the presence and absence of the β4-peptide (two-way repeated-measures ANOVA with Bonferroni post hoc; n = 3–7). D, Remaining current fraction (fraction of maximal peak current) at 50 ms following a series of depolarizing voltage steps from −100 mV to 40 mV. Inset, Averaged raw traces for WT (black) and L245V (red) in response to a single voltage step. *p < 0.05, individual comparisons. p = 0.0026, WT versus L245V group comparison (two-way repeated-measures ANOVA with Bonferroni post hoc; n = 12–16). E, Peak slow-inactivation currents were obtained for L245V (red) and WT (black) by applying an extended conditioning pulse (10 s) ranging from −100 mV to 30 mV before stepping to −120 mV for 100 ms to recover channels from the fast inactivated state followed by a test pulse at −10 mV for 20 ms. Analysis shows peak currents, normalized to maximal current, obtained at the final −10 mV test pulse plotted against the prepulse potential. Data are fitted with a Boltzmann equation (see Materials and Methods) where: for the WT channels, V_0.5 = −31.43 ± 1.8 mV and k = 9.9 ± 0.6; and for the L245V channels, V_0.5 = −41.36 ± 0.9 mV and k = 11.3 ± 1.7 (n = 8–16). Comparisons are shown in Table 1. F, Rate of channel deactivation following a brief depolarizing step to 20 mV (0.4 ms) from a holding potential of −100 mV before going to a final potential ranging from −120 mV to −40 mV. Inset, Averaged raw traces for WT (black) and L245V (red) when a final voltage step to −60 mV is applied. *p < 0.05, individual comparisons. p < 0.0001, WT versus L245V group comparison (two-way repeated-measures ANOVA with Bonferroni post hoc; n = 10–14). G, Analysis of inward currents during a depolarizing ramp stimulus from −140 mV to 70 mV. Inset, Average raw traces for WT (black) and L245V (red) in response to the test pulse applied. *p < 0.05, individual comparisons. p = 0.0266, WT versus L245V group comparison (two-way repeated-measures ANOVA with Bonferroni post hoc; n = 12–15).