SCN5A mutation G615E results in NaV1.5 voltage-gated sodium channels with normal voltage-dependent function yet loss of mechanosensitivity

Figures

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  Figure 1.. Shear sensitivity of voltage-gating of G615E NaV1.5 compared to WT NaV1.5. (a, c), Na+ current traces from wild-type (WT, a) or G615E (c) NaV1.5 channels elicited by a voltage step from −120 to −50 mV, before (–) or during shear stress (▬) by flow of extracellular solution at 10 mL/min. Insets show the current traces at peak. (b,d), Normalized peak current densities (IPEAK) of WT (b) or G615E (d) Na+ currents before (●) or during (○) shear stress. (e-f), Voltage of half-activation (V1/2A) during control or shear stress (e) and the average shift in V1/2A with shear (f, ΔV1/2A) for WT (black) and G615E (red). (g-h), Peak conductance (GMAX) during control or shear stress (g) and the average change in GMAX with shear (h, ΔGMAX) for WT (black) or G615E (red). (b,d,e,g), n = 12 cells each, *P < 0.05 shear vs. control and P < 0.05 interaction between genotype and shear by a three-way ANOVA with Tukey post-test. (f,h), n = 12 cells each, *P < 0.05% to 0% by a two-tailed one-sample t-test; †P< 0.05 to WT by a two-tailed unpaired t-test.

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  Figure 2.. Shear sensitivity of voltage-dependent gating kinetics of G615E NaV1.5 compared to WT NaV1.5. (a-f), Time constants of activation (a, d; τA) and two inactivation components (b-c, e-f) from wild-type (a-c, WT) or G615E (d-f) NaV1.5 currents, before (●) or during shear stress (○) by flow of extracellular solution at 10 mL/min [n = 12 cells each; *P < 0.05 shear stress vs. baseline controls and P < 0.05 interaction between voltage and shear (τA) or between genotype and shear (τF, τS) by a three-way ANOVA with Tukey post-test; †P< 0.05 to WT by a two-tailed unpaired t-test]. (g-i), Average change in time constants of activation (g) and two inactivation components (h-i) of WT (black) or G615E (red) NaV1.5 currents at −30 mV (n = 12 cells each; *P < 0.05% to 0% by a two-tailed one-sample t-test; †P < 0.05 to WT by a two-tailed unpaired t-test).

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  Figure 3.. Pressure sensitivity of WT NaV1.5 compared to G615E NaV1.5. (a-b), Voltage-clamp (a) and pressure-clamp (b) protocols were used to elicit macroscopic Na+ currents. Pressure was off during voltage step 1 (vehicle) and on during step 2 (pressure). (c-d), Representative macroscopic patch currents in HEK293 cells transfected with WT- (c) or G615E- NaV1.5 (d), elicited by depolarizations to −60, −50, −40, or −30 mV during voltage step 2 with the pressure indicated by the color gradient defined in (b). (e-f), Steady-state activation curves of macroscopic patch currents for WT (e) or G615E-NaV1.5 (f) during voltage step 2 with the pressure indicated by the color gradient. P < 0.05 effect of pressure or voltage for WT- (d) and G615E-NaV1.5 (e) by a two-way ANOVA with Dunnett post-test. (g), Change in voltage of half-activation (ΔV1/2A) from WT (black) or G615E- NaV1.5 (red), the difference between V1/2A during voltage step 2 and V1/2A during step 1, plotted as a function of the pressure during step 2. P < 0.05 effect of pressure and P > 0.05 effect of genotype by a three-way ANOVA with Tukey post-test. (h), Change in peak current (ΔIPEAK) from WT (black) or G615E- NaV1.5 (red), the %…

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  Figure 4.. Mechanosensitivity of WT and G615E NaV1.5-induced spontaneous electrical excitability. (a-b), Gap-free current clamp recording of spontaneous potentials from an HEK293 cell expressing WT NaV1.5 (a) or G615E (b), before, during (underline), or 35 s after shear stress. (c), Average frequencies of spontaneous potentials from HEK293 cells expressing WT (black) or G615E (red) NaV1.5 channels, before (CTRL), during (SHEAR), or after (POST) shear stress (n = 6 cells each, *P < 0.05 vs. CTRL and P < 0.05 effect of genotype or shear by a two-way ANOVA with Dunnett post-test). (d), Average change in frequency of spontaneous potentials in HEK293 cells expressing WT (black) or G615E (red) NaV1.5 channels (n = 6 cells each, *P < 0.05 WT to 0% by a two-tailed one-sample t-test, NS for G615E).

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  Figure 5.. Mechanosensitivity of WT and G615E NaV1.5-induced elicited electrical excitability. (a-b), Potentials evoked by a 50-ms current stimulus before (–) or during (▬) shear stress from HEK293 cells expressing either WT (a) or G615E (b) NaV1.5 channels. (c-d), 20 overlapping sweeps of potentials from WT (c) or G615E-NaV1.5 (d) transfected HEK cells, induced by a current stimulus protocol (inset) with one super-threshold (Δ15 pA) 50-ms stimulus followed by four sub-threshold 50-ms stimuli (Δ10 pA), before (control) or during (shear) mechanical stimulation. (e-f), Fraction of potentials from WT (e) or G615E-NaV1.5 (f) transfected HEK cells evoked at the super-threshold 15-pA stimulus or four sub-threshold 10-pA stimuli before (●) or during (○) application of shear stress (n = 6 cells each; *P < 0.05 WT control vs. WT shear and P < 0.05 interaction between stimulus, genotype, and shear by a three-way ANOVA with Tukey post-test). (g-h), Time to peak potential (tPEAK) from cells expressing WT (g) or G615E-NaV1.5 (h) channels evoked at the 15-pA (■) or 10-pA (□) stimulus before (CTRL) or during shear stress (SHEAR) (n = 6 cells each; *P < 0.05, WT control vs. WT shear; P < 0.05, effect of…