Resurgent and Gating Pore Currents Induced by De Novo SCN2A Epilepsy Mutations

Figures

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  Figure 1.. Mutation locations. Secondary structure of hNav1.2 is shown. Locations of the R853Q and R1882Q mutations are labeled with red stars and red arrows.

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  Figure 2.. The R1882Q mutation has no effect on transient current size and impairs inactivation. A, Representative raw current trace families elicited by voltage steps from –80 to +60 mV. B, Average transient current densities elicited by voltage steps from –80 to +60 mV (n = 40 WT cells, n = 25 R1882Q cells). C, Voltage stimulation protocols used for collection of activation (top) and inactivation (bottom) data from HEK cells. D, Activation curves. Conductance was calculated as G/Gmax, where G = I/(Vm-Vrev), Vrev = reversal potential, and Gmax = maximum inward conductance across all tested voltages (n = 41 WT cells, n = 27 R1882Q cells). E, Inactivation curves. Fraction available was calculated as I/Imax for each cell at each voltage step (n = 40 WT cells, n = 23 R1882Q cells). F, Time constants of fast inactivation were calculated by fitting the decay portion of each current trace to a single-exponential equation in PulseFit (HEKA; n = 41 WT cells, n = 27 R1882Q cells). WT data are represented by black circles, and R1882Q data by red triangles, in B, D–F. Error bars (SEM) are included for every data point in parts B, D–F; please note that many error bars are small and are thus obscured by…

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  Figure 3.. The R1882Q mutation enhances persistent and resurgent currents. A, Representative raw current traces from a voltage step to –15 mV. Persistent current (box and inset) was measured as the average current over the last 10% (i.e., 5 ms) of the voltage step. B, Average persistent current densities during the last 10% of voltage steps from –80 to +60 mV (n = 39 WT cells, n = 25 R1882Q cells). C, Average persistent current amplitudes normalized to maximum peak transient current amplitudes (n = 40 WT cells, n = 27 R1882Q cells). D, Resurgent current voltage protocol (top) and representative resulting raw current trace families (middle and bottom). E, Average peak resurgent current densities over a range of voltages from –80 to +60 mV (n = 39 WT cells, n = 25 R1882Q cells). F, Average peak resurgent current amplitudes normalized to maximum peak transient current amplitudes (n = 39 WT cells, n = 25 R1882Q cells). For B, C, E, F, values were calculated for each individual cell and averaged for each group. WT data are represented by black circles, and R1882Q data by red triangles, in B, C, E, F. Error bars (SEM) are included for every data point in parts B, C, E, F; please note that many…

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  Figure 4.. The R853Q mutation reduces transient current size and enhances inactivation. A, Representative raw current trace families elicited by voltage steps from –80 to +60 mV. B, Average transient current densities elicited by voltage steps from –80 to +60 mV (n = 40 WT cells, n = 16 R853Q cells). C, Voltage stimulation protocols used for collection of activation (top) and inactivation (bottom) data from HEK cells. D, Activation curves. Conductance was calculated as G/Gmax, where G = I/(Vm-Vrev), Vrev = reversal potential, and Gmax = maximum inward conductance across all tested voltages (n = 41 WT cells, n = 16 R853Q cells). E, Inactivation curves. Fraction available was calculated as I/Imax for each cell at each voltage step (n = 40 WT cells, n = 16 R853Q cells). F, Time constants of fast inactivation were calculated by fitting the decay portion of each current trace to a single-exponential equation in PulseFit (HEKA; n = 41 WT cells, n = 16 R853Q cells). WT data are represented by black circles, and R853Q data by red squares, in B, D–F. Error bars (SEM) are included for every data point in parts B, D–F; please note that many error bars are small and are thus obscured by the symbols.…

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  Figure 5.. The R853Q mutation reduces persistent and resurgent currents. A, Representative raw current traces from a voltage step to –15 mV. Persistent current (box and inset) was measured as the average current over the last 10% (i.e., 5 ms) of the voltage step. B, Average persistent current densities during the last 10% of voltage steps from –80 to +60 mV (n = 39 WT cells, n = 16 R853Q cells). C, Average persistent current amplitudes normalized to maximum peak transient current amplitudes (n = 40 WT cells, n = 16 R853Q cells). D, Resurgent current voltage protocol (top) and representative resulting raw current trace families (middle and bottom). E, Average peak resurgent current densities over a range of voltages from –80 to +60 mV (n = 39 WT cells, n = 16 R853Q cells). F, Average peak resurgent current amplitudes normalized to maximum peak transient current amplitudes (n = 39 WT cells, n = 16 R853Q cells). For B, C, E, F, values were calculated for each individual cell and averaged for each group. WT data are represented by black circles, and R853Q data by red squares, in B, C, E, F. Error bars (SEM) are included for every data point in parts B, C, E, F; please note that many error bars…

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  Figure 6.. R853Q creates a gating pore and reduces channel surface expression. A, B, Nav1.2 channel in resting state (Domains I and III, N-terminal domain, and C-terminal domain not shown). A, In the resting state, the WT Nav1.2 is essentially impermeable. B, In the resting state, the mutation of arginine 853 to glutamine creates a small aqueous pathway that is blocked when the channel transitions to an open state. Since this channel (i.e., the gating pore) is structurally distinct from the central pore of Nav1.2, current through it can be isolated by blocking the central pore with TTX. C, Average gating charge (Q) values measured from oocytes expressing either WT (black squares) or R853Q (red circles) hNav1.2. The Qmax for each group was calculated as the average Q value at +40 (n = 5 WT cells, n = 8 R853Q cells). D, Voltage dependence of gating charge movement is shown as a normalized gating charge-voltage relationship (n = 5 WT cells, n = 8 R853Q cells). Average gating charge (Q) across a range of membrane potentials were normalized to the average Qmax value for each group. WT data are represented by black squares, and R853Q data by red circles, in C, D. Error bars (SEM) are included for…

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  Figure 7.. R853Q creates nonlinear leak current. Single-cell representative data (n = 1 WT, n = 1 R853Q). A, B, Representative raw leak current traces measured from oocytes expressing either WT (black) or R853Q (red) hNav1.2 show inward rectification at hyperpolarized potentials for R853Q that was not observed for WT hNav1.2. Voltage steps over a range from –120 mV to +40 mV were applied from a holding potential of –100 mV. C, D, Leak I-V relationships of the representative cells. Dashed line shows the background linear leakage current, estimated by fitting a line to the steady-state IV plot in the range –10 to +10 mV. Notice that the holding current at –100 mV is entirely from the linear nonspecific leak in WT, whereas for the representative R853Q oocyte the holding current is almost entirely from the contribution of the non-linear inward rectifying component. E, F, Subtraction of the linear background current reveals strong inward rectification for R853Q (red) and low-amplitude nonspecific variance for WT (black). WT data are shown in black (A, C, E); R853Q data in red (B, D, F). Figure Contributions: Fenfen Wu performed data collection and analysis.

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  Figure 8.. R853Q-induced gating pore is permeable to guanidinium. Single-cell representative data (n = 1 WT, 1 R853Q). Leak currents in oocytes expressing either WT (black squares) or R853Q (red circles) hNav1.2 in the absence (empty symbols) and presence (filled symbols) of guanidinium (Gu), a cation believed to be permeant to the gating pore created by the R853Q mutation in hNav1.2. Figure Contributions: Fenfen Wu performed data collection and analysis.

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  Figure 9.. R853Q creates gating pore current. A, Distance of gating charge movement (Qmax) was plotted against the gating pore current amplitude measured at –120 mV for each cell (n = 6 WT cells, n = 11 R853Q cells). B, Average leak-subtracted (i.e., gating pore) current was normalized to Qmax for each cell and the normalized gating pore I-V relationships for WT (black squares) and R853Q (red circles) were plotted (n = 6 WT cells, n = 11 R853Q cells). Error bars (SEM) are included for every data point in part B; please note that many error bars are small and are thus obscured by the symbols. Asterisks indicate significance level (*p < 0.05, ***p < 0.001, ****p < 0.0001). Figure Contributions: Fenfen Wu performed data collection and analysis.