Hsu EJ , Zhu W , Schubert AR , Voelker T , Varga Z , Silva JR .

T32 GM099608 NIGMS NIH HHS

	Figure 1. Ionic currents and voltage dependence of WT-LFS and IQM-LFS channels. Na+ currents were recorded from human NaV1.5 channels for WT-LFS and IQM-LFS channels. The mean ± SEM for groups of five to seven cells is reported. To obtain steady-state activation data, cells were held at a potential of −120 mV for 100 ms, depolarized in 20-mV increments for 50 ms where maximum current was measured, and then returned to the original −120-mV holding potential for 50 ms. To obtain the voltage dependence measurements of SSI, cells were held at a potential of −120 mV and depolarized to conditioning potentials in increments of 10 mV for 200 ms. The available channel fraction was assessed by a 20-ms depolarizing pulse to −20 mV. (A, left) Ionic currents from WT channels were recorded during 50-ms-long depolarizing pulses ranging from −150 to 30 mV in 20-mV steps. Current traces corresponding to only −130, −70, −30, and 30 mV are shown for clarity. (right) Voltage dependence curves of steady-state activation (GV, black circles) and SSI (black squares) for WT channels were created. (B, left) Ionic currents from IQM channels were recorded during 50-ms-long depolarizing pulses ranging from −160 to 20 mV in 20-mV steps. Current traces corresponding to only −160, −100, −40, and 20 mV are shown for clarity. (right) Voltage dependence curves of steady-state activation (GV, black circles) and SSI (black squares) for IQM channels were created. Dashed lines represent the corresponding curves for the WT channel for comparison. (A and B) Curves were constructed as described in the Materials and methods section. See Table 1 for Boltzmann fit parameters.
	Figure 2. Steady-state and kinetic properties of ionic currents and fluorescent signals from the DI- to DIV-IQM mutants. (A–D) Na+ currents and fluorescence signals were recorded from human NaV1.5 channels carrying DI-IQM (A, orange), DII-IQM (B, green), DIII-IQM (C, blue), or DIV-IQM (D, purple) mutations. GV and SSI curves were obtained as described in Fig. 1. FV curves were measured by the maximum change in ΔF during the depolarizing pulse, and SSI-FV curves were found by measuring the maximum change during the test pulse to −20 mV. Ionic currents (left, top) and fluorescence signals (left, bottom) from DI- to DIV-IQM channels were recorded during 50-ms-long depolarizing pulses ranging from −160 to 80 mV in 20-mV steps. Current and fluorescence traces corresponding to only −160, −100, −40, and 20 mV are shown for clarity. The amounts of current produced and fluorescent signal (in units of ΔF/F) acquired are displayed to the left of their respective traces. Cells with the greatest amount of current produced and fluorescence magnitude are shown. Dashed lines represent fluorescence changes corresponding to DI-, DII-, DIII-, or DIV-LFS channel at 20-mV depolarization (Varga et al., 2015). (right) The mean ± SEM for groups of three to six cells is reported. Voltage dependence curves of steady-state activation (GV, black circles), SSI (black squares), and the corresponding fluorescence signals (colored circles and squares, respectively) for each channel were created. Curves were constructed as described in the Materials and methods section. Dashed lines represent the corresponding curves for the respective DI- to DIV-LFS channel for comparison (Varga et al., 2015). See Table 1 for both LFS and mutant Boltzmann fit parameters.
	Figure 3. Kinetic properties of deactivation of the DIII-VSD in both WT and IQM mutant channels. (A) DIII-LFS fluorescence signals measured after the step back to −120 mV from depolarizing steps to 50 mV for 1 ms (light blue) or 200 ms (dark blue). The exponential fits to the fluorescence traces are shown in red. Deactivation time constants for this cell after 1-ms and 200-ms, −120-mV depolarizations were respectively 3.83 ms and 8.28 ms. (B) DIII-IQM fluorescence signals measured after the step back to −120 mV from depolarizing steps to 50 mV for 1 ms (light blue) or 200 ms (dark blue). The exponential fits to the fluorescence traces are shown in red. Deactivation time constants for this cell after 1-ms and 200-ms, −120-mV depolarizations were respectively 10.91 ms and 5.03 ms. Note the reversal of the 1- and 200-ms rates of recovery when compared with A. (C) 10–90% deactivation time of the fluorescence signal from exponential fits after the step back to −120 mV from depolarizing steps to 50 mV for durations ranging from 1 to 200 ms. DIII-LFS WT times are in light blue, whereas DIII-IQM times are in dark blue. The mean ± SEM for groups of six cells is reported.
	Figure 4. Steady-state and kinetic properties of ionic currents and fluorescent signals from the DIII-N1325S long QT syndrome type 3 mutant. Na+ currents and fluorescence signals were recorded from human NaV1.5 channels carrying DIII-N1325S mutations. The mean ± SEM for groups of three to six cells is reported. Activation, inactivation, and fluorescence curves were obtained via the protocols described in Figs. 1 and 2. To obtain recovery data, cells were held at a potential of −120 mV for 50 ms and depolarized to −20 mV for either 10 or 200 ms. The potential was then returned to −120 mV for varying recovery durations of 2–500 ms before being depolarized to −20 mV again for 20 ms. The fraction of current recovered was measured as the peak current during this −20-mV hold divided by the peak current of the first −20-mV hold. The potential was finally returned to the original −120-mV holding potential for 50 ms. (A, left) Ionic currents from DIII-N1325S channels were recorded during 50-ms-long depolarizing pulses ranging from −160 to 20 mV in 20-mV steps. Current traces corresponding to only −160, −100, −40, and 20 mV are shown for clarity. (right) Voltage dependence curves of steady-state activation (GV, black circles), SSI (black squares), and the corresponding fluorescence signals (colored circles and squares, respectively) for DIII-N1325S channels were created. See Table 1 for Boltzmann fit parameters. (B, left) DIII-N1325S fluorescence signals measured after the step back to −120 mV from depolarizing steps to 50 mV for 1 (light blue) or 200 ms (dark blue). (right) 10–90% deactivation time of the fluorescence signal from exponential fits after the step back to −120 mV from depolarizing steps to 50 mV for durations ranging from 1 to 200 ms. DIII-LFS WT times are in light blue, whereas DIII-N1325S times are in dark blue. (C, left) Ionic currents from DIII-N1325S channels were recorded with the protocol used for the IQM mutations. The initial −20-mV depolarizing pulse was held for either 10 (top) or 200 ms (bottom). (right) Time dependence of fraction of current recovered for DIII-N1325S after a 10-ms depolarizing pulse (gray) or 200-ms depolarizing pulse (black). The smaller subplot only shows the fitted curves for time dependence of recovery, with solid lines representing DIII-N1325S recovery. See Table 2 for exponential parameters. (A and C) Curves were constructed as described in the Materials and methods section. Dashed lines represent the corresponding curves for the DIII-LFS channel for comparison.
	Figure 5. Steady-state and kinetic properties of ionic currents and fluorescent signals from the DIII-A1330P long QT syndrome type 3 mutant. Na+ currents and fluorescence signals were recorded from human NaV1.5 channels carrying DIII-A1330P mutations. The mean ± SEM for groups of three to six cells is reported. Activation, inactivation, and fluorescence curves were obtained via the protocols described in Figs. 1 and 2. Recovery curves were obtained via the protocols described in Fig. 4. (A, left) Ionic currents from DIII-A1330P channels were recorded during 50-ms-long depolarizing pulses ranging from −160 to 20 mV in 20-mV steps. Current traces corresponding to only −160, −100, −40, and 20 mV are shown for clarity. (right) Voltage dependence curves of steady-state activation (GV, black circles), SSI (black squares), and the corresponding fluorescence signals (colored circles and squares, respectively) for DIII-A1330P channels were created. See Table 1 for Boltzmann fit parameters. (B, left) DIII-A1330P fluorescence signals measured after the step back to −120 mV from depolarizing steps to 50 mV for 1 (light blue) or 200 ms (dark blue). The exponential fits to the fluorescence traces are shown in red. Deactivation time constants for this cell after 1-ms and 200-ms, −120-mV depolarizations were respectively 3.37 ms and 6.82 ms. (right) 10–90% deactivation time of the fluorescence signal from exponential fits after the step back to −120 mV from depolarizing steps to 50 mV for durations ranging from 1 to 200 ms. DIII-LFS WT times are in light blue, whereas DIII-A1330P times are in dark blue. (C, left) Ionic currents from DIII-A1330P channels were recorded with the protocol described in Fig. 4. The initial −20-mV depolarizing pulse was held for either 10 (top) or 200 ms (bottom). (right) Time dependence of fraction of current recovered for DIII-A1330P after a 10-ms depolarizing pulse (gray) or 200-ms depolarizing pulse (black). The smaller subplot only shows the fitted curves for time dependence of recovery, with solid lines representing DIII-A1330P recovery. See Table 2 for exponential parameters. (A and C) Curves were constructed as described in the Materials and methods section. Dashed lines represent the corresponding curves for the DIII-LFS channel for comparison.
	Figure 6. Steady-state and kinetic properties of ionic currents and fluorescent signals from the DIII-N1659A and DIV-N1659A mutant. Na+ currents and fluorescence signals were recorded from human NaV1.5 channels carrying DIII-N1659A or DIV-N1659A mutations. The mean ± SEM for groups of three to six cells is reported. Activation, inactivation, and fluorescence curves were obtained via the protocols described in Figs. 1 and 2. Recovery curves were obtained via the protocols described in Fig. 4. (A, left) Ionic currents from DIII-N1659A channels were recorded during 50-ms-long depolarizing pulses ranging from −160 to 20 mV in 20-mV steps. Current traces corresponding to only −160, −100, −40, and 20 mV are shown for clarity. (right) Voltage dependence curves of steady-state activation (GV, black circles), SSI (black squares), and the corresponding fluorescence signals (colored circles and squares, respectively) for DIII-N1659A channels were created. (B, left) DIII-N1659A fluorescence signals measured after the step back to −120 mV from depolarizing steps to 50 mV for 1 (light blue) or 200 ms (dark blue). The exponential fits to the fluorescence traces are shown in red. Deactivation time constants for this cell after 1-ms and 200-ms, −120-mV depolarizations were respectively 1.04 ms and 3.97 ms. (right) 10–90% deactivation time of the fluorescence signal from exponential fits after the step back to −120 mV from depolarizing steps to 50 mV for durations ranging from 1 to 200 ms. DIII-LFS WT times are in light blue, whereas DIII-N1659A times are in dark blue. (C, left) Ionic currents from DIII-N1659A channels were recorded with the protocol described in Fig. 4. The initial −20-mV depolarizing pulse was held for either 10 (top) or 200 ms (bottom). (right) Time dependence of fraction of current recovered for DIII-N1659A after a 10-ms depolarizing pulse (gray) or 200-ms depolarizing pulse (black). The smaller subplot only shows the fitted curves for time dependence of recovery, with solid lines representing DIII-N1659A recovery. See Table 2 for exponential parameters. (A and C) Curves were constructed as described in the Materials and methods section. Dashed lines represent the corresponding curves for the DIII-LFS channel for comparison. (D, left) Ionic currents from DIV-N1659A channels were recorded during 50-ms-long depolarizing pulses ranging from −160 to 20 mV in 20-mV steps. Current traces corresponding to only −160, −100, −40, and 20 mV are shown for clarity. (right) Voltage dependence curves of steady-state activation (GV, black circles), SSI (black squares), and the corresponding fluorescence signals (colored circles and squares, respectively) for DIV-N1659A channels were created. Curves were constructed as described in the Materials and methods section. Dashed lines represent the corresponding curves for the DIV-LFS channel for comparison. (A and D) See Table 1 for Boltzmann fit parameters.
	Figure 7. Comparison of DIII-VSD deactivation kinetics with recovery kinetics. As described in the Materials and methods section, time dependence of recovery saw fit using a sum of exponentials with the following equation: fraction recovered y = C − Af*exp(−t/τf) − As*exp(−t/τs). (A) Representative recovery from WT, displaying each component. Af and τf more heavily contribute to recovery after shorter recovery durations, whereas As and τs more heavily contribute to recovery after longer recovery durations. (B) All four of the above parameters for each mutant were plotted against DIII-VSD deactivation time after 10-ms (open circles) and 200-ms (closed circles) pulses. The legend describes the color that each mutant’s deactivation or recovery parameters correspond to on each graph.
	Figure 8. A working model for DIII-VSD and DIV-VSD regulation of inactivation. (A) At rest, the DIII- and DIV-VSDs are in the resting conformation, the DIII–DIV linker is unbound, and the channel is not inactivated. (B) Upon depolarization, the DIII- and DIV-VSDs activate within 10 ms (Fig. 2), facilitating interaction between the DIII–DIV linker and the DIV-VSD. (C) After ∼100 ms, the DIII–DIV linker undergoes a second, IFM-dependent interaction with the DIII-VSD that stabilizes its activated conformation. (D) Once the cell returns to its resting potential, the DIII–DIV linker first dissociates from the DIV-VSD. (E) Later, the DIII–DIV linker dissociates from the DIII-VSD, allowing recovery from inactivation to occur.

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