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.