Fig. 3. Recovery from inactivation in the absence and presence of 100 µM riluzole in F1579A mutant channels.
Peak amplitudes are plotted against hyperpolarizing gap length. Thin black lines – normalized control data from n = 7 cells, thin red lines – 100 µM riluzole from the same 7 cells, normalized each to the control in the same cell. Averaging (thick lines) was performed as described in Methods.
Fig. 4. Effect of riluzole perfusion as monitored with the 3PT protocol on F1579A mutant channels. The voltage protocol is the same as in Fig. 2.
A Example of sodium currents evoked in a typical F1579A mutant channel expressing cell by the three depolarizations of the 3PT protocol. Subsequent traces are overlaid on each other. Blue traces show control, dark to light red show successive traces during riluzole application, light gray to black traces show successive traces during washout.
B The same currents after subtraction of capacitive and leakage artifacts.
C Illustration of the first 29 consecutive trains (protocol in the upper panel), and the currents evoked by them (lower panel). Peaks of 1st, 2nd, and 3rd pulse-evoked currents are marked with blue, red, and green circles, respectively. Shaded area indicates the perfusion of 100 μM riluzole.
D Amplitude plots for 9 individual cells. Connecting peak amplitudes of 1st (blue), 2nd (red), or 3rd (green) pulse-evoked currents gives a plot of peak amplitudes throughout the whole experiment. To help compare the extent of inhibition, 1st, 2nd, and 3rd pulse-evoked currents were normalized each to its own control (peak amplitudes recorded during the first train).
E Onset and offset time constants (mean ± SEM) for the amplitude plots of the nine cells shown in panel D. Data from one individual cell (the same as in panels A to C) is shown for illustration. Mono- or bi-exponential functions were fitted to traces after correcting for slow inactivation (see Methods). Dashed lines show exponentials fitted to this particular cell.
Fig. 5. Effect of conformation-selective photolabeling by azido-riluzole on gating kinetics of WT channels. A Experimental protocol. RFI , SSI , and SDO protocols were run before and after azido-riluzole perfusion and pulsed UV illumination. During azido-riluzole perfusion, 90 ms UV illumination pulses were used at a specific time within each cycle (shown by purple shaded areas) in all three voltage protocols.
B, C, D Assessment of gating kinetics and equilibrium before and after azido-riluzole perfusion and UV irradiation. Black lines show control data before azido-riluzole perfusion and UV illumination, colored lines show data measured after stopping UV pulses and washing out azido-riluzole. Blue lines: after resting-state-illumination protocol; teal lines: after V-half-illumination protocol; green lines: after inactivated-state-illumination protocol. Amplitudes were normalized to the maximal amplitude in control. Insets: Amplitudes normalized each to its own maxima. B: Plot of 2nd pulse-evoked peak amplitudes (mean ± SEM) against hyperpolarizing gap duration. C: Plot of channel availability against pre-pulse potential. D: Plot of 2ndpulse-evoked amplitudes against 1st pulse duration.