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.