2.3 Voltage protocols
The ”steady-state inactivation” (SSI ) protocol investigated the availability of channels at different membrane potentials, the ”recovery from inactivation” (RFI ) protocol investigated the dynamics of channels regaining their availability after inactivation, and the state-dependent onset (SDO ) protocol investigated the dynamics of developing inhibition. These same protocols have been used before (Lukacs et al., 2018); and they are illustrated in Fig. 5A.
The ”three pulse train” (3PT ) protocol (Fig. 2A and D) was similar to the one we called by this name before (Lukacs et al., 2018); but in this study, it was optimized for maximal time resolution. The protocol is designed to monitor changes in both gating kinetics and gating equilibrium. For the sake of high time resolution, it uses only a single interpulse interval: 2 ms (instead of several interpulse intervals as in RFI) , and only a single membrane potential: -65 mV (instead of several membrane potentials as in the ”steady-state inactivation” protocol – SSI ). Thus we could record changes at 26 ms resolution (38.5 Hz). In each experiment, we delivered 100 consecutive trains: after 6 initial control trains we perfused riluzole throughout 10 trains (i.e., for 260 ms), and then monitored washout for the remaining 84 trains. Perfusion was started and stopped during the 10 ms intervals between two trains, which was amply enough for complete exchange of the solution with the theta tube system.
Peak amplitudes of evoked currents have been determined after subtracting capacitive and leakage artifacts. Recordings from a typical cell throughout a 100 train experiment are shown in Fig. 2B. The 6 control traces are shown in blue, the 10 traces recorded during riluzole perfusion are in red (dark to light red indicates consecutive traces), and currents evoked during washout are shown in light gray to black. Fig. 2C illustrates the same currents after subtraction of artifacts. Fig. 2D shows the same corrected current traces are shown in sequential order, together with the voltage protocol, as they were evoked in the experiment (only the first 29 of the 100 trains, for the sake of clarity). Peak amplitudes are marked by circles: blue (1st pulses), red (2nd pulses), and green (3rd pulses). Connecting blue, red, and green circles we get different onset and offset characteristics, as shown for n = 7 cells in Fig. 2E. All amplitudes were normalized to the 1st pulse-evoked amplitude at the start of the experiment (1st, 2nd, and 3rd pulse-evoked amplitudes each to its own control, to help comparison of inhibited fractions). Current traces for the same seven cells, as well as for the nine recorded binding site mutant cells are shown in supporting information Fig. S1, together with a detailed discussion of fitted time constants.
A small fraction of channels (11.4 ± 0.1% by the end of the 100th train) underwent slow inactivation during the test; this was a necessary tradeoff for high temporal resolution. In order to appropriately calculate the extent of inhibition by riluzole, we corrected for the slow inactivated fraction: A sum of an exponential and a linear component was fit to the first 6 and last 3 points of 1st pulse-evoked current amplitudes in each cell (supporting information Fig. S1B and H). This fitted function represented the non-slow-inactivated fraction fnsi(t) of the channel population. Measured amplitude plots (AM(t) ), then were transformed into corrected amplitude plots (AC(t) ) by expressing it as a fraction of non-slow-inactivated channels: AC(t) = AM(t) / fnsi(t) . This is illustrated in supporting information Fig. S1B and C, where panel C shows the same data as panel B, after correction. Measurement of the extent of inhibition and determination of onset and offset time constants were performed on the corrected plots.