3.2 Fast and slow recovery upon removal of the drug.
We investigated the onset and offset kinetics using the 3PTprotocol (Fig. 2, see Methods for the details of the protocol). The
offset of inhibition upon actual removal of the drug was more than
hundredfold slower than recovery from inactivation in the presence of
riluzole: it occurred with a time constant of 824 ± 99 ms (Fig. 2F).
Paradoxically, within each cycle, the recovery was almost complete
within each 10 ms of between-trains hyperpolarization (see the minimal
inhibition at blue traces), but then the inhibition was reestablished
before the 2nd depolarization, and became even
stronger during the subsequent 8 ms spent near the V1/2(-65 mV), causing a massive inhibition at the 3rd pulse of each train.
This pattern repeated itself during riluzole perfusion, and also for 1-2
s after riluzole had been washed out.
The extent of inhibition in 1st,
2nd, and 3rd pulse-evoked currents
was 16.1 ± 4.0%, 73.4 ± 3.9%, and 95.4 ± 1.2%. Apparent affinity
(IC50 ) values can be determined from the extent
of inhibition, as described in Methods; these values corresponded with
606 ± 137 µM, 33.5 ± 6.8 µM, and 3.98 ± 1.32 µM, respectively. Mean ±
SEM of time constants for the 7 cells are shown in Fig. 2F.
In conclusion, two distinct recovery processes coexisted with completely
different kinetics. Recovery is conventionally explained by
dissociation, but the two recovery processes with more than hundredfold
different time constants obviously cannot both reflect dissociation. To
investigate what physical processes underlie the progression of both
fast and slow recovery, we first studied the effect of mutating the most
important residue of the local anesthetic binding site, F1579. The
presence of this aromatic residue is crucial in determining the affinity
of binding (Mike & Lukacs, 2010; Zhang et al., 2015), and also in
coupling drug binding to altered voltage sensor movements (Hanck et al.,
2009; Muroi & Chanda, 2009), which is the basis of gating modulation.