3.5 State-dependence of binding.
Riluzole, like most sodium channel inhibitors, is known to preferentially bind to inactivated channels (Catterall et al., 2019). We first intended to investigate how the conformation of the channel influences the ability of the drug to bind. We used the three voltage and illumination protocols as described in Methods. Before drug perfusion and after washout we assessed changes in gating kinetics and equilibrium of the channels by using the RFI , SSI , andSDO voltage protocols. Covalent binding-induced changes in channel gating kinetics (RFI and SDO ) and gating equilibrium (SSI ) are shown in Fig. 5B, C, D. Blue, teal, and green colors indicate resting-state-, V-half-, and inactivated-state-illumination, respectively. Insets show normalized data, where amplitudes were normalized to the maximal amplitude after azido-riluzole perfusion and irradiation. This gives a clearer picture of how the gating has been modulated. Similarly to our earlier data (Lukacs et al., 2018) obtained using continuous illumination, the population of ion channels that were still conducting showed modulated gating: delayed recovery from inactivation (Fig. 5B), shifted steady-state availability (Fig. 5C), and accelerated onset of inhibition (Fig. 5D). Note that the SDO curves were also affected by covalently bound ligand; in the case of the inactivated-state-illumination protocol, the onset proceeded with a time constant of 1.21 ± 0.12 ms. Since there was no possibility of binding or unbinding, this must correspond with a conformational rearrangement of the ligand-channel complex.
Interestingly, 90 ms UV pulses delivered at every 400 ms during inactivated conformation were as effective as continuous illumination had been (Lukacs et al., 2018), in spite of the fact that the total illumination time was only 23.75% of it. It seems that repeatedly allowing azido-riluzole molecules to diffuse to their most favorable location (probably the binding site) without activating them, and then delivering the UV pulse only when they are at the right location, actually works as effectively as continuous illumination.
In contrast, when UV pulses were delivered during resting state, no significant decrease of current amplitudes was observed, and changes in recovery kinetics (RFI protocol) and equilibrium of inactivation (SSI protocol) were also non-significant (p = 0.2 and p = 0.25 for changes in the predominant time constant of recovery and V1/2, respectively, n = 7). This may either suggest that the central cavity or the binding site itself is inaccessible at resting conformation, or that the binding site has very low affinity at resting state. UV pulses delivered at the approximate half-inactivation voltage were between the other two illumination protocols in effectiveness.