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.