2.5 Combined voltage and illumination protocols
To perform photolabeling-coupled electrophysiology experiments, we used the photoreactive analog of riluzole, azido-riluzole (Lukacs et al., 2018). During drug perfusion, we used one of three illumination protocols, to compare the possibility of binding to different conformations (Fig. 5A). While one of the three protocols was repeated at 2.5 Hz (every 400 ms), 90 ms UV light pulses were applied within each cycle. The UV pulses were delivered either during hyperpolarizations, (”resting-state-illumination”); during depolarizations (”inactivated-state-illumination”); or while cells were kept at the approximate half-inactivation voltage (”V-half-illumination”), i.e., when channels were distributed roughly equally between the two conformations. To each cell, a maximum of 450 UV pulses was delivered (i.e. , for up to 3 min), it was stopped earlier whenever evoked currents were inhibited to ~20% of their original amplitude. (Note that an 80% inhibition did not mean an 80% occupancy of binding sites, as we will discuss below.) Gating kinetics and equilibrium of the channels were investigated both before drug perfusion and after washout, using the ”recovery from inactivation” (RFI ), ”steady-state inactivation” (SSI ) and ”state-dependent onset” (SDO ) voltage protocols.
Variations in the combined voltage and illumination protocols provided further insight in the mechanism, regarding the relative contribution of modulation and channel block (supporting information Fig. S2), and regarding the sequence of recovery from inactivation and unbinding (supporting information Fig. S3).