1 INTRODUCTION
Hyperexcitability is at the core of a rather diverse set of disorders
affecting the heart, skeletal muscles, and the nervous system.
Out-of-control electric activity is involved in the development of
several types of epilepsies, chronic pain syndromes, neuromuscular
disorders, cardiac arrhythmias, and even psychiatric disorders (Rogawski
& Löscher, 2004). These conditions can originate from genetic
conditions (mutations altering the operation of channels themselves, or
proteins involved in their modulation), or can be due to damages caused
by mechanical injury, inflammation or ischemia.
To suppress hyperexcitability, sodium channels are the primary target
because they are responsible for the fast onset of action potentials, as
well as their all-or-none and self-regenerating nature. A small fraction
(0.5 to 5%) of sodium channels evades rapid inactivation and produces a
current component named persistent or late sodium current. This
component plays a crucial role in the initiation of action potentials,
and its enlargement has been observed in several
hyperexcitability-related pathologies (Cannon, 2018; Lampert et al.,
2006; Makielski, 2016; Meisler, 2019; Stafstrom, 2007; Tang et al.,
2015; van Zundert et al., 2012).
In order to design therapeutically useful sodium channel inhibitors, one
encounters the seemingly impossible task of having to prevent
pathological hyperexcitability, while maintaining the normal
physiological activity of nerves and muscles. Interestingly, there are
compounds, which are able to carry out this feat – at least to a
certain extent. These compounds include antiarrhythmic, antiepileptic,
antispastic, etc. compounds. The trick that enables them to do so is
state-dependence, i.e., their preference for certain conformational
states of the channel protein. Most sodium channel inhibitors prefer
inactivated state to resting state, they bind to it more rapidly, and/or
dissociate from it more slowly. The fact that an inhibitor has a higher
affinity to inactivated state, means that drug-bound inactivated
channels form an energetically favorable complex. This implies both
slower dissociation of the ligand from this conformation and slower
recovery from inactivated to resting state. The latter effect – called
modulation of channel gating – is an inseparable element of
state-dependent inhibition, as described by the modulated receptor
hypothesis (Hille, 1977). High affinity to inactivated state ensures
delayed dissociation, while modulation ensures delayed conformational
transition to the low affinity resting state, thus restraining both
possible pathways to recovery (dissociation followed by recovery, or
recovery followed by dissociation).
Pathological states induced by injury, inflammation, ischemia, tumor, or
epilepsy alter the electrical characteristics of excitable cells, which
may include a depolarized membrane potential due to energy failure,
increased leakage currents, left-shifted voltage sensitivity of sodium
channels, and increased persistent component of the sodium current
(Fischer et al., 2017; Hammarström & Gage, 2002; Ma et al., 2006;
Morris & Joos, 2016; Q. Zhang et al., 2019; Zheng et al., 2012). These
changes make sodium channels more likely to be in open and inactivated
conformations, therefore state-selectivity alone is enough for
preferential inhibition of pathological tissue. On a first impression,
one could suppose that the stronger the state-preference is, the better
the drug will be.
However, the temporal aspect must also be considered. Action potentials
are fired repetitively, and pathological behavior of neurons is often
manifested as high-frequency firing. The extent of state-dependence is,
therefore, not the only crucial aspect, equally important is the
onset/offset dynamics of state-dependent binding. Its significance is
obvious in the case of Class 1 antiarrhythmics, where subclasses a, b,
and c differ in their association/dissociation kinetics, but the same is
true for the much higher firing rates in central and peripheral neurons.
For selective inhibition of cells firing at pathologically high
frequency, the ideal drug should work as a low pass filter, with a steep
frequency response, to be able to distinguish pathological and
physiological rates of firing. This, however, presents a theoretical
limit for state-dependent binding, because fast dissociation precludes
high affinity. One would want both fast binding/unbinding dynamics, and
high state dependence. However, high state-dependence requires high
affinity to inactivated state, and high affinity means slow
dissociation, which means that binding/unbinding dynamics cannot be
fast.
Intriguingly, riluzole seems to be able to elude this limitation. Here
we examine how this is possible.
We first describe the peculiar pattern of inhibition, we observed during
and after riluzole perfusion. We use a voltage-clamp protocol where
block and modulation of the channels are monitored in parallel.
Paradoxically, two distinct recovery processes seem to coexist, with
rates differing by more than two orders of magnitude. We presume that
this peculiarity may be the key to being able to feature fast kinetics
and high affinity at the same time. A fast recovery allows channels to
regain their ability to conduct ions within ~10 ms. In
spite of their ability to conduct, however, channels remain modulated by
the drug for a much longer time: it requires ~2 s for
the channels to recover from the modulatory influence. This mechanism
enables riluzole to function as a low pass filter with an exceptionally
steep frequency response, which is probably a key element of its
distinctive therapeutic efficacy.
We set out to identify the physical processes that underlie fast and
slow recovery processes with the help of conformation-selective
photolabeling-coupled electrophysiology, and in silico docking
experiments.