3.3 The two recovery processes in binding site mutant channels.
We repeated the same experiments using the RFI and 3PTprotocols in cells expressing binding site mutant (F1579A) channels. We
found that both the fast and the slow processes were radically altered
by the mutation. In mutant channels fitting the recovery required a
biexponential function, the major fast component had a time constant of
0.30 ± 0.02 ms, and it contributed 85.5 ± 2.5% to the amplitude. The
time constant of the minor, slow component was 4.36 ± 0.62 ms. Riluzole
only slightly delayed fast component, to 0.43 ± 0.02 ms (p = 0.012,
paired t-test, n = 7) (Fig. 3). The minor, slow component was not
changed significantly.
The effect of the mutation on the fast recovery was also indicated by
the decreased inhibition of 2nd pulse-evoked currents
in the 3PT protocol (Fig. 4), see the minimal difference
between blue and red traces in Fig. 4D. Interestingly,
3rd pulse-evoked currents were still considerably
inhibited (by 37.2 ± 6.6%). Apparent affinity
(IC50 ) values, calculated from the extent of
inhibition, were 1590 ± 180 µM, 895 ± 126 µM, and 114 ± 20.0 µM for
1st, 2nd, and 3rdpulse-evoked currents, respectively. Compared to the apparent affinity
values of WT channels, 1st pulse-evoked responses were
least sensitive to the mutation (2.62-fold decrease in affinity; p =
0.012, paired t-test, n = 9), while 2nd and
3rd pulse-evoked responses much more sensitive
(26.7-fold and 28.6-fold decrease in affinity, respectively; p
< 1*10-7 for both).
The slow recovery process was also accelerated, (compare Fig. 4E to Fig.
2F). For a detailed analysis of time constants see supporting
information Fig. S1.
3.4 Conformation-selective photolabeling. The problem of
interrelated binding and gating .
To summarize, we observed two distinct recovery processes, and they were
both accelerated by mutation of the local anesthetic binding site. What
physical processes may underlie them?
In the presence of riluzole, the rate of the fast recovery process may
be determined either by modulated gating (i.e., recovery from
inactivation is slowed down by the bound drug) or by the dissociation of
the drug – which is the conventional explanation.
The modulated receptor hypothesis (Hille, 1977) predicts that, since the
drug has different affinities to different conformational states, drug
binding must alter the energetics of conformational transitions, making
higher affinity states more stable. Any mutation that changes binding
affinities to different states must also change the effect of the drug
on rates of transitions between these states (i.e., the gating of
drug-bound channels). In other words, decreased affinity must
necessarily cause decreased modulation of gating. Mutation-induced
changes in the fast and slow recovery processes, therefore, could both
be manifestations of decreased affinity, on the level of gating and
binding, respectively. Mutation-induced acceleration of the fast
recovery process (compare Fig. 1 and Fig. 3) may be due to decreased
modulation of channel gating, while acceleration of the slow recovery
process (compare Fig. 2F and 4E) may be caused (at least partly) by
faster ligand unbinding. If we accept this explanation, we must suppose
that channel gating alone (without dissociation of the inhibitor) can
make the channel available for activation and conduction, in other
words, drug-bound channels can conduct, therefore ”non-blocking
modulation” (Lukacs et al., 2018) is possible.
The complex problem of interrelated gating and binding/unbinding can be
simplified using photolabeling-coupled electrophysiology, as we have
previously demonstrated (Lukacs et al., 2018). By binding the
photoreactive riluzole analog, azido-riluzole, covalently to the
channel, we could exclude the processes of binding and unbinding. We
found that covalently bound channels were still able to conduct ions,
but with modulated gating. In the 3PT protocol modulated gating
was reflected as the difference between 1st and
2nd pulse-evoked currents (Fig. 2). This difference
almost disappeared when the key residue of the local anesthetic binding
site was mutated (Fig. 4). It was logical to investigate if mutation of
the same residue would cause the same lack of modulation in
photolabeling-coupled electrophysiology experiments. In addition, we
also investigated whether the binding was conformation-dependent. We
have improved the method of photolabeling-coupled electrophysiology by
synchronizing UV light pulses with the voltage protocol, instead of the
continuous illumination we had used previously (Lukacs et al., 2018).
This allowed us to target specific conformations of the channel, and
also to verify that the channel-ligand complex indeed assumes a
drug-bound resting conformation before unbinding occurs. By precise
timing of the UV light pulse, it was possible to test how soon this
unfavorable drug-bound resting conformation was ended by unbinding (see
supporting information). These data helped us identify the physical
processes underlying both fast and slow recovery processes.