stabilized by orders of magnitude after light-off
(Fig. 4D). The time-constant of channel closure
(toff) for SwiChRCT was 7.3 s, compared with 24 ms
for the parent iC1C2 (Fig. 4E). Beyond stability,
another feature of step-function variants is the
ability to quickly convert to the closed state upon
redshifted light application (21), and indeed,
SwiChRCT channel closure was accelerated by application of 632 nm of light (SwiChRCT toff-632 =
375 ms) (Fig. 4E). Another feature of step-function
variants is increased light sensitivity of expressing
cells, which effectively become photon integrators for long light pulses (23). Indeed, SwiChRCT-
expressing cells showed a 25-fold increase in light
sensitivity as compared with that of iC1C2, and a
200-fold increase compared with that of the pump-based inhibitor NpHR (Fig. 4F). Similar results
were observed in neurons; SwiChRCT generated
outward current at AP threshold in neurons with
reversal potential of –61 mV and −68 mV for
SwiChRCA (Fig. 4G and fig. S3). This sufficed to
stably and reversibly inhibit spiking (Fig. 4H and
fig. S3) with minimal directly driven current (Fig.
4G and fig. S3) or membrane potential change
(Fig. 4H and fig. S3), presenting desirable properties for optogenetic investigation.
We have demonstrated structure-guided conversion of a cation-selective ChR into a light-activated Cl– channel. The iC1C2 mechanism
provides more physiological inhibition that does
not require a major membrane potential change,
and variants enable improvement of stability and
light sensitivity by orders of magnitude over existing inhibitory tools. Depolarization-block strategies
with excitatory tools (18, 36, 37), although useful
in some settings, may not reliably inhibit all targeted cells because light intensities are highly
variable in scattering tissue (18, 36–38); in contrast, iC1C2-based tools can only depolarize membranes to Vrev of ~–64 mV (well below VAP) and
hyperpolarize when membrane potential is above
Vrev (Fig. 3F).
Although aspects of final functionality arose
by design (for example, removal of acidic res-
idues and introduction of basic residues) (Fig.
2A), other properties remain to be fully explored.
For example, iC1C2 showed dependence on ex-
ternal pH; we hypothesize that one or more basic
residues within the ion-conducting pathway are
protonated and positively charged at physiolog-
ical and lower external pH, which in turn facilitates
association and permeation of anions. Subsequent
improvements in iC1C2 and SwiChR variants by
using structure-guided engineering strategies may
further enhance photocurrent properties and may
be easily ported to complementary and closely re-
lated ChR backbones, such as the potent chimeric
red (23, 39) and two-photon/infrared (40) light-
activated ChRs. The new Cl– permeability of iC1C2
not only provides an unexpectedly effective illustra-
tion of cation-channel to anion-channel conver-
sion (41–43) but also demonstrates structure-guided
design of ChRs for new classes of functionality.
References and Notes
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A B D
Fig. 4. Fast and bistable inhibition of neuronal spiking with iC1C2 and
SwiChR. (A and B) Representative voltage traces of iC1C2-expressing neurons stimulated with either (A) a continuous electrical pulse (3s) or (B) pulsed
current injections (10Hz/3s). Electrically evoked spikes were inhibited by 475 nm
of light (blue bar) at 5 m W/mm2. (C) Distribution of spike-inhibition probability
for iC1C2-expressing cells (n = 18 neurons; fraction of spikes blocked shown).
(D) Inward and outward photocurrents of SwiChRCT in HEK cell upon 475 nm of
light (blue bar). (E) Channel off-kinetics (t) for iC1C2, SwiChRCT, and SwiChRCT
exposed to red light during channel closure. (F) Light sensitivity of SwiChRCT
compared with that of iC1C2 and NpHR. iC1C2 and SwiChRCT were activated
with 470 nm, and NpHR was activated with 560 nm. Photocurrents were
measured at light intensities between 0.0036 and 5 m W/mm2, and holding
potential was –80 mV. Amplitudes were normalized to the maximum value for
each construct (n = 6 to 8 cells). (G) Reversal potential of iC1C2, SwiChRCT,
and SwiChRCA relative to VAP and Vrest (n = 10 to 22) (left). Photocurrent
amplitudes at VAP are shown at right (n = 9 to 15 cells). (H) Bistable spiking
modulation by SwiChRCT. Spiking was induced with a continuous electrical
pulse (3 s) and stably inhibited with 475 nm light (blue bar). Spiking
resumed after 632 nm of light application (red bar). Light power density was
5 m W/mm2. Error bars indicate SEM.