TRPswitch - A Step-Function Chemo-optogenetic Ligand for the Vertebrate TRPA1 Channel

Article abstract of DOI:10.1021/jacs.0c06811

Chemo-optogenetics has produced powerful tools for optical control of cell activity, but current tools suffer from a variety of limitations including low unitary conductance, the need to modify the target channel, or the inability to control both on and off switching. Using a zebrafish behavior-based screening strategy, we discovered "TRPswitch", a photoswitchable nonelectrophilic ligand scaffold for the transient receptor potential ankyrin 1 (TRPA1) channel. TRPA1 exhibits high unitary channel conductance, making it an ideal target for chemo-optogenetic tool development. Key molecular determinants for the activity of TRPswitch were elucidated and allowed for replacement of the TRPswitch azobenzene with a next-generation azoheteroarene. The TRPswitch compounds enable reversible, repeatable, and nearly quantitative light-induced activation and deactivation of the vertebrate TRPA1 channel with violet and green light, respectively. The utility of TRPswitch compounds was demonstrated in larval zebrafish hearts exogenously expressing zebrafish Trpa1b, where the heartbeat could be controlled using TRPswitch and light. Therefore, TRPA1/TRPswitch represents a novel step-function chemo-optogenetic system with a unique combination of high conductance, high efficiency, activity against an unmodified vertebrate channel, and capacity for bidirectional optical switching. This chemo-optogenetic system will be particularly applicable in systems where a large depolarization current is needed or sustained channel activation is desirable.

Full text of DOI:10.1021/jacs.0c06811

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Article
TRPswitch — a step function chemo-optogenetic
ligand for the vertebrate TRPA1 channel
Pui-Ying Lam, Aditya Raymond Thawani, Enrique Balderas, Andrew J.
P. White, Dipayan Chaudhuri, Matthew J Fuchter, and Randall Peterson
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c06811 • Publication Date (Web): 23 Sep 2020
Downloaded from pubs.acs.org on September 24, 2020
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TRPswitch — a step function chemo-optogenetic ligand for the
vertebrate TRPA1 channel
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Pui-Ying Lam1, Aditya R. Thawani2, Enrique Balderas3, Andrew J.P. White2, Dipayan Chaudhuri3,
Matthew J. Fuchter2 and Randall T. Peterson1, *
1 Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT 84112 USA
2 Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, White City Campus,
Wood Lane, London W12 OBZ, UK
3 Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University
of Utah, Salt Lake City, UT 84112 USA
ABSTRACT: Chemo-optogenetics has produced powerful tools for optical control of cell activity, but current tools suffer
from a variety of limitations including low unitary conductance, the need to modify the target channel, or the inability to
control both on and off switching. Using a zebrafish behavior-based screening strategy, we discovered “TRPswitch”, a
photoswitchable non-electrophilic ligand scaffold for the transient receptor potential ankyrin 1 (TRPA1) channel. TRPA1
exhibits high unitary channel conductance, making it an ideal target for chemo-optogenetic tool development. Key
molecular determinants for the activity of TRPswitch were elucidated and allowed for replacement of the TRPswitch
azobenzene with a next-generation azoheteroarene. The TRPswitch compounds enable reversible, repeatable, and nearly
quantitative light-induced activation and deactivation of the vertebrate TRPA1 channel with violet and green light,
respectively. The utility of TRPswitch compounds was demonstrated in larval zebrafish hearts exogenously expressing
zebrafish Trpa1b, where heartbeat could be controlled using TRPswitch and light. Therefore, TRPA1/TRPswitch represents
a novel step-function chemo-optogenetic system with a unique combination of high conductance, high efficiency, activity
against an unmodified vertebrate channel, and capacity for bidirectional optical switching. This chemo-optogenetic
system will be particularly applicable in systems where a large depolarization current is needed or sustained channel
activation is desirable.
Introduction
conductance actuators should also decrease the amount
of channel expression needed for sufficient light-
controlled activity. Some progress has been made toward
these goals, including the development of an endogenous
protein-targeting photoswitch that confers light-
sensitivity on endogenous neuronal ion channels,
proposed to have the potential to restore vision to the
blind.3-5
Optogenetics has proven to be a transformative
technology for various fields of basic research, particularly
in neuroscience. It allows for a non-invasive, localized,
and temporally selective optical modulation of selected
cells within an animal. Optogenetic technologies hold
great promise for clinical applications. For example,
preclinical animal models have demonstrated potential
utility in treating retinitis pigmentosa with AAV-delivered
channelrhodopsin2 (ChR2).1 However, high expression of
the light-gated ion channel ChR2, originating from green
algae, has cytotoxic effects.2 It remains unclear whether or
not expression of ChR2 in humans will result in immune
rejection or inflammation. Therefore, to advance the
utility of optogenetics in clinical applications,
development of optogenetic actuators that are vertebrate
in origin or endogenous to humans, as well as those based
on ion channels with high unitary conductance, might be
advantageous. The use of vertebrate protein actuators
might reduce the risk of immunological reactions due to
long-term expression of exogenous proteins. High
Discovery of new optogenetic actuators that possess
novel and unique properties will undoubtedly enhance
our ability to dissect biological systems such as the
complex neuronal networks of the brain. For example,
optogenetic experiments that require long activation
periods would benefit greatly from “step function”
optogenetic tools that allow stable, bi-directional on and
off switching of channels. Step function opsins (SFOs) are
engineered versions of ChR2 that persist in their open
state after illumination ceases.6-8 As such, they are useful
in modifying the spontaneous firing rate of neurons, as
well as in applications where behavioral analysis without
continuous optic fiber tethering is desired.9-10 However, as
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mentioned above, the non-vertebrate origin of these corresponding ion channel either in their E or Z
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opsins present potential challenges in clinical applications.
As a complementary approach, chemo-optogenetic tools
that combine chemicals and optogenetics have been
under rapid development. Using a chemical design
approach, photochromic soluble ligands (PCLs) for various
wild-type ion channels have been synthesized to allow for
configuration. Examples include PCLs for the TRPV1
channel,11 NMDA receptor,16-17 and kainate receptor.18-19
TRPA1 is a member of the Transient Receptor Potential
(TRP) channel family. It is a non-selective cation channel
that plays an important role in inflammatory and
neuropathic pain, itch, and respiratory diseases.20-22 We
have previously reported
light-controlled
channel
activity.11-15
These
photoswitchable PCLs block and unblock their
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Figure 1. Photoswitching of TRPswitch-A activates the Trpa1b channel. (a) Schematic diagram of the behavioral
screening assay setup. Screening was performed using a 96-well plate format. 3 wild-type zebrafish larvae at 3
days post fertilization (dpf) were placed in each well. Individual small molecules were added to each well. A series
of different wavelengths of light from WL1 – WL4 were applied to each well and the motion activity from the well
was recorded. WL1, 450 nm – 500 nm; WL2, 415 nm – 455 nm; WL3, 352 nm – 402 nm; WL4, white light. The
screening assay was performed on a motorized inverted compound microscope. A hit is defined as having motion
in the well after photoactivation. (b) Image of one well on the 96-well plate with 3 zebrafish larvae. (c) Line graph
showing the zebrafish behavioral response of TRPswitch-A treated animals. The vertical bars indicate the timing
of a 1 s light stimulus at the indicated wavelength. (d) Electrophysiology analysis of TRPswitch-A in HEK293T cells
expressing zebrafish Trpa1b. Current density-Voltage relationships of Trpa1b current without treatment (black),
10 µM TRPswitch-A with violet light stimulus (magenta), and then followed by green light stimulus (green). (e)
Isomerization of TRPswitch-A as indicated by the absorbance profile of TRPswitch before irradiation (black dotted
line), after violet light (352 - 402 nm) irradiation (magenta line), and subsequently after green light (500 - 600
nm) irradiation (green line). (f) Basal zTrpa1b currents (black) are activated (blue trace) and subsequently
desensitize (orange trace) with AITC.
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photoactivable ligands for TRPA1, such as optovin, and
demonstrated their ability to act as chemo-optogenetic
unitary conductance, make it a complementary alternative
to existing chemo-optogenetic tools.
tools.23-24 TRPA1 has
a
channel conductance of
Result
approximately 100 pS,25 1000 times greater than ChR2,
making it ideal for applications where high conductance
or low expression levels are desired. Importantly however,
while photochemical stimulation of optovin-class chemo-
optogenetic ligands activate TRPA1 rapidly (in low
millisecond time scales), channel deactivation depends
upon spontaneous (i.e. non-photochemical) reversal of
ligand action, which occurs on the time scale of seconds.
In other words, the approach is somewhat akin to
photodecaging, where a ‘protected’ ligand is activated
(‘deprotected’) following a light stimulus,26 but where
deactivation requires spontaneous and often slow
Zebrafish behavior-based chemical screening
identifies TRPswitch-A
With the discovery of photochromic soluble ligands
(PCLs) as a means to control ion channel function using
light,11-15 we reasoned that molecular photoswitches for
TRPA1 could be discovered using small molecule
screening. To achieve this goal, we developed a modified
version of the behavioral assay that we previously utilized
to identify photoactivable, but non-photoreversible,
ligands of TRPA1.23 This medium-throughput, semi-
automated screening assay was performed using a 96-
well plate format where three larvae per well were
incubated with small molecules. We screened a library of
1,000 structurally diverse small molecules enriched for
molecular photoswitch moieties such as acylhydrazone,27
azobenezene,28-31 azoheteroaryl32-33 and stilbene.31, 34 We
particularly focused on molecular photoswitches that
operate via a reversible E/Z isomerization using different
wavelengths of light. In this new assay, each well of a 96-
well plate is illuminated with a series of different
wavelengths of light (450-500 nm, 415 – 455 nm, 352-402
nm and white light) for one second to induce
isomerization of the photoswitchable compounds (Fig. 1a).
Each illumination event is separated by a dark period of 5
s and the motion of larvae during this light illumination
sequence is recorded and analyzed (Fig. 1b). Wild-type 3-
days-post-fertilization larvae (dpf) were used, as they have
a relatively developed central nervous system and show
no motion response to light exposure at this stage of
development. Zebrafish larvae express Trpa1b in a subset
of trigeminal and Rohon-Beard sensory neurons.35
dissipation of the ligand.
It would be a great
advancement to develop a chemo-optogenetic system
that preserves the high conductance of TRPA1 and the
rapid activation of optovin-class ligands, but enables
rapid,
light-controlled
channel
deactivation:
a
photoreversible/photoswitchable system.
In this study, we used a customized, light-responsive
chemical library coupled with a behavior-based screening
assay with zebrafish larvae to discover ‘TRPswitch’
azoarene photoswitchable ligands for the TRPA1 channel.
Our TRPswitch molecules allow for optical control of both
the activation and deactivation of the TRPA1 channel. Our
analysis suggests that the zebrafish Trpa1b channel is
necessary and sufficient for TRPswitch light-induced
activity. Channel activation and deactivation can be
controlled by violet light and green light illumination,
respectively. The TRPswitch/light-induced TRPA1 channel
activity is reversible and repeatable in vivo, and sustained
channel activation is achieved after only a short pulse of
light illumination. To our knowledge, this is the first time
a photoswitchable TRPA1 system has been described. Our
data show that this TRPA1/TRPswitch system is a robust
chemo-optogenetic tool that can be applied to both
neuronal and non-neuronal cells. The TRPA1/TRPswitch
system’s step function properties, along with its high
Activation of Trpa1b induces a reproducible and robust
35-36
motion response.23-24,
A light-induced motion
response, due to the presence of a photoactivated ligand
for ion channels such as Trpa1b, is used as the readout for
the assay. DMSO treated larvae on each screening plate
served as negative controls. Using this screening assay,
we identified TRPswitch-A, an azobenzene containing
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small molecule with no previously annotated biological is incomplete (Fig. 1e). We sought to determine the
1
activity. The presence of TRPswitch-A led to a light-
induced motion response across multiple wavelengths,
with the most activity in a bandwidth between 415 nm -
455 nm (WL2 in Fig. 1c). Using whole-cell patch-clamp
recordings of HEK293T cells expressing zebrafish Trpa1b,
we characterized the photocurrents elicited by the
Trpa1b/TRPswitch-A pair upon light stimulation. Violet
light stimulation of TRPswitch-A-primed Trpa1b channels
generated high amplitude currents when compared to
baseline measurements (Fig. 1d). When the same cell was
subsequently stimulated with green light, Trpa1b
photocurrent recovered to its baseline current magnitude
(Fig. 1d). This reversible light response corresponds to the
reversible E/Z isomerization of TRPswitch-A observed
upon illumination with violet and green light, as judged
by UV-Vis absorbance measurements (Fig. 1e). Expression
of functional zTrpa1b channels in HEK293T cells was
confirmed by the observance of allyl isothiocyanate
(AITC)-activated currents (Fig. 1f), as AITC is a potent
TRPA1 agonist. We also observed desensitization of AITC-
induced current over time, further confirming the activity
of zTrpa1b channels (Fig. 1f, S1). Overall, these data
indicate that TRPswitch-A is a light-dependent and light-
reversible activator of Trpa1b.
chemical features responsible for TRPswitch-A’s biological
activity and how photoswitch performance correlates to
the biological effects observed. We therefore designed,
synthesized and analyzed the structure activity
relationships of key derivatives of TRPswitch-A. We found
that the para amide is needed for Trpa1b activation as
suggested by the reduced activity of compound 2 (Fig. 2).
Substituting the furan for thiophene was tolerated (Fig. 2,
compound 3), albeit with a slightly lower photoresponse
which might be due to the overall chemical structural
change. Derivatives bearing para electron-withdrawing
groups – to increase the “push-pull” character 37-38 – were
prepared (compounds 4, 5, 6, 7) and found to be far less
active in the assay (Fig. 2). This poor activity may, in part,
be ascribed to the incomplete E-Z photoswitching of
these compounds (Fig. S2 and Supp. Table 1). We have
previously found that azoarene performance can be
improved/tuned by substituting one of the benzene rings
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in
a conventional azobenzene for a 5-membered
heteroaromatic ring.32, 39-40 Specifically, we have found the
azopyrazoles show near quantitative photoswtiching in
both directions and exhibit long Z-isomer thermal half-
lives. Replacement of the phenyl ring in compound 1
(TRPswitch-A) and compound 3 with a trimethylpyrazole
generates compounds (8 and 9) that undergo quantitative
photoswitching in both directions (Fig. 3f) and that have
long thermal half-lives. The thermal Z-isomer half-life of
compound 9 (TRPswitch-B), in DMSO-d6, is 17 hours (Fig.
3a). While we observed some aqueous solubility issues
for compound 8, which likely limits its biological response,
compound 9 (TRPswitch-B) has a comparable biological
response to TRPswitch-A.
TRPswitch-A structure activity relationship analysis
The TRPswitch-A chemical structure contains an
azobenzene and a 2-furamide group in both the ortho and
para positions of one of the benzene rings (Fig. 2,
compound 1).
It is clear that while TRPswitch-A
undergoes reversible E/Z isomerization upon illumination
with violet and green light, the Z-E photoswitching event
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Figure 2. TRPswitch structure activity relationship analysis. The structure of individual compounds and
their in vivo activity are shown in the bar graph side by side for comparison. The assay was performed as
described in Fig. 1. Each bar in the graph represents the motion response after photoactivation using
individual wavelength sets as indicated in the lower x-axis. WL1, 450 nm – 500 nm; WL2, 415 nm – 455 nm;
WL3, 352 nm – 402 nm; WL4, white light. 1% DMSO was used as a control. Values are means ± SEM from
at least 3 experimental setups. Parenthetical values represent the number biological replicates. Compounds
with robust biological activity are labeled in red.
Table 1. Photostationary state (PSS) of TRPswitches at 365 nm and 495 nm determined by UV-Vis spectroscopy in
30% water:DMSO.
Estimated 365 nm PSS
Estimated 495 nm PSS
Compound
(E-Z)
(Z-E)
TRPswitch-A
TRPswitch-B
86% Z
92% Z
73% E
100% E
To further characterize the thermal half-lives of
TRPswitches in conditions that more closely resemble
those used in biological experiments, we measured their
thermal half-lives at higher water contents (30%
water:DMSO solutions). We found that the thermal half-
lives of TRPswitch-A and -B are 43 min and 1 hour,
respectively. When measurements were made in DMSO-
d6, the half-lives for TRPswitch-A and -B were 11 and 17
hours, respectively.
To further characterize the photochemical properties of
TRPswitches, the photostationary state (PSS) of
TRPswitches in 30% water:DMSO solutions were examined
via UV-Vis. By extrapolating a pure Z spectrum, using
methodology outlined in previous publications,32, 39, 41 we
approximate the PSS compositions to be 86% Z at 365 nm
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and 73% E at 495 nm for TRPswitch-A; and 92% Z at 365 superior, near quantitative photoswitching compared to
1
2
3
4
nm and 100% E at 495 nm for TRPswitch-B (Table 1). In
addition, we also examined the PSS of TRPswitches in
DMSO-d6 at 365, 420 and 495 nm (Supp. Table 2, Figs.
S11-16). From our analysis, TRPswitch-B demonstrates
the azobenzene analogue, at least under the conditions
tested. These data suggest that photoswitch performance,
particularly Z-isomer half-life, contributes to the activity
and that both azobenzene
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Figure 3. Properties of TRPswitch. (a) Thermal kinetics of TRPswitch-A and TRPswitch-B at room temperature. The
natural log of the Z isomer percentage is plotted against time. (b) Activity of TRPswitch requires Trpa1b. Bar graph
showing the average light-induced motion response of the different treatment groups. WT, Wild-type; Trpa1b-/-, Trpa1b
mutant. Values are means ± SEM from 3 experiments. (c) Line graph indicating the positive correlation between the
percentage of wild-type larvae that were pretreated with TRPswitch and showed light-induced motion response with
increasing duration of light stimulus. Values are means ± SEM from 3 experiments. (d) Bar graph showing the response
latency (time from the beginning of light pulse to the first motion response) with various intensities of light stimulation
as indicated. Values are means ± SEM 3 experiments. (e) Dose response curve showing TRPswitch’s effects on animal
behavior (n = 3). (f) Isomerization of TRPswitch-B as indicated by the absorbance profile of TRPswitch before
irradiation (black dotted line), after violet light (352 - 402 nm) irradiation (magenta line), and subsequently after green
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light (500 - 600 nm) irradiation (green line). (g-h) Electrophysiology analysis of TRPswitch. Representative peak
whole cell Trpa1b current densities measured at -100 mV (blue trace) and +100 mV (black trace) with TRPswitch-A
(g) and TRPswitch-B (h) treatment. Violet light and green light illumination were applied at the time indicated by the

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Table 2. Activity of TRPswitch-B on Trpa1b ohnolog and orthologs.
1
2
3
Trpa1b-/- with transient expression of
Trpa1b-/-
WT
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5
6
7
8
9
zTrpa1b
38.5
zTrpa1a
mTRPA1
hTRPA1
% response
N
0
0
0
0
65
20
26
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16
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Rescue experiments were performed in Trpa1b-/- zebrafish with transient mosaic re-expression of zebrafish Trpa1b
(zTrpa1b), zebrafish Trpa1a (zTrpa1a), mouse TRPA1 (mTRPA1) or human TRPA1 (hTRAP1) in Rohon-beard neurons.
Embryos were pre-treated with 20 µM TRPswitch-B and decapitated right before light stimulation to elimination the light
response mediated by the retina. % response is the percentage of embryos exhibited light-induced motion response
within 5 s after the 1 s WL3 illumination. Only the re-expression of zTrpa1b in Rohon-beard neurons in Trpa1b-/- embryos
rescued the TRPswitchB-mediated light-induced motion response. N, number of embryos tested.
(TRPswitch-A) and azoheteroaryl (TRPswitch-B) moieties
can be used as PCLs of Trpa1b, with the latter appearing
to have an improved intrinsic photoswitch performance.
Additionally, we have obtained the X-Ray crystal structure
assay, the light-induced motion response does not
require sustained channel activation, i.e., the duration of
motion does not directly correlate with the half-life of the
TRPswitch Z state. To characterize the stability and
kinetics of Trpa1b/TRPswitch-dependent photocurrents,
we recorded zTrpa1b activity in HEK293T cells. The light-
induced activation and deactivation of Trpa1b/TRPswitch
was triggered with violet and green light pulses,
of TRPswitch-B (Fig. S3).
Notably, this compound
crystallizes in a chiral space group, with the azo bridge
orientated to take advantage of a favorable hydrogen
bonding interaction from the ortho amide.
respectively (Fig. 3g and 3h).
Importantly, the
TRPswitch is a reversible photoswitch ligand for
Trpa1b
photocurrents were sustained after the initial 10 sec pulse
of violet light illumination and did not require continuous
illumination. The photocurrents could be converted back
to baseline using a subsequent 10 sec pulse of green light.
The current density fold increments of TRPswitch-A and
TRPswitch-B upon violet light illumination were 2.4 ± 0.5
and 2.3 ± 0.6 at +100 mV and 4.4 ± 1.4 and 2.3 ± 0.4 at -
100 mV, respectively. A subsequent pulse of green light
reduced the current density fold increments of TRPswitch-
A and TRPswitch-B to 1.2 ± 0.2 and 1.3 ± 0.3 at +100 mV
and 3.7 ± 1.3 and 1.4 ± 0.4 at -100 mV, respectively.
Together, these data indicate that TRPswitch-A and
TRPswitch-B are novel, reversible photoswitches that act
on the Trpa1b channel. Overall, the light-activated
currents with TRPswitches are comparable to those
induced by the canonical TRPA1 agonist AITC (Fig. 3i).
We previously identified optovin as a photoactivable
ligand for the Trpa1b channel, and confirmed that its
activity is abolished in Trpa1b mutant zebrafish.23-24 To
determine if Trpa1b is necessary for TRPswitch’s biological
activity, we performed a behavioral assay using Trpa1b
mutant larvae.35 When Trpa1b mutant larvae were used,
the TRPswitch light-induced motion response was
abolished (Fig. 3b). This suggests that Trpa1b is required
for the activity of TRPswitch in vivo, consistent with our
electrophysiology analysis (Fig. 1d). Both TRPswitch-A-
and TRPswitch-B-treated larvae showed
a
higher
probability of light-induced motion response as the light
stimulation duration increased (Fig. 3c). The probability
for larvae to respond to light reached its maximum with a
<1 s light-pulse length. The biological response triggered
by TRPswitch/Trpa1b activation was very rapid (Supp.
Movie 1). The latency to motion response from the
introduction of light was in the range of milliseconds and
To test the potential cross activity of TRPswitch-B on the
zebrafish ohnolog Trpa1a and on mammalian orthologs,
we transiently re-expressed zebrafish Trpa1a, mouse
TRPA1 or human TRPA1 in the Rohon-beard neurons of
mutant Trpa1b-/- zebrafish and performed light-induced
motion response experiments in the presence of
TRPswitch-B. Only re-expression of Trpa1b rescued the
light-induced motion response of Trpa1b-/- mutants
(Table 2). Our results suggest that TRPswitch-B is specific
to the zebrafish Trpa1b channel.
decreased with increasing light intensity (Fig. 3d).
A
positive correlation was observed between the activity of
TRPswitch and an increase in compound concentration,
with maximum activity achieved at concentrations
between 10 – 20 µM (Fig. 3e). Similar to TRPswitch-A,
TRPswitch-B undergoes reversible E/Z isomerization upon
illumination with violet and green light. However, unlike
TRPswitch-A, the Z-to-E conversion by green light leads
to complete return to the pre-illumination “off” state (Fig.
3f).
TRPswitch’s mechanism of action
Most known activators of the TRPA1 channel are
electrophilic ligands that covalently modify cysteines in
TRPA1’s cytoplasmic domain.42 Optovin, our previously
identified photoactivable TRPA1 ligand, reacts with those
Next, we turned to characterize the kinetics of Trpa1b
channel activity. However, in the behavioral screening
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cysteines through a photochemical reaction involving the reversible photoswitches for the Trpa1b channel and have
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generation of singlet oxygen species. DABCO, a singlet
oxygen quencher and triplet energy acceptor can
completely suppress the optovin response.23 To
determine whether or not TRPswitch is also
photoactivated in a singlet oxygen-based mechanism, we
tested the ability of DABCO to suppress TRPswitch activity
and found that it did not (Fig. 3j). These data suggest that
light-induced generation of singlet oxygen is not
necessary for TRPswitch’s behavioral effect. As the
TRPswitches contain either azobenzene or azopyrazole
groups, it is likely that their activity is due to the E/Z
isomerization of the compounds upon light illumination
(Fig. 1e and 3f), without the production of radicals. These
observations suggest that both TRPswitch-A and -B are
a distinct mechanism of activation as compared to
electrophilic ligands.
Trpa1b/TRPswitch allows cellular activation in non-
neuronal cells
To demonstrate the practical utility of TRPswitch in vivo,
we performed a heartbeat interruption experiment using
a
zebrafish transgenic line expressing Trpa1b in
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cardiomyocytes, Tg(cmlc2:Trpa1b-2A-EGFP), and by
applying pulses of violet and green light to the zebrafish
heart. Since proper calcium handling is important for
normal heartbeat, we used heartbeat as the biological
readout for the activation and deactivation of Trpa1b
channels. During regular
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larvae in vivo. Experiments were performed on Trpa1b-/-
larvae expressing Trpa1b in cardiomyocytes. Larvae were
pre-incubated with TRPswitch for 1 h in the dark before
experimental manipulation. (a) Image of zebrafish heart
showing the ventricle chamber and the position where
ventricle width measurements were made and displayed in
panel b. (b) Representative line graph showing the
ventricle width over time in larvae treated with TRPswitch-
B. The magenta and green vertical rectangles indicate
photoactivation with 1 s violet light and green light,
respectively (Supp. Movie 2). (c-d) Repetitive stopping and
re-starting of ventricle heart contractions by violet and
green light, respectively. Dotted plot showing the ventricle
contraction frequency in individual larvae illuminated with
a series of light flashes as indicated in the x-axis. Each dot
represents measurement from one larva. Larvae were
treated with 20 µM TRPswitch-A (c, Supp. Movie 3) or
TRPswitch-B (d, Supp. Movie 4). (e) TRPswitch induced a
sustained heart contraction after a brief pulse of violet light.
Transgenic zebrafish expressing Trpa1b in cardiomyocytes
were pre-treated with 20 µM TRPswitch. 1 s violet light was
applied to the heart and the ventricle heart diastolic width
ratio (comparing the diastolic width before and after
photoactivation) was quantified. Values are means ± SEM
from 3 experiments. In all panels, parenthetical values
represent the number biological replicates.
heartbeat cycles, an increase in intracellular calcium is
required for cardiomyocyte contraction as calcium
binding to troponin C leads to a conformational change
that displaces tropomyosin from the actin binding sites.
Calcium levels must then decrease for cardiomyocyte
relaxation as calcium prevents tropomyosin from
returning to its original conformation. We hypothesize
that a sustained elevation of intracellular calcium, such as
with TRPA1 activation, will result in a sustained tetanic
contraction of the heart and interrupt rhythmic beating.
In transgenic larvae treated with either TRPswitch-A or
TRPswitch-B, we found that the ventricle heartbeat could
be stopped and would persist in a sustained systolic state
(tetany) after a brief 1 s illumination with violet light (Fig.
4a-d, Supp. Movie 2). Normal ventricular rhythm was
restored after briefly illuminating the heart with 1 s of
green light (Fig. 4a-d, Supp. Movie 2). These data suggest
that the reversible E/Z isomerization of the TRPswitches
Figure 4. Reversible and repeatable activity of TRPswitch.
(a-d) Light-controlled heartbeat interruption in zebrafish
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Journal of the American Chemical Society
by violet and green light induced in vivo activation and
deactivation of Trpa1b channels, respectively. The ability
photoswitchable
compound.
Importantly,
the
TRPswitches allow for sustained channel activation after
only a brief pulse of violet light illumination, but the
channel can also be rapidly deactivated with green light
illumination. As only short pulses of light are required to
control the activity of the TRPA1 channel, cells subjected
to the TRPA1/TRPswitch chemo-optogenetic system are
less prone to photo-toxicity. This requirement of a short
photoactivation period for target activation is a unique
and advantageous feature among current PCLs for
unmodified ion channels11-15 and receptors.44
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for TRPswitch/light to induce tetany depends on the
number of cardiomyocytes with Trpa1b expression. In
larvae with transient mosaic expression of Trpa1b in
cardiomyocytes treated with TRPswitch-B, ventricle tetany
can only be induced when at least around 15% of
cardiomyocytes in the ventricle have Trpa1b expression
(Fig. S4). Furthermore, there was no significant difference
in heart rate among transgenic larvae treated with DMSO
control or either TRPswitch in the dark (Fig. S5a),
suggesting that the TRPswitches have no effect on Trpa1b
channels without light illumination. Treating larvae that
lack exogenous expression of Trpa1b in cardiomyocytes
with TRPswitch-B and light illumination resulted in similar
negative results (Fig. S5b), indicating that the switching off
and on of heartbeat with violet and green light
illumination was due to TRPswitch isomerization and the
subsequent effect on Trpa1b channel activity.
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The TRPA1 channel’s high conductance combined with
the step function property of the TRPA1/TRPswitch
chemo-optogenetic
system
offers
certain
new
opportunities for basic research. As TRPswitch activity is
specific to zebrafish Trpa1b, TRPswitch can be used in
heterologous applications by expressing zTrpa1b in
animals or cells with endogenous mammalian TRPA1
expression, without interference from the endogenous
channel. This new tool will be particularly beneficial in
applications where a large depolarization current is
needed, such as in large primary motor neurons, or when
sustained channel activation is desirable. In addition,
tools for manipulating TRPA1 activity are relevant for
medical research since TRPA1 is involved in inflammatory
and neuropathic pain, itch, and respiratory diseases.20-21, 45
TRPswitch may prove useful as a research tool to help
dissect the mechanism of TRPA1-related disease, as well
as to identify disease-modifying agents.
Photoswitchable control of Trpa1b activity by the
TRPswitches was repeatable.
Cyclical rounds of
illumination using violet and green light induced the
stopping and restarting of the ventricle heartbeat (Fig. 4c
and 4d; Supp. Movie 3 and 4). Additionally, channel
activation after a brief pulse of violet light was sustained
on a timescale of minutes, as measured in the gradual
relaxation of ventricle width over time (Fig. 4e). As a
further demonstration of the utility of the heterologously
expressed zTrpa1b/TRPswitch system, we expressed
zTrpa1b in human colonic fibroblast cells, CCD-18Co,
which express human TRPA1 endogenously.43 The
expression of human TRPA1 channel in CCD-18Co cells
was verified with RT-PCR (data not shown). CCD-18Co
cells responded to AITC, confirming the functional
expression of human TRPA1 channel (Fig. S6a). As
expected, TRPswitch showed no light induced
photocurrent in CCD-18Co cells without the expression of
The TRPA1/TRPswitch chemo-optogenetic system
offers several advantages over existing tools, and we have
shown it to be robust and easy to use in cultured
mammalian cells and in zebrafish. Nevertheless, its
suitability for other in vivo and clinical applications
remains unknown. TRPswitch is a freely diffusible small
molecule, and exposing zebrafish larvae by incubating
them in a solution containing TRPswitch is sufficient for
robust activity. However, its uptake and distribution in
rodents and larger mammals remains to be determined.
In zebrafish larvae incubated with TRPswitch for several
hours, no obvious adverse physiological phenotypes were
observed. The TRPswitches show minimal activity on
TRPA1 before photoactivation, highlighting the specificity
of the light-induced effect of TRPswitch. On the other
hand, as the TRPswitches display a wide range of spectral
activity, it may be difficult to combine a TRPswitch with
other optogenetic tools or fluorescence-based biosensors
due to potential spectral overlap. The use of violet light
for TRPswitch activation may also limit the tissue depth of
its activation; however, future studies could explore the
possibility for multi-photon 46 or longer near infrared (NIR)
light excitation.47
zTrpa1b (Fig. S6b).
When CCD-18Co cells were
transfected with zTrpa1b and incubated with TRPswitch-
B, they exhibited violet light-induced photocurrent and
green light-induced decrease in photocurrent (Fig. S6c).
These data provide further evidence that TRPswitch-B has
specific activity on zTrpa1b and does not cross react with
endogenously expressed human TRPA1 channel. Taken
together, these data suggest that the TRPA1/TRPswitch
pair constitutes a reversible and repeatable chemo-
optogenetic system that is compatible with use in
zebrafish and mammalian cells.
Discussion
These studies have identified TRPswitch A and B, two
photoswitchable small molecules that enable optical
control of currents in Trpa1b expressing cells in vivo. Our
data suggest that the TRPswitches specifically target
Trpa1b channel and enable repeatable optical control of
both neuronal and non-neuronal cells. This is the first
Our analysis suggests that both TRPswitch-A and
TRPswitch-B have thermal half-lives in the scale of hours
in DMSO. However, the half-life of the biological
response to these compounds was measured to be in the
example of TRPA1 channel activation by
a
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timescale of minutes during in vivo cardiac experiments intact animals, hit compounds identified are, by definition,
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(Fig. 4e). Several factors may account for the shorter half-
life observed in vivo. Firstly, the thermal half-life of
TRPswitch would be different under the conditions of the
biological assays, for example in aqueous solution with
biologically active and likely to have minimal general
toxicity. In the case of TRPswitches, it is noteworthy that
they are specific for the zebrafish Trpa1b channel and are
inactive at the mammalian channels tested. It remains to
be seen whether modification of the TRPswitch scaffold
can yield ligands with different species specificity, but the
current specificity for zebrafish Trpa1b has significant
advantages, including the ability to use the
TRPswitch/Trpa1b system in mammalian cells without
unwanted cross-reactivity with endogenous channels.
only 1% DMSO.
In fact, the thermal half-life of
TRPswitches become shorter when measured in 30%
water:DMSO (43 min and 1 hour for TRPswitch-A and -B,
respectively).
Secondly, although we found that
9
TRPA1/TRPswitch light-induced activity is reversible and
repeatable, TRPswitch does not covalently bind to TRPA1
and is therefore free to diffuse away from the channel over
time. Thirdly, there might be biological adaptation during
TRPA1 activation, such as receptor internalization, that
leads to a shorter time of biological effect compared to
the half-life measured by UV-Vis in vitro.
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It should also be noted that despite having excellent
and tunable switching properties, heteroaryl azo motifs
are still significantly underexplored compared to their
azobenzene
counterparts
in
photoaddressible
applications including as PCLs.54 Our discovery that the
azopyrazole-containing TRPswitch-B exhibits a longer
half-life and more efficient photoswitching than the
azobenzene-containing TRPswtich-A is a good indication
that heteroaryl azo photoswitches are good alternatives
for photoswitch optimization. Indeed, this is the first
application of the high performance arylazopyrazoles 32, 40
for in vivo photopharmacology, showcasing the potential
of this scaffold for future studies.
Most of the known TRPA1 ligands are electrophiles that
activate TRPA1 via the covalent modification of cysteine
residues present in the channel’s cytoplasmic ankyrin
repeat domain.48 This includes our previously identified
TRPA1 photoactivatable ligand, optovin.23-24 Although
both optovin and TRPswitch target TRPA1 and are
activated by violet light, they are unique in their
mechanism of action, reversibility, and kinetics. Unlike
optovin and its derivatives, which act though light-
induced
covalent
modification,
TRPswitch’s
photoreversible TRPA1 activity depends on differential
activity of its E and Z isomers. How isomerization triggers
opening and closing of the TRPA1 channel remains
unknown. Mechanistically, the fifth transmembrane (TM5)
domain of TRPA1 has been shown to determine channel
sensitivity to non-electrophilic agonists such as menthol
and anethole.49-50 Considering that menthol and anethole
are structurally distinct molecules, we speculate that
TRPA1’s TM5 may act as a general binding site for non-
electrophilic agonists and may therefore be a candidate
site for the binding of TRPswitch. Alternatively, a
peptidergic scorpion toxin (WaTx) was recently discovered
that activates TRPA1 by binding to the same allosteric
nexus that is covalently modified by electrophilic
irritants.51 It is possible that TRPswitch binds to this
allosteric nexus on TRPA1 channel as well.
The development of PCLs for endogenous targets offers
theoretical advantages in clinical applications, since the
introduction of exogenous gene products is not needed.
The common approach for PCL discovery is to modify a
known biologically active molecule with photoswitchable
functionality, such as ‘azologization’.52 Although this
approach has had some success,11-15 it is limited by the
need for already identified active molecules for specific
targets, which have a defined mechanism of activation
and that contain a chemical structure amenable to
incorporation of a photoswitch. The screening strategy
described here offers an alternative, phenotypic approach
for the discovery of photoswitchable ligands of novel
endogenous targets.53 Since screening is performed using
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excitation of samples and bright field acquisition. Schott
Materials and Methods
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2
3
longpass absorption glass (RG610; Chroma) was added in
the transmitted light path to reduce unwanted excitation
to the well and to provide sufficient light for video
recording. All behavioral experiments were conducted at
room temperature. For Fig. 3c and 3d, experiments were
performed using an inverted compound microscope
(AxioObserver A1; Zeiss) equipped with an EMCCD
camera (C9100; Hamamatsu), a violet LED light source
(415 nm) with a CW 310 mW maximum output power
source (BLS-LCS-0415-03-22; Mightex) which was
controlled by a BioLED light source control module (BLS-
SA02-US; Mightex), and a pulse master multi-channel
stimulator (A30; World Precision Instruments). Bright field
time-lapse was captured for 500 frames at ~103 FPS. A
light pulse was applied at frame 20. For Fig. 3c,
percentage responding was quantified based on whether
there was any motion in the entire acquisition period. For
Fig. 3d, response time is the duration from the beginning
of the light pulse to the first movement of the larvae. Light
intensity was measured using a hand-held laser meter
(LaserCheck, Coherent 1098293). For Trpa1b ohnolog
and paralog rescue experiments, Trpa1b-/- embryos were
injected with ngn1:zTrpa1b-2A-mCherry, ngn1:zTrpa1a-
2A-mCherry, ngn1:hTRPA1-2A-EGFP or ngn1:mTRPA1-2A-
mCherry at the 1-cell stage for mosaic Rohon-beard
neuron expression. Embryos were screened at 2 dpf for
fluorescent expression in Rohon-beard neurons,
incubated with 20 µM TRPswitch-B for 1 hr and
decapitated posterior to the eyes right before
experiments. NA 0.25 5x objective and 1 s WL3 light
illumination was used.
Zebrafish
Zebrafish (Danio rerio) wild-type TuAB or trpa1b mutant
35
larvae were used for all experiments. Zebrafish embryos
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were produced using group mating of adult zebrafish.
Larvae were raised in E3 media and maintained in an
incubator with a 14 h light/10 h dark cycle at 28.5C until
experiments.
The maintenance of adult animals,
9
obtaining of embryos and larvae, and all experimental
procedures were carried out according to protocols
approved by the University of Utah’s Institutional Animal
Care and Use Committee (IACUC).
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Chemical libraries and treatments
A total of 1,000 compounds were selected from the
ChemBridge Corp. catalog. Compounds were dissolved in
DMSO at a stock concentration of 1 µg/µl (~1.5 mM). The
library was screened at a 1:100 dilution in E3 solution for
a final concentration of ~15 µM. Negative controls were
treated with an equal volume of DMSO. Groups of 3
larvae at 3 days post fertilization (dpf) were distributed
into the wells of 96-well clear bottom black microplates
(07-200-567; Fisher Scientific) in 150 µl E3 before the
addition of small molecules.
Stock solutions of
compounds were added directly to zebrafish in the wells
of a 96 well plate (Corning 3631), mixed, and allowed to
incubate for 1 h in the dark at 28.5C prior to behavioral
evaluation in the behavioral assay. Ordering information:
1 (5533696; ChemBridge); 3 (5538408; ChemBridge).
Other compound synthesis and characterization
information are described in the supplementary note.
Behavioral assay
Behavioral analysis
Larvae in each of the wells were exposed to four 1
second flashes of light stimulus in the following order:
450-500 nm (WL1), 415 – 455 nm (WL2), 352-402 nm
(WL3), white light (WL4), with a 5 second inter-stimulus
interval. Digital video recording was performed for 5
seconds before the first light stimuli and continued
throughout the stimulation sequence. Band pass filters
(Semrock FF02-475/50, FF02-435/40, FF01-377/50) were
used to restrict the excitation light to the indicated
wavelengths. Light-induced motion response was used as
the assay readout as DMSO treated control larvae do not
respond to light stimulus. 290 frames of digital video
were recorded per well at 10 FPS using an EMCCD camera
(C9100; Hamamatsu) mounted on an inverted compound
microscope (AxioObserver A1; Zeiss) with a NA 0.03 1.25x
objective and a barrier with a 0.7 mm diameter opening
to restrict light scattering to the sample. MetaMorph
software (Molecular Devices) was used to control the
execution of TTL signals and camera capture using the
built-in stream acquisition with trigger function. Each
video was saved for review. Light stimuli were generated
with an ozone free 300 Watt xenon bulb housed in a
Lambda LS illuminator (Sutter Instruments). A dichroic
mirror (T510LPXRXT; Chroma) was used for appropriate
To analyze digital video recordings, custom MetaMorph
software scripts were used to automatically threshold the
video to identify the area of larvae in each frame.
Threshold images of the frames after each light activation
event were overlaid to calculate a new combined
threshold area. The motion index was calculated as the
percentage change between the threshold area in the
frame right before light activation and the new combined
threshold area. This motion index correlates with the
overall amount of motion in the well.
Electrophysiology analysis
Immortalized HEK293T cells or human colonic fibroblast
cells (CCD-18Co; ATCC) were used. HEK293T cells were
plated on 12 mm cover slips and transiently transfected
with 10 μg of Trpa1b plasmid (pCMV-zTrpa1b-FLAG) and
5 μg of mNeonGreen cDNA (Allele Biotechnology), then
grown in a 6 cm plate for 24-48 h. CCD-18Co were
cultured in Eagle’s minimal essential medium with 10%
FBS, 1x penicillin and streptomycin. CCD-18Co cells were
plated on 12 mm cover slips and transiently transfected
with 5 μg of zTrpa1b plasmid (pCMV-zTrpa1b-FLAG)
together with 5 μg of mNeonGreen cDNA (Allele
Biotechnology), then grown in a 10 cm plate for 48-72 h.
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After this time the cover slips were transferred to a and UV-Vis absorbance was measured using a NanoDrop
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recording chamber containing an extracellular solution
composed of (in mM): 145 NaGluconate, 4 KCl, 3 MgCl2,
10 D-glucose, 10 HEPES; pH 7.4 adjusted with NaOH. The
internal solution contained (in mM): 122 Cesium methane
sulfonate, 1.8 MgCl2, 9 EGTA, 14 creatine phosphate (Na-
salt), 4 MgATP, 0.3 NaGTP, 10 HEPES, pH 7.2 adjusted with
CsOH. Borosilicate glass pipettes with a resistance of 3–5
MΩ were used. Whole cell currents were measured on an
Axopatch 200B amplifier (Molecular Devices) under the
control of pClamp software. Signals were digitized
through a Digidata1550B interface (Molecular Devices).
Currents were filtered at 5 kHz (lowpass, Bessel) and
sampled at 10 kHz prior to analysis with Clampfit software
(Molecular Devices). All the plots and statistical tests were
performed on Excel (Microsoft corp.). Basal Trpa1b whole
cell currents were measured with a ramp protocol (-100 to
+100 mV, at a holding potential of 0 mV). Photoactive
compounds were perfused into the recording chamber
and a fluorescent light generated by mercury vapor short
arc (U-HGLGPS, OLYMPUS) filtered through a ET-ECFP
434/17 nm filter and through a ET-mCherry 560 nm filter
(Chroma Technology) was switched ON and OFF for 10 s.
All experiments were performed at room temperature.
The p values were calculated using a two-tailed, paired
Student’s t-test.
1000 (Thermo Scientific).
Statistical Analyses
All results are expressed as means ± SEM. Unless
otherwise indicated, a two-tailed unpaired student’s t test
with
determine p values.
significance was p < 0.05.
Mann-Whitney
post-test
criterion
Statistical analysis was
was
used
statistical
to
The
for
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performed using Prism (GraphPad Software).
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Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website.
Additional experimental data (Figures S1-6, thermal
isomerization kinetics (Figures S7-10), photostationary
state determination (Figures S11-18), supplemental tables
1-2, general methods of compound synthesis, synthetic
route of compounds, NMR spectra, and supplemental
movie legends as described in the text) (PDF)
Movie 1 (AVI)
Movie 2 (AVI)
Movie 3 (AVI)
Movie 4 (AVI)
Heart experiments
AUTHOR INFORMATION
Heartbeat interruption experiments were performed in
vivo on Tg(cmlc2:Trpa1b-2A-EGFP)24 larvae or Tol2-cmlc2-
Trpa1b-2A-EGFP plasmid24 (Addgene plasmid #106405)
injected larvae in a Trpa1b-/- background. Expression of
the plasmid was obtained by microinjecting 2 nL of a
solution containing 7 ng/µl of DNA plasmid and 6.5 ng/µL
in vitro transcribed (Ambion) Tol2 transposase mRNA into
the cytoplasm of one-cell stage embryos. At 2 dpf, larvae
were pretreated with 20 µM of TRPswitch-A or TRPswitch-
B in E3 at a final concentration of 1% DMSO for 1 h in the
dark at 28.5C before experimental manipulation.
Immediately prior to the onset of experiments, E3 solution
was replaced with 0.2 mg/mL tricaine (Sigma, A5040) in E3
for anesthetizing the larvae. Violet light (352-402 nm) and
green light (500-600 nm) were used for E to Z and Z to E
Corresponding Author
* randall.peterson@pharm.utah.edu
ACKNOWLEDGMENT
This work was supported by the National Institute Of
Neurological Disorders And Stroke of the National
Institutes of Health under Award Number K99NS112599
(P.Y.L.), an American Heart Association Postdoctoral
Fellowship #16POST29680003 (P.Y.L.), R01HL141353 (D.C),
R00HL124070 (D.C.), R01DK082971 (R.T.P.) and by funding
from the L. S. Skaggs Presidential Endowed Chair (R.T.P).
We would also like to thank the UK Engineering and
Physical Sciences Research Council for funding, including
an Established Career Fellowship to M.J.F. (EP/R00188X/1).
Mouse TRPA1 cDNA plasmid used for subcloning was
kindly provided by Greg Lamb and Christopher Reilly.
isomerization, respectively.
Violet and green light
excitation were achieved using a band pass filter FF01-
377/50 and an et500lp long pass barrier filter, respectively,
together with a t600plxxr dichroic mirror and a 300 Watt
xenon bulb light source (Sutter Instrument). Experiments
were performed using a NA 0.6 40x air objective at room
temperature. The zebrafish heart in the whole field of
view was illuminated with 1 s violet light followed by
various lengths of green light as indicated. Ventricle width
measurement was performed using the ImageJ ‘measure’
function on the widest outer ventricle width every 100 ms.
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