Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches

Article abstract of DOI:10.1021/ja5026326

Synthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution.

Full text of DOI:10.1021/ja5026326

Article
pubs.acs.org/JACS
Two-Photon Neuronal and Astrocytic Stimulation with Azobenzene-
Based Photoswitches
Merce Izquierdo-Serra,†,+ Marta Gascon-Moya,‡,+ Jan J. Hirtz,§ Silvia Pittolo,† Kira E. Poskanzer,§
̀
́
Eric Ferrer,†,‡ Ramon Alibes,‡ Felix Busque,‡ Rafael Yuste,*,§ Jordi Hernando,*,‡ and Pau Gorostiza*,†,∥,⊥
̀
́
́
́
†Institut de Bioenginyeria de Catalunya, 08028 Barcelona, Spain
‡Departament de Química, Universitat Auton
oma de Barcelona, 08193 Cerdanyola del Valles
§Department of Biological Sciences, Columbia University, New York, New York 10027, United States
̀
̀
, Spain
∥Centro de Investigacion
́
Biomed
́
ica en Red en Bioingeniería, Biomateriales y Nanomedicina, 50018 Zaragoza, Spain
ts, 08010 Barcelona, Spain
⊥Institucio
́
Catalana de Recerca i Estudis Avanca
̧
S
* Supporting Information
ABSTRACT: Synthetic photochromic compounds can be
designed to control a variety of proteins and their biochemical
functions in living cells, but the high spatiotemporal precision
and tissue penetration of two-photon stimulation have never
been investigated in these molecules. Here we demonstrate two-
photon excitation of azobenzene-based protein switches and
versatile strategies to enhance their photochemical responses.
This enables new applications to control the activation of
neurons and astrocytes with cellular and subcellular resolution.
protein.19−22 Remarkably, two-photon stimulation of synthetic
photoswitchable proteins has not been investigated despite the
advances of neurotransmitter uncaging23 and optogenetics24,25
using pulsed NIR illumination.
INTRODUCTION
■
The large number of photoswitchable biomolecules discovered
and developed in recent years covers a great variety of cellular
functions, like catalysis of metabolic processes,1,2 cytoskeletal
polymerization3 and motors,2,4 nucleic acids dynamics,5−7
intracellular signaling,8,9 and, perhaps most dazzling, membrane
excitability, which has been at the focus of optogenetics10 and
optopharmacology.11 The dream of precisely and remotely
photocontrolling every aspect of the cell’s inner workings in
intact tissue appears within reach and offers the promise of
interrogating complex cellular processes to discover their
molecular mechanisms.12
In order to take full advantage of light-regulated proteins,
multiphoton excitation with near-infrared (NIR) light provides
sub-micrometric resolution in three dimensions,13 deep
penetration into tissue,14 and patterned illumination.15,16
However, to be adapted to two-photon stimulation technology,
the light response of natural photoswitchable proteins like
Channelrhodopsin-2 (ChR2) must often be adjusted by
mutating the tight binding pocket of the natural chromophore,
which has fixed photochemical characteristics.17,18 In contrast,
synthetic photoswitches developed by optochemical genetics
and optopharmacology are based on chromophores that act on
the protein surface and thus offer excellent opportunities for
rationally tuning their photochemical behavior by chemical
substitutions that do not affect the functional properties of the
To demonstrate the multiphoton activation of synthetic
photoswitches, we chose ion channels because they constitute
highly sensitive transducers of chromophore isomerization
(potentially up to the single channel level). In particular, we
focused on the well-characterized light-gated glutamate
receptor (LiGluR),26,27 a GluK2 kainate receptor-channel that
is chemically conjugated to a maleimide−azobenzene−gluta-
mate photoswitch (MAG 1, Figure 1a). Azobenzene trans−cis
photoisomerization28 of this photoswitchable tethered ligand
(PTL) allows the efficient activation of the receptor upon one-
photon absorption of violet or blue radiation (open LiGluR,
Figure 1b), a process that can be reverted back either by
absorption of green light or thermal relaxation in the dark
(closed LiGluR, Figure 1b).21,26,27
To control LiGluR using multiphoton excitation, here we
have investigated the performance of MAG and two new MAG
derivatives (2 and 3, Figure 1a) upon pulsed NIR illumination.
Compounds 2 and 3 were devised to enhance the two-photon
excitation response of the symmetrically substituted azoben-
Received: March 19, 2014
Published: May 23, 2014
© 2014 American Chemical Society
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single-wavelength operation of the switch. This behavior is also
expected for 3 containing the same azobenzene core as MAG2p.
However, a novel scheme was exploited in this compound to
enhance its nonlinear optical response, which consists in the
introduction of a light-harvesting antenna to sensitize the
trans−cis isomerization of the system by absorption of NIR
radiation and subsequent resonant electronic energy transfer
(RET) to the trans-azobenzene group.33 Because of its
maleimide−azobenzene−glutamate−antenna structure, we
named compound 3 as MAGA2p. A naphthalene derivative
was selected as antenna because of (i) its high two-photon
absorption cross-section,34 (ii) the large spectral overlap
between its emission and the absorption of the trans isomer
of the aminoazobenzene group in 3, and (iii) its reduced size, to
minimize potential steric hindrance effects on the glutamate-
binding site of the receptor.
RESULTS AND DISCUSSION
■
Synthesis of MAG2p and MAGA2p. The preparation of
compounds MAG2p and MAGA2p was achieved via a multistep
modular synthetic sequence allowing structural diversity in the
final compounds as well as the additional incorporation of a
photo-harvesting antenna in 3 (Scheme 1). In both cases, we
took the N,N-orthogonally diprotected L-lysine 4 as scaffold, to
which the different functional fragments of the target
compounds were sequentially introduced: O-protected amino-
azobenzene 5, fully protected glutamate derivative 6,
naphthalene derivative 7, and furan-protected maleimide 8.
These fragments were obtained from commercial products as
described in the Supporting Information. With respect to the
synthesis of MAG,26 several changes were realized in our
procedure. First, a branching point was inserted between the
glutamate and azobenzene moieties to facilitate the incorpo-
ration of additional functional units to the PTL structure.
Second, we introduce herein the use of 6 and 8 as more robust,
versatile, and convenient precursors of glutamate and
maleimide moieties during the multistep synthesis of novel
MAG derivatives.
The synthesis of both MAG2p and MAGA2p began by the
coupling reaction of 4 with aminoazobenzene 5 to afford the
common intermediate 9, from which the synthetic pathways
diverged. For the synthesis of MAG2p, acid removal of the tert-
butyl carbamate protection of 9 was followed by the coupling
reaction of the resulting amine with glutamate derivative 6,
basic deprotection of the terminal amine, and its acetylation to
furnish intermediate 12a. In the case of MAGA2p, the best
results were obtained by deprotecting first the amino terminus
and proceeding through its reaction with the antenna fragment
7 to deliver 11. Removal of the carbamate protection and
coupling with 6 then furnished compound 12b. From
intermediates 12a and 12b, the next synthetic steps were
analogous for both ligands: removal of the allyl protecting
group, introduction of the furan-protected maleimide 8 under
Mitsunobu conditions, release of the maleimide moiety via a
retro-Diels−Alder reaction, and cleavage of the tert-butyl
carbamate and ester protections, thus finally affording the
target compounds MAG2p and MAGA2p.
Figure 1. (a) Structures of the photoswitchable tethered ligands
applied to the two-photon control of LiGluR: MAG (1), MAG2p (2),
and MAGA2p (3). (b) Operating mode of different PTLs on LiGluR.
Violet (one-photon) or NIR (two-photon) light excitation induces
glutamate recognition and channel opening via trans−cis isomerization,
which results in ion flow across the membrane. This process is
reverted by illumination with visible light (one-photon excitation) for
LiGluR-MAG and by thermal back-isomerization for LiGluR-MAG2p
and LiGluR-MAGA2p.
zene chromophore in MAG, which is expected to be poor.29,30
We tested two design concepts using a modular architecture. In
compound 2 we introduced an asymmetric aminoazobenzene
with sufficiently strong push−pull character as to enhance its
two-photon absorption cross-section (MAG2p).29−32 In addi-
tion, the presence of the electron-donating tertiary amine in the
4-position should dramatically decrease the thermal stability of
its cis state in physiological conditions,21 thus resulting in fast
spontaneous cis−trans back-isomerization and, as such, enabling
Photochemical Characterization of MAG2p and
MAGA2p. Figure 2a plots the electronic absorption spectra of
the initial trans state of compounds 1−3 and of the photo-
harvesting antenna tethered to MAGA2p (see also Figures S1
and S2 in the Supporting Information). Owing to the 4-amino
substituent introduced in the azobenzene core of trans-MAG2p
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a
Scheme 1. Total Synthesis of MAG2p (2) and MAGA2P (3)
a
Reagents and conditions: (a) 5, HATU, DIPEA, THF (89%); (b) 37% HCl, MeOH (93%); (c) 6, EDCI, HOBt, DIPEA, THF (88%); (d) 20%
piperidine/DMF (87%); (e) 7, EDCI, DIPEA, THF (81%); (f) 20% piperidine/DMF (64%); (g) ClCOCH3, pyridine, THF (69%); (h) 37% HCl,
MeOH (93%); (i) 6, EDCI, HOBt, DIPEA, THF (71%); (j) RhCl(PPH3)3, EtOH/H2O, reflux; (k) HgO, HgCl2, acetone/H2O, reflux; (l) 8, Ph3P,
DIAD, THF (81%, over the three steps, for 13a, 27% for 13b); (m) toluene, reflux; (n) TFA, CH2Cl2 (81% over the two steps for 2, 86% for 3).
Abbreviations: HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; DIPEA, diisopropylethylamine; EDCI, N-
ethyl-N′-(3-dimethyldiaminopropyl)-carbodiimide HCl; HOBt, 1-hydroxybenzotriazole hydrate; DIAD, diisopropyl azodicarboxylate.
and trans-MAGA2p,21,35 the absorption maximum of the
azoaromatic π→π* electronic transition of these compounds
clearly bathochromically shifts with respect to trans-MAG (∼50
nm in DMSO). This allows the trans−cis photoisomerization of
MAG2p and MAGA2p to occur upon illumination with blue light
instead of violet radiation. As shown in Figure 2b, excitation of
both ligands at 473 nm led to a noticeable decrease of their π→
π* absorption band, a typical signature of photoinduced cis
spontaneous thermal cis−trans isomerization plays a significant
role in the recovery of the initial state of the ligands, and it
strongly competes with cis−trans photoisomerisation due to the
lower stability of their cis isomers. This effect is ascribed to the
introduction of a 4-amino substituent in the azobenzene moiety
of MAG2p and MAGA2p,21,35 and it is expected to be
dramatically enhanced in aqueous media.36 Thus, while the
lifetimes of cis-MAG2p and cis-MAGA2p in the dark at room
temperature are ∼75 min in DMSO (see Figure S4 in the
Supporting Information), they further drop off down to the
millisecond time scale in aqueous buffer (τ = 118 and 96 ms in
80% PBS: 20% DMSO, respectively; Figure 2c). This allows
repetitive trans−cis isomerization of MAG2p and MAGA2p at
high frequencies in aqueous media with a single irradiation
source, which we have exploited to demonstrate the high
isomer formation.35 This was further confirmed by H NMR
1
measurements in DMSO-d6, which revealed that the relative
concentration of cis-MAG2p and cis-MAGA2p in the resulting
photostationary mixtures was 58% in both cases. Such
photoproducts can be transformed into their corresponding
trans isomers by irradiation with green light, as previously
reported for MAG26,27 (Figure S3 in the Supporting
Information). In the case of MAG2p and MAGA2p, however,
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Figure 3. (a) Fluorescence emission spectra in 80% PBS:20% DMSO
of trans-MAGA2p and the photo-harvesting antenna tethered to this
ligand. The spectra are normalized relative to the excitation intensity
and the absorption at the excitation wavelength (λexc = 355 nm). (b)
trans−cis photoconversion efficiency of trans-MAG2p and trans-
MAGA2p upon irradiation at λ = 355 nm in DMSO, which allows
nearly selective excitation of trans-MAGA2p sensitizer (see Figure 2a).
Forster radius calculated for this donor−acceptor pair (see
̈
Figure S6 in the Supporting Information). Consequently,
photosensitized trans−cis isomerization should take place in
this ligand, as demonstrated in Figure 3b: ∼60% increase in
trans−cis photoconversion was determined for MAGA2p with
respect to MAG2p upon selective irradiation of the naphthalene
antenna at λexc= 355 nm.
Electrophysiological Characterization of MAG,
MAG2p, and MAGA2p under One- and Two-Photon
Stimulation. We next tested MAG, MAG2p, and MAGA2p to
photoswitch LiGluR in living cells using one- and two-photon
stimulation. We expressed GluK2-L439C-eGFP in HEK293
cells and incubated them in MAG, MAG2p, or MAGA2p in order
to allow the selective conjugation of the PTLs to the cysteine
introduced at position L439C of the receptor. For each
compound, we recorded the corresponding photocurrents
generated upon light-induced opening of LiGluR channels
using whole-cell patch clamp26,27 (see the Supporting
Information).
Figure 2. (a) Absorption spectra in DMSO of trans-MAG, trans-
MAG2p, trans-MAGA2p, and the free naphthalene photo-harvesting
antenna. Each spectrum was normalized to its maximum. (b)
Absorption spectra of trans-MAG2p and trans-MAGA2p (solid lines),
and the photostationary states obtained upon photoexcitation of these
compounds in DMSO at λexc = 473 nm (dashed lines). (c) Variation of
the absorption at λ = 450 nm of trans−cis mixtures of MAG2p and
MAGA2p in the dark at 25 °C in 80% PBS:20% DMSO. At these
conditions, thermal cis−trans back-isomerization takes place, thus
restoring the initial concentration of the trans state of the ligands,
which presents a larger extinction coefficient at λabs = 450 nm. Solid
lines correspond to the experimental data, while dashed lines were
obtained from monoexponential fits.
photostability of these light-responsive ligands (Figure S5 in the
Supporting Information).
Although the incorporation of a photo-harvesting antenna
negligibly affects the intrinsic photochemical behavior of the
azobenzene group of MAGA2p with respect to MAG2p, it
provides ligand 3 with some additional optical properties. Thus,
trans-MAGA2p displays an extra band in the absorption
spectrum (λmax = 385 and 380 nm in DMSO and 80% PBS:
20% DMSO, respectively), which arises from the naphthalene
sensitizer (Figure 2a and Figure S1 in the Supporting
Information). The fluorescence emission of this group is
however strongly quenched upon covalent attachment to the
ligand, with a ∼20-fold decrease in fluorescence quantum yield
One-photon LiGluR currents were obtained when the
receptor was conjugated with the new compounds (Figures 4
and 5a). The magnitude of the photocurrent response was not
reduced after repeated stimulations, demonstrating the photo-
stability of these compounds after protein conjugation (Figure
4b,c and Figure S8 in the Supporting Information). Figure 4
shows that for one-photon excitation, the wavelength depend-
ence of current amplitude measured is different for each PTL.
Photocurrent amplitudes at different wavelengths were
quantified from electrophysiological recordings obtained for
the three compounds (Figure 4a−c), and the corresponding
one-photon action spectra were calculated (Figure 4d).
Introduction of the 4-amino substituent in the azo core allows
the one-photon action spectra of MAG2p and MAGA2p to red-
shift ∼60 nm with respect to that of MAG, as recently reported
measured in aqueous buffer (Φantenna = 0.43 and Φtrans‑MAGA2p
=
0.02; Figure 3a). This indicates the occurrence of efficient RET
processes from the photoexcited naphthalene antenna to the
azo moiety of trans-MAGA2p, in agreement with the large
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Figure 5. (a) One- and (b) two-photon whole-cell voltage-clamp
recordings on HEK293 cells expressing LiGluR conjugated with MAG
(black), MAG2p (blue), and MAGA2p (red). Bars indicate stimulation
pulses applied to open (one-photon pulses in violet and blue, two-
photon pulses in gray) and close LiGluR (one-photon pulses in
green). Irradiation wavelengths are given in each case. Two-photon
excitation conditions: MAG (λ = 820 nm, 10 scans of 0.4 s, 38 mW on
sample), MAG2p (λ = 900 nm, 0.4 s scan, 30 mW on sample), and
MAGA2p (λ = 880 nm, 0.4 s scan, 42 mW on sample).
for a similar compound21 (Figure 4d and Table S1 in the
Supporting Information). An additional peak is observed for
MAGA2p at λ = 360 nm, which lies very close to the absorbance
band of the naphthalene moiety (see Figure 2a). Thus,
sensitization of the azobenzene photoisomerization by the
antenna also occurs when the photoswitch is conjugated to
LiGluR. In addition, the time course of the MAG2p and
MAGA2p one-photon currents (blue and red traces in Figure
5a) confirms that fast spontaneous cis−trans back-isomerization
and channel closure takes place after the illumination is
switched off, while it requires irradiation with green light for
MAG (black trace in Figure 5a). By fitting the one-photon
current decays in the dark with monoexponential functions, the
lifetimes of cis-MAG2p and cis-MAGA2p tethered to LiGluR
were determined to be 150 and 265 ms, respectively (Table S1
in the Supporting Information). These values are larger than
those measured in solution (see above), which suggests that the
ligand-binding site interaction slows down the thermal cis−trans
isomerization of the azobenzene-based switches. This effect is
enhanced for MAGA2p probably due to additional hydrophobic
interactions and/or steric hindrance effects arising from the
tethered naphthalene antenna.
Using a custom-built multiphoton setup where a tightly
focused fs laser is raster scanned over the cells of interest, all
three PTLs display robust and LiGluR-specific photocurrents in
living cells that first demonstrate two-photon stimulation with
NIR light of a synthetic photoswitchable protein (Figure 5b and
Figures S9 and S10 in the Supporting Information). The
amplitude of the responses follows the characteristic power
dependence of two-photon absorption processes (Figure S11 in
the Supporting Information) and corresponds to 10−20% of
the photocurrent under one-photon excitation (Table S2 in the
Supporting Information). In order to optimize the multiphoton
Figure 4. Whole-cell voltage-clamp current recordings in HEK293
cells expressing GluK2-L439C after conjugation to (a) MAG, (b)
MAG2p, and (c) MAGA2p. Current responses to one-photon light
pulses of wavelengths ranging from 325 to 575 nm were quantified.
(Note that in (a) resting λ = 500 nm induces LiGluR deactivation, and
in (b) and (c) resting λ = 690 nm is not absorbed and allows thermal
relaxation of these photoswitches.) (d) Normalized one-photon action
spectra corresponding to MAG (black), MAG2p (blue), and MAGA2p
(red) (N = 2 cells, N = 3−8 cells, and N = 4−10 cells, respectively).
Before averaging over different cells, wavelength-dependent current
amplitudes were normalized to the maximum photocurrent along the
spectral range measured for each cell. Errors are SEM.
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stimulation conditions we characterized the two-photon action
spectrum of each PTL (Figure 6a). The wavelength that yields
the end of each stimulus, with time constants similar to those
obtained with one-photon illumination (Table S2 in the
Supporting Information), which enable fast, repeated activation
of the receptor without requiring a second irradiation source for
deactivation. Thus, the novel compounds MAG2p and MAGA2p
enable single-wavelength, multiphoton gating of LiGluR.
However, MAG achieves higher two-photon current amplitudes
than MAG2p and MAGA2p in the long term (Figure 6b),
because the thermal stability of its cis isomer allows building up
a larger population of open-state channels upon repeated cell
raster scans (Figure S12 in the Supporting Information). To
compare the efficacy of LiGluR activation between PTLs, we
calculated the ratio between two-photon and one-photon
maximal responses (Figure 6c). Noticeably, MAG2p and
MAGA2p (both via direct and sensitized azobenzene excitation)
display a higher ratio than MAG, thereby demonstrating that
the efficiency of multiphoton isomerization was enhanced by
the design of the new photoswitches.
After characterizing the two-photon stimulation of LiGluR,
we pursued physiological applications that exploited the ability
of this receptor to rapidly activate neurons37 and trigger
calcium-regulated processes.38,39 The stimulation of individual
neurons in micrometric volumes and millisecond time scales
has been demonstrated using two-photon neurotransmitter
uncaging23 and optogenetics.24,25 To complement this set of
tools for investigating brain connectivity, we applied two-
photon activation of LiGluR in neuronal and non-neuronal cells
of the brain using the high photocurrents provided by MAG
and MAG2p. We expressed GluK2-L439C-eGFP in cultured
hippocampal neurons, incubated them in MAG2p and recorded
neuronal activity using whole-cell patch clamp (Figures 7).
Excitation of the soma with 900 nm light elicits inward currents
in voltage-clamp experiments (Figure 7c). In current-clamp
mode, these photocurrents triggered action potentials in two
Figure 6. (a) Two-photon action spectra of LiGluR-MAG (black) and
LiGluR-MAG2p (blue) and two-photon activation of LiGluR-MAGA2p
(red) at selected wavelengths. Photocurrent amplitudes were corrected
for the different power densities used (PD), averaged over all cells
measured, and normalized to the spectral maximum. (b) Absolute two-
photon (2P) responses at the optimal wavelength. For MAGA2p values
are given for sensitized (λ = 740 nm) and direct (λ = 880 nm)
azobenzene excitation. (c) Ratio between the two- and one-photon
responses (2P/1P). To compare between different LiGluR-tethers,
two-photon responses were corrected for the distinct power densities
and excitation times used and averaged over all cells measured. In all
spectra here, N = 1−6 cells and errors are SEM.
maximal two-photon responses of MAG is around 820 nm.
Repeated cell raster scans are required to get a saturating
photocurrent from all available receptors (black trace in Figure
5b). Then, the current remains stable without laser illumination
until LiGluR is closed with 500 nm light via one-photon cis−
trans back-photoisomerization. The MAG2p two-photon action
spectrum is red-shifted and yields maximum current amplitude
around 900 nm. The reduced currents obtained from MAGA2p
hindered the acquisition of a detailed action spectrum, but are
sufficient to identify two spectral ranges allowing two-photon
activation of LiGluR: the first can be found at ∼880 nm
(corresponding to the direct absorbance of azobenzene, as in
MAG2p), and the second is located around 740 nm and is
consistent with the naphthalene-sensitized photoisomeriza-
tion.34
Figure 7. (a) Two-photon image (λ = 1000 nm) of a cultured LiGluR-
MAG2p hippocampal neuron filled with Alexa Fluor 594. Red square
defines the raster scan area of two-photon stimulation. Scale bar is 20
μm. (b) Voltage-clamp recording during one-photon stimulation (blue
bar). (c,d) Two-photon raster scan (gray bars) of the same neuron
during (c) voltage-clamp recording, which shows two-photon current
with a transient current spike (two-photon mean current amplitude:
21 3 pA, 19 2% of one-photon current, N = 3), and (d) current-
clamp recording (resting potential = −45 mV). Two-photon excitation
conditions: λ = 900 nm, 0.25 s scan, and 24 mW on sample.
Remarkably, multiphoton currents mediated by MAG2p and
MAGA2p completely saturate after a few laser scans of the
recorded cell (blue and red traces in Figure 5b). In addition,
their rapid relaxation allows LiGluR to close immediately after
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Figure 8. (a) Two-photon image (λ = 1000 nm) of a cultured LiGluR-MAG astrocyte filled with Alexa Fluor 594. Red and blue squares define the
whole cell and subcellular raster scan areas, respectively. Locations of point stimulations are depicted by orange dots. Scale bar is 10 μm. (b) Whole-
cell voltage-clamp recording of the astrocyte during one-photon stimulation (LiGluR opening, purple bar; LiGluR closing, green bar). (c−h) Two-
photon currents measured on the same astrocyte (LiGluR opening, gray bar; one-photon LiGluR closing, green bar): (c) cell scan stimulation at λ =
820 nm, 10 scans of 0.7 s and 37 mW on sample (mean current amplitude: 60 20 pA, 30 10% of one-photon current, N = 2); (d) subcellular
scan stimulation at λ = 820 nm, 10 scans of 0.25 s and of 68 mW on sample (mean current amplitude: 17 9 pA, N = 2); (e−g) single-point
stimulation (gray bar) at λ = 820 nm, 50 ms and 68 mW on sample (points 1 and 2 are on the cytoplasm, and point 3 is out of the cell as control);
(h) LiGluR closing at the end of the stimulation protocol (green bar). (i−k) Two-photon calcium imaging of cultured astrocytes loaded with Fura-2
(in purple, λ = 800 nm) overlapped with an image of GFP fluorescence (in green, λ = 900 nm) to identify astrocytes expressing LiGluR-MAG. Scale
bar is 20 μm, except 50 μm in (i). (j) Images at 1.55, 4.65, and 7.75 s after targeting the astrocyte to which the arrow points in (i) with two-photon
stimulation (20 targets, 16-pixel diameter, 10 ms per point at λ = 800 nm, 60 mW on sample). (k) Recovery of intracellular calcium levels 38.75 s
after stimulation.
out of three tested neurons (Figure 7d). Although several
properties of LiGluR-MAG2p must be improved in order to
reliably photocontrol whole neurons and individual presynaptic
terminals (lifetime of the cis isomer, receptor expression level,
and subcellular localization), these results indicate that it is
possible to activate neurons using two-photon stimulation of
synthetic photoswitchable proteins.
In the same experimental conditions, no spikes were elicited
by two-photon stimulation of LiGluR-MAG, probably due to
the slow photoresponses shown in Figure 5b. However, the
large, step-function photocurrents provided by MAG and the
calcium permeability of GluK240 make LiGluR-MAG more
attractive to trigger calcium-regulated processes39,41 including
astrocyte activation38 (see also Figure S7 in the Supporting
Information). In cultured astrocytes expressing LiGluR-MAG
(Figures 8), two-photon excitation at 820 nm triggered bistable
currents (Figure 8c). Interestingly, whole-cell photocurrents
can also be measured during the stimulation of a subcellular
region (Figure 8d) or a spot (Figures 8e−g), and these
responses are reversible by illuminating the cell at 500 nm
(Figures 8c,d,h). In order to verify whether such stimuli were
enough to activate an intracellular calcium response in the
astrocyte,38,42 we performed two-photon calcium imaging
together with two-photon stimulation of astrocytes expressing
LiGluR-MAG. When we stimulated an expressing astrocyte,
LiGluR activation caused a calcium increase that propagated to
neighboring cells, generating a calcium wave that expanded to
astrocytes throughout the field of view (Figure 8i−k and Movie
S1 in the Supporting Information). This effect, which is not
observed when locally stimulating non-expressing astrocytes
(Figure S13 and Movie S2 in the Supporting Information),
demonstrates that two-photon LiGluR activation can be used to
manipulate cytosolic calcium levels in cultured astrocytes.
CONCLUSIONS
■
We have demonstrated the two-photon activation of
azobenzene-based photoswitches in living cells expressing the
light-gated receptor LiGluR.26 Although a symmetrically
substituted azobenzene was reported to photoisomerize under
continuous-wave NIR excitation,43 in general these chromo-
phores present low two-photon absorption cross sections.29,30
However, synthetic PTLs like MAG26 offer great flexibility to
adjust their photochemical properties without altering protein
function. We have rationally designed MAG derivatives with
visible absorption, fast thermal relaxation, and high two-photon
isomerization efficacy based on push−pull substitu-
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tions31,32,44,45 and sensitization33 of the azobenzene photo-
isomerization. These modifications and the reported multi-
photon excitation conditions should be directly applicable to all
azobenzene-based bioactive ligands,11 including intracellular
photoswitches known to penetrate into cells directly46 or
through specific ion channels,47 and hyperpolarizing step-
function photoswitchable channels like SPARK48 or LiGABA.11
Our findings thus enable the use of synthetic photoswitches to
manipulate extra- and intracellular biochemical processes with
the spatiotemporal precision provided by two-photon stim-
ulation.
frames, 1.55 s/frame) at 800 nm and 40 mW on sample with a 20x/
0.5-NA objective for recording the activity of astrocytes and stimulated
single nonexpressing or GFP-positive astrocytes using custom written
software,49 with a protocol of 20 stimulation targets on the cell with
a16-pixel diameter, corresponding to approximately 11 μm diameter.
Data Analysis. Amplitudes of LiGluR currents were analyzed using
IgorPro (Wavemetrics), and closing time constants of LiGluR were
determined with a custom-made software using LabView. In the two-
photon action spectrum of each compound, every set of data from one
cell was normalized to the action spectrum integral from a chosen
representative before cell average. Finally, we normalized each action
spectrum to its maximum. Calcium imaging of astrocytes was analyzed
using custom written software (Caltracer) and ImageJ.
EXPERIMENTAL SECTION
■
ASSOCIATED CONTENT
* Supporting Information
General materials and methods, detailed description of the
synthesis of MAG2p and MAGA2p, and additional photo-
chemical (Figures S1−S6) and biological (Figures S7−S13,
Tables S1 and S2, and Movies S1 <ja5026326_si_003.avi> and
S2 <ja5026326_si_004.avi>) measurements. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
Synthesis. A detailed description of the synthesis of target
photoswitchable tethered ligands is given in the Supporting
Information.
Photochemical Characterization. Trans−cis isomerization of
MAG2p and MAGA2p in solution was investigated by (i) 1H NMR for
the elucidation of the photostationary-state mixtures; (ii) steady-state
UV−vis absorption spectroscopy for trans−cis photoisomerization and
slow cis−trans thermal back-isomerization processes; and (iii) transient
absorption spectroscopy for fast cis−trans thermal back-isomerization
processes.
LiGluR on Cultured Cells. HEK293 tsA201 cell line, cultured
hippocampal neurons, and astrocytes plated on glass coverslips were
transfected with GluK2-L439C-eGFP. Prior to each experiment, they
were incubated with one of the PTLs to allow the chemical
conjugation with the receptor channel and light sensitization. A
second incubation with concavalin A was done in order to inhibit
desensitization of the glutamate receptor.
S
AUTHOR INFORMATION
■
Corresponding Authors
rmy5@columbia.edu
jordi.hernando@uab.cat
pau@icrea.cat
Author Contributions
+M.I.-S. and M.G.-M. contributed equally.
Electrophysiology. For two-photon stimulation, voltage-clamp
and current-clamp recordings under whole-cell configuration were
done with an Axon Multiclamp 700B amplifier (Molecular Devices),
and data were acquired at 10 kHz. Borosilicate glass pipettes were
pulled with a typical resistance of 4−6 MΩ for HEK293 tsA201 cells
and neurons or 7−8 MΩ for astrocytes. Bath solution was composed
of 140 mM NaCl, 1 mM MgCl, 2.5 mM KCl, 10 mM HEPES, 2.5 mM
CaCl2, and 10−20 mM glucose to fix osmolarity to 310 mOsm·kg−1,
pH 7.42 adjusted with NaOH. For HEK293 tsA201 cell line, pipet
solution contained 120 mM cesium methanosulfonate, 10 mM TEA-
Cl, 5 mM MgCl2, 3 mM Na2ATP, 1 mM Na2GTP, 20 mM HEPES,
and 0.5 mM EGTA, 290 mOsm·kg−1, pH 7.2 adjusted with CsOH. For
neurons and astrocytes it consisted of 130 mM potassium gluconate, 5
mM NaCl, 10 mM HEPES, 0.1 mM EGTA, 2 mM MgSO4, 4 mM Mg-
ATP, 0.4 mM NaXGTP, 7 mM Na2-phosphocreatine, 2 mM pyruvic
acid, and 0.1 mM Alexa Fluor 594 (Molecular Probes), pH 7.3
adjusted with KOH.
Two-Photon Stimulation. All two-photon experiments were
performed in the Yuste laboratory with a custom-made two-photon
laser scanning microscope based on a modified Olympus BX50WI
microscope with a Ti:sapphire laser as light source (Coherent
Chameleon Ultra II, 140 fs pulses, 80-MHz repetition rate). Laser
power was modulated by a Pockels cell (Conoptics) and adjusted for
each wavelength to be close to 40 mW on sample for MAG2p and
MAGA2p and 50 mW on sample for MAG, if not specified otherwise.
In experiments with MAG, we used a 20x/0.5-NA objective
(Olympus), and with the red-shifted compounds, we used a 20x/
0.95-NA objective (Olympus) in Figures 5 and 6 and a 40x/0.8-NA
objective (Olympus) in Figures 7 and 8. For two-photon stimulation
we defined a ROI and applied a unidirectional raster scan using
FluoView software, or we performed point stimulations with custom-
written LabView Software.49
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dirk Trauner for providing us the MAG compound;
Ehud Isacoff for supplying us with the GluK2-L439C-eGFP
■
́ ́
́
construct; Ariadna Perez-Jimenez and Nuria Camarero for
subcloning it into pcDNA3; Yeonsook Shin, Alexa Semonche,
and Masayuki Sakamoto for preparation of primary cultures
̀ ́
and cell transfection; and Sonia Pares for synthesis at initial
stages of the project. We are grateful to Darcy S. Peterka and
Artur Llobet for helpful discussions. We acknowledge financial
support from the RecerCaixa foundation (2010ACUP00378),
́
the “Marato de TV3” foundation (111531), the Human
Frontier Science Program (CDA022/2006), the European
Research Council (ERC-2007-StG-210355 and ERC-2012-
PoC-335011), the European Commission (FP7-ICT-2009-
270483), the Catalan government (grant 2009-SGR-277 and
̀
fellowship 2012-FI_B 01122), the Universitat Autonoma de
Barcelona (predoctoral fellowship of M.G.-M.), and the
Spanish government (grants CTQ2008-06160, CTQ2012-
30853, and CTQ2010-15380 and FPU fellowship AP2008-
03313). The Yuste laboratory is supported by NIDA
(R21DA034195), NIHM (R01MH101218) and Deutsche
Forschungsgemeinschaft (Research fellowship HI 1728/1-1).
This material is based upon work supported by the U.S. Army
Research Laboratory and the U.S. Army Research Office under
contract no. W911NF-12-1-0594 (MURI). This article is
dedicated to Prof. Josep Font Cierco on the occasion of his
75th birthday.
Calcium Imaging of Astrocytes. First, 50 μL of DMSO was
added to a 50 μg aliquot of Fura-2-AM (Life Technologies). Next, 0.2
μL of this solution and 0.2 μL of pluronic acid (20% in DMSO) in 2
mL supplemental media were added to the culture dish and incubated
at 37 °C for 30 min, before washing and LiGluR conjugation with
MAG and concanavalin A treatment. We raster-scanned Fura-2 (100
REFERENCES
(1) Harvey, J. H.; Trauner, D. ChemBioChem 2008, 9, 191.
■
8700
dx.doi.org/10.1021/ja5026326 | J. Am. Chem. Soc. 2014, 136, 8693−8701

Journal of the American Chemical Society
Article
(2) Yamada, M. D.; Nakajima, Y.; Maeda, H.; Maruta, S. J. Biochem.
2007, 142, 691.
(3) Wu, Y. I.; Frey, D.; Lungu, O. I.; Jaehrig, A.; Schlichting, I.;
Kuhlman, B.; Hahn, K. M. Nature 2009, 461, 104.
(4) Umeki, N.; Yoshizawa, T.; Sugimoto, Y.; Mitsui, T.; Wakabayashi,
K.; Maruta, S. J. Biochem. 2004, 136, 839.
(36) Schanze, K. S.; Mattox, T. F.; Whitten, D. G. J. Org. Chem. 1983,
48, 2808.
(37) Szobota, S.; Gorostiza, P.; Del Bene, F.; Wyart, C.; Fortin, D. L.;
Kolstad, K. D.; Tulyathan, O.; Volgraf, M.; Numano, R.; Aaron, H. L.;
Scott, E. K.; Kramer, R. H.; Flannery, J.; Baier, H.; Trauner, D.; Isacoff,
E. Y. Neuron 2007, 54, 535.
(38) Li, D.; Herault, K.; Isacoff, E. Y.; Oheim, M.; Ropert, N. J.
Physiol. 2012, 590, 855.
(39) Izquierdo-Serra, M.; Trauner, D.; Llobet, A.; Gorostiza, P.
Biophys. J. 2013, 104, 497A.
(40) Egebjerg, J.; Heinemann, S. F. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 755.
(41) Izquierdo-Serra, M.; Trauner, D.; Llobet, A.; Gorostiza, P.
Biochim. Biophys. Acta 2013, 1830, 2853.
(42) Cornell-Bell, A. H.; Finkbeiner, S. M.; Cooper, M. S.; Smith, S.
J. Science 1990, 247, 470.
(43) Zhang, Y.; Erdmann, F.; Fischer, G. Nat. Chem. Biol. 2009, 5,
724.
(44) Venkataramani, S.; Jana, U.; Dommaschk, M.; Sonnichsen, F.
D.; Tuczek, F.; Herges, R. Science 2011, 331, 445.
(45) Sakamoto, R.; Kume, S.; Sugimoto, M.; Nishihara, H. Chem.
Eur. J. 2009, 15, 1429.
́
(46) Nevola, L.; Martín-Quiros, A.; Eckelt, K.; Camarero, N.; Tosi,
S.; Llobet, A.; Giralt, E.; Gorostiza, P. Angew. Chem., Int. Ed. 2013, 52,
7704.
(47) Mourot, A.; Fehrentz, T.; Le Feuvre, Y.; Smith, C. M.; Herold,
C.; Dalkara, D.; Nagy, F.; Trauner, D.; Kramer, R. H. Nat. Methods
2012, 9, 396.
(48) Banghart, M.; Borges, K.; Isacoff, E.; Trauner, D.; Kramer, R. H.
Nat. Neurosci. 2004, 7, 1381.
(5) Schierling, B.; Noel, A. J.; Wende, W.; Hien, l. T.; Volkov, E.;
̈
Kubareva, E.; Oretskaya, T.; Kokkinidis, M.; Rompp, A.; Spengler, B.;
̈
Pingoud, A. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1361.
(6) Asanuma, H.; Liang, X.; Nishioka, H.; Matsunaga, D.; Liu, M.;
Komiyama, M. Nat. Protoc. 2007, 2, 203.
(7) Ito, H.; Liang, X.; Nishioka, H.; Asanuma, H. Org. Biomol. Chem.
2010, 8, 5519.
(8) Airan, R. D.; Thompson, K. R.; Fenno, L. E.; Bernstein, H.;
Deisseroth, K. Nature 2009, 458, 1025.
(9) Oh, E.; Maejima, T.; Liu, C.; Deneris, E.; Herlitze, S. J. Biol.
Chem. 2010, 285, 30825.
(10) Miesenbock, G. Science 2009, 326, 395.
(11) Kramer, R. H.; Mourot, A.; Adesnik, H. Nat. Neurosci. 2013, 16,
816.
(12) Toettcher, J. E.; Gong, D.; Lim, W. A.; Weiner, O. D. Nat.
Methods 2011, 8, 837.
(13) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73.
(14) Papagiakoumou, E.; Begue, A.; Leshem, B.; Schwartz, O.; Stell,
B. M.; Bradley, J.; Oron, D.; Emiliani, V. Nat. Photonics 2013, 7, 274.
(15) Oron, D.; Papagiakoumou, E.; Anselmi, F.; Emiliani, V. Prog.
Brain Res. 2012, 196, 119.
(16) Watson, B. O.; Nikolenko, V.; Yuste, R. Front. Neural Circuits
2009, 3, 6.
(17) Yizhar, O.; Fenno, L. E.; Prigge, M.; Schneider, F.; Davidson, T.
J.; O’Shea, D. J.; Sohal, V. S.; Goshen, I.; Finkelstein, J.; Paz, J. T.;
Stehfest, K.; Fudim, R.; Ramakrishnan, C.; Huguenard, J. R.;
Hegemann, P.; Deisseroth, K. Nature 2011, 477, 171.
(18) Wen, L.; Wang, H.; Tanimoto, S.; Egawa, R.; Matsuzaka, Y.;
Mushiake, H.; Ishizuka, T.; Yawo, H. PLoS One 2010, 5, e12893.
(19) Gorostiza, P.; Isacoff, E. Y. Physiology 2008, 23, 238.
(20) Mourot, A.; Kienzler, M. A.; Banghart, M. R.; Fehrentz, T.;
Huber, F. M.; Stein, M.; Kramer, R. H.; Trauner, D. ACS Chem.
Neurosci. 2011, 2, 536.
(49) Nikolenko, V.; Poskanzer, K. E.; Yuste, R. Nat. Methods 2007, 4,
943.
(21) Kienzler, M. A.; Reiner, A.; Trautman, E.; Yoo, S.; Trauner, D.;
Isacoff, E. Y. J. Am. Chem. Soc. 2013, 135, 17683.
(22) Fehrentz, T.; Kuttruff, C. A.; Huber, F. M.; Kienzler, M. A.;
Mayer, P.; Trauner, D. ChemBioChem 2012, 13, 1746.
(23) Judkewitz, B.; Roth, A.; Hausser, M. Neuron 2006, 50, 180.
̈
(24) Packer, A. M.; Peterka, D. S.; Hirtz, J. J.; Prakash, R.; Deisseroth,
K.; Yuste, R. Nat. Methods 2012, 9, 1202.
(25) Prakash, R.; Yizhar, O.; Grewe, B.; Ramakrishnan, C.; Wang, N.;
Goshen, I.; Packer, A. M.; Peterka, D. S.; Yuste, R.; Schnitzer, M. J.;
Deisseroth, K. Nat. Methods 2012, 9, 1171.
(26) Volgraf, M.; Gorostiza, P.; Numano, R.; Kramer, R. H.; Isacoff,
E. Y.; Trauner, D. Nat. Chem. Biol. 2006, 2, 47.
(27) Gorostiza, P.; Volgraf, M.; Numano, R.; Szobota, S.; Trauner,
D.; Isacoff, E. Y. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10865.
(28) Szymanski, W.; Beierle, J. M.; Kistemaker, H. A. V.; Velema, W.
A.; Feringa, B. L. Chem. Rev. 2013, 113, 6114.
(29) De Boni, L.; Rodrigues, J. J.; dos Santos, D. S.; Silva, C.; Balogh,
D. T.; Oliveira, O. N.; Zilio, S. C.; Misoguti, L.; Mendonca
Chem. Phys. Lett. 2003, 374, 684.
̧
, C. R.
(30) De Boni, L.; Misoguti, L.; Zilio, S. C.; Mendonca
ChemPhysChem 2005, 6, 1121.
̧
, C. R.
(31) Antonov, L.; Kamada, K.; Ohta, K.; Kamounah, F. S. Phys.
Chem. Chem. Phys. 2003, 5, 1193.
(32) Magennis, S. W.; Mackay, F. S.; Jones, A. C.; Tait, K. M.; Sadler,
P. J. Chem. Mater. 2005, 17, 2059.
(33) Jiang, D. L.; Aida, T. Nature 1997, 388, 454.
(34) Kim, H. M.; Cho, B. R. Acc. Chem. Res. 2009, 42, 863.
(35) Bandara, H. M. D.; Burdette, S. C. Chem. Soc. Rev. 2012, 41,
1809.
8701
dx.doi.org/10.1021/ja5026326 | J. Am. Chem. Soc. 2014, 136, 8693−8701