Tuning photochromic ion channel blockers

Article abstract of DOI:10.1021/cn200037p

Photochromic channel blockers provide a conceptually simple and convenient way to modulate neuronal activity with light. We have recently described a family of azobenzenes that function as tonic blockers of Kv channels but require UV-A light to unblock and need to be actively switched by toggling between two different wavelengths. We now introduce red-shifted compounds that fully operate in the visible region of the spectrum and quickly turn themselves off in the dark. Furthermore, we have developed a version that does not block effectively in the dark-adapted state, can be switched to a blocking state with blue light, and reverts to the inactive state automatically. Photochromic blockers of this type could be useful for the photopharmacological control of neuronal activity under mild conditions.

Full text of DOI:10.1021/cn200037p

RESEARCH ARTICLE
pubs.acs.org/acschemicalneuroscience
Tuning Photochromic Ion Channel Blockers
||
Alexandre Mourot,†, Michael A. Kienzler,‡,§,|| Matthew R. Banghart,‡ Timm Fehrentz,§ Florian M. E. Huber,§
Marco Stein,§ Richard H. Kramer,† and Dirk Trauner*,§
†Department of Molecular and Cell Biology and ‡Department of Chemistry, University of California, Berkeley, California 94720,
United States
§Department of Chemistry and Pharmacology, Ludwig-Maximilians-Universit€at, M€unchen, and Center for Integrated Protein Science,
81377 Munich, Germany
S Supporting Information
b
ABSTRACT: Photochromic channel blockers provide a conceptually simple and
convenient way to modulate neuronal activity with light. We have recently
described a family of azobenzenes that function as tonic blockers of Kv channels
but require UV-A light to unblock and need to be actively switched by toggling
between two different wavelengths. We now introduce red-shifted compounds
that fully operate in the visible region of the spectrum and quickly turn themselves
off in the dark. Furthermore, we have developed a version that does not block
effectively in the dark-adapted state, can be switched to a blocking state with blue
light, and reverts to the inactive state automatically. Photochromic blockers of this
type could be useful for the photopharmacological control of neuronal activity
under mild conditions.
KEYWORDS: photopharmacology, ion channel blockers, voltage-gated potassium channels, photochromic molecules, azoben-
zenes
he merger of artificial photoswitches with natural receptor
proteins has proven to be an effective way to control neural
different wavelengths of light that greatly favor one isomer or the
other. Alternatively, one could use photoswitches that are ac-
tively switched to one isomer with light but thermally revert to
the more stable form in the dark. This obviates the need to work
with two different wavelengths but requires switches that turn
themselves off at appropriate rates.
T
activity with light.1ꢀ4 This approach combines the virtually
limitless repertoire of synthetic chemistry with a detailed under-
standing of the transmembrane proteins that underlie the gen-
eration and modulation of action potentials. As such, it provides
a useful alternative to naturally occurring light-gated ion channels
and light-powered pumps, which are currently driving the field of
optogenetics.5 Most of these are based on a single photoswitch,
retinal, which is often endogenously produced and does not need
to be added externally. While this provides advantages in terms
of practicality, it confines the tuning of these systems to muta-
tions in the protein surrounding the chromophore.6 By contrast,
a combination of synthetic photoswitches and natural receptors
should allow for more flexibility, since both components can
be manipulated through chemistry and genetic engineering,
respectively.
Recently, we introduced a family of simple azobenzene deri-
vatives that function as PCLs for tetrameric voltage-gated ion
channels, in particular potassium channels.7,9 These molecules,
represented by AAQ and BzAQ (Figure 1), operate as photo-
chromic open channel blockers, that bind in a light-dependent
manner to the tetraethylammonium (TEA) binding site located
in the inner cavity of potassium channels.7 In their extended trans
form, AAQ and BzAQ fit into this cavity, but in their bent cis form
their apparent affinity drops sharply. To reach their binding site,
these amphiphilic molecules can either partition into the mem-
brane or they can be imported through a patch pipet, whose
content can rapidly exchange with the cytosol. Although many
details of this reversible molecular encapsulation of azobenzenes
by channel proteins remain to be clarified, they have already
proven themselves as effective modulators of neural activity. For
instance, Purkinje neurons and pacemaker neurons in the heart
Artificial photoswitches can be combined with endogenous
neural receptors through covalent or noncovalent bonding.7 So-
called photochromic ligands (PCLs) bind noncovalently and
contain a photoswitchable moiety whose configuration can be
changed upon irradiation.7ꢀ10 As such, they change their efficacy
in a light-dependent way and essentially function as photochro-
mic neurotransmitters or neuromodulators. Since they are dis-
tributed like small-molecule drugs, they are able to photosensitize
naïve tissues within minutes, as opposed to days in the case of
optogenetic probes. PCLs can be rapidly switched by using two
Received: April 14, 2011
Accepted: June 9, 2011
Published: June 09, 2011
r
2011 American Chemical Society
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Figure 1. (a) Molecular structures of AAQ and BzAQ, two PCLs for Kv channels; (b) AAQ, BzAQ, and related PCLs are membrane permeable and
function as photochromic open-channel blockers.
Figure 2. (a) Potential photochromic Kv blockers investigated in this study. (b) UV/Vis spectra of the azobenzenes investigated. Spectra were recorded
at room temperature in phosphate buffered saline solution at pH 7.4.
of Hirudo medicinalis could be controlled with photochromic
neuromodulators of this type.9
One of the greatest advantages of azobenzene photoswitches
is the well-understood effect of substitutions and molecular
extensions on their photophysical properties and thermal
stability.11 So-called “regular azobenzenes”, represented by
the parent molecule, as well as the bis-acylated azodianilines
AAQ and BzAQ, are thermodynamically more stable in their
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Scheme 1. Synthesis of BENAQ, PhENAQ, DENAQ, DENAQ-F4, and AFM 2-10a
a Reagents and conditions: (a) 4-nitroaniline, isoamylnitrite, HCl, MeOH (87% for 1, 79% for 2, 62% for 3); (b) Na2S, H2O, 1,4-dioxane, 90 °C (88% for
4, 42% for 5, 88% for 6); (c) 2-triethylammonium acetic acid chloride, DIPEA, DMF, 0 °C to RT (67% for BENAQ, 66% for PhENAQ, 92% for
DENAQ); (d) BF3 Et2O, isoamylnitrite, THF ꢀ40 to ꢀ5 °C; (e) diethylaniline, NaOAc, 0 °C to RT (36% over two steps); (f) piperidine, Et2O, 0 °C to
RT (85%); (g) 2-triethylammonium acetic acid chloride, DIPEA, DMF, 0 °C to RT (38%); (h) DMF, 80 °C (92%).
trans-configuration, which completely predominates in the dark-
adapted state. Their photostationary cis/trans ratios (PSRs) as-
sume their maximum values in the UV-A region of the electro-
magnetic spectrum (315ꢀ380 nm). At these wavelengths, cis/
trans ratios exceeding 9:1 can be observed.12 Once the light is
turned off, the cis-isomers of regular azobenzenes are thermally
relatively stable. The half-life of AAQ in physiological solution at
room temperature, for instance, is 7ꢀ8 min.
dialkylamino or aryl alkylamino group on one side and a mildly
electron-withdrawing acylamino moiety that terminates in a
quaternary ammonium ion on the other side of the azobenzene.
This positively charged “head group” interacts with the TEA
binding site in the inner lumen of the channel, which blocks the
flow of ions. The “tail” of the molecules, that is, the electron-
donating substituent, determines the spectral properties of the
photoswitch as well as the thermal stability of the cis-isomer.
Spectroscopic Characterization of Red-Shifted PCLs.
AAQ, a representative of our original type, which is marked by
a bis-acylated azodianiline core, serves as a point of reference
(Figure 2). Spectroscopically, it can be classified as a “regular azo-
benzene” with a strong πꢀπ* band at 362 nm in water (trans
isomer). Replacement of the lower acylamino group with an
alkylamino group increases the electron density of one side and
shifts the absorption spectrum of the trans-isomer toward the red.
For instance, the absorption spectrum of trans-PhENAQ, which
bears a phenylethylamino group, is shifted to 456 nm in phos-
phate buffered saline solution. BENAQ, which bears a benzy-
lethylamino substituent, has a similar absorption spectrum peak-
ing at 459 nm. The diethylamino derivative DENAQ is shifted
to an larger extent, as its absorption spectrum peaks at 470 nm.
To increase the pushꢀpull effect without significantly altering
the sterics of the azobenzene moiety, we also explored fluori-
nated derivatives, such as DENAQ-F4. This compound shows
a relatively small bathochromic shift with respect to DENAQ
(484 nm). Our most red-shifted compound, AFM2-10, which
peaks at 580 nm, is in essence an azobenzene version of the well-
known fluorescent styrene dye FM2-10. The synthesis of these
compounds follows standard protocols and is summarized in
Scheme 1 (for further details see the Supporting Information).
In general, the absorption spectra of the trans-isomers of azo-
benzenes are correlated with their PSRs at different wavelengths.
Hence, red-shifting of the absorption maximum of their trans
isomer through appropriate substitutions should also red-shift
Azobenzenes with strongly electron-donating substituents on
both rings are known to absorb at increased wavelengths and have
an increased rate of thermal back-isomerization from cis to trans.11,13
In addition to these, one could use azobenzenes that feature an
electron-donating substituent on one end and an electron-with-
drawing one on the other. These “pushꢀpull” azobenzenes,
which are also referred to as “pseudo-stilbenes”, are marked
by greatly red-shifted absorption spectra. They are also thermally
instable in their cis form and revert at room temperature to the
thermodynamically more stable trans form on a millisecond to
second time scale. The rate of this reversal is greatly dependent
on the solvent, with polar protic solvents promoting very fast
isomerization.14 As such, pushꢀpull azobenzenes are ideally
suited as photochromic ion channel blockers that can be acti-
vated with visible light and turn themselves off once the light
intensity drops below a certain level. Studies using red-shifted
blockers would generally benefit from the deeper tissue penetra-
tion of light with longer wavelength. Since longer wavelengths
are also associated with less phototoxicity, photochromic com-
pounds of this type would be particularly useful in chemical
approaches toward restoring vision.
’ RESULTS AND DISCUSSION
We now present a family of pushꢀpull azobenzenes that have
red-shifted action spectra and decreased thermal stability and
function as photochromic blockers of voltage-gated ion channels
(Figure 2). These molecules feature a strongly electron-donating
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Figure 4. Effects of PhENAQ on Shaker-IR expressed in HEK 293 cells.
Cells were treated with 50 μM PhENAQ. (a) Kv current was measured
using a 200 ms depolarization from ꢀ60 to +40 mV, in the dark and
under 480 nm light. (b) Current versus voltage dependence of block and
unblock, under 480 nm light and in the dark, respectively. Steady state
current was plotted as a function of membrane potential. (c) Reversi-
bility of PhENAQ using cycles of 480 nm light and dark. Peak current
was measured using the protocol described in (a) looped at 0.5 Hz.
(d) Apparent thermal relaxation rate of PhENAQ measured by electro-
physiology. Shaker-IR peak current is plotted as a function of time after
the light is switched off. Three light/dark cycles are averaged for this
single cell. Datapointswerefitted with a monoexponential decay equation:
y = y0 + A exp(ꢀx/τ) with y0 = 838 ( 3 pA, A = ꢀ306 ( 5 pA, and τ =
3.9 ( 0.2 s.
Figure 3. Effect of DENAQ on Kv3.1 expressed in HEK293 cells.
(a) Cells were treated with 100 μM DENAQ. Kv current was measured
in whole cell mode using a 200 ms depolarization from ꢀ60 to +40 mV,
in the dark and under 480 nm light. Capacitive currents have been cut off
for clarity. (b) Membrane-voltage dependence of block and unblock.
Steady-state current (at the end of the 200 ms depolarization) is plotted
as a function of membrane potential. (c) Reversibility of photoswitching
and action spectrum of DENAQ on Kv3.1. Potassium current was
measured using the protocol described in (a) looped at 1 Hz. Peak
current is plotted as a function of time. Cycles of dark and illumination
(420ꢀ500 nm) are indicated. (d) Unblock as a function of wavelength.
Unblock was normalized to 100% for 480 nm light (n = 3ꢀ4 cells).
(e) Apparent thermal relaxation rate of DENAQ measured by electro-
physiology. Kv3.1 peak current is plotted as function of time after light is
switched off. Four light-dark cycles are averaged for this single cell. Data
points were fitted with a biexponential decay equation: y = y0 + A1
exp(ꢀx/τ1) + A2 exp(ꢀx/τ2) with y0 = 1122 ( 23 pA, A1 = 1148 (
59 pA, τ1 = 392 ( 37 ms, A2 = 422 ( 39 pA, and τ2 = 3.5 ( 0.8 s.
similar PSRs in DMSO (see Supporting Information Figure 1),
but it is difficult to extrapolate this to aqueous buffer solutions.
Electrophysiological Characterization of Red-Shifted PCLs.
The electrophysiological action spectrum of photochromic chan-
nel blockers should mirror their absorption spectra and PSRs in
solution, provided the interaction with the channel protein does
not greatly influence these. Pushꢀpull azobenzenes, such as
DENAQ or PhENAQ, with their red-shifted absorption spectra, are
therefore expected to show a red-shifted action spectrum and fully
operate in the visible region of the electromagnetic spectrum. This
is indeed the case. Figure 3 shows the effects of 100 μM DENAQ
on the conductance of a voltage-gated potassium channel at dif-
ferent wavelengths. Recordings were performed in HEK 293 cells
transiently expressing Kv3.1 in whole-cell voltage clamp mode
after transient treatment with the PCL. Our data show that DE-
NAQ is a much better blocker in the dark adapted-state than at
480 nm (Figure 3a), which is true at all membrane potentials
tested (Figure 3b). Unblock could be achieved at all wavelengths
between 380 and 540 nm, with maximum unblock observed
around 480 nm (Figure 3c and d). At this wavelength, percent
photoswitching, as determined by the difference between the
maximum current under irradiation and in the dark, divided by the
maximum current under irradiation, is 63.2 ( 7.2% (n = 5 cells).
Thus, the action spectrum of DENAQ is shifted by ca. 100 nm
with respect to AAQ and BzAQ. Remarkably, the blocking effect
of trans-DENAQ is restored within seconds once the light is
turned off (t1/2 = 305 ( 57 ms, averaged over n = 4 cells,
Figure 3e). DENAQ is therefore a trans-blocker with a strongly
the PSR maximum as a function of the wavelength. This is dif-
ficult to measure with our pushꢀpull azobenzenes because their
thermal relaxation is too fast to enrich the cis isomer in aqueous
solution for detection by standard spectrophotometric methods.15
Similar observations were made by Uyeda et al.16 who investi-
gated structurally related azobenzenes that bore a dimethylamino
substituent on one side and acylamino substituents on the other.
In this case, thermal cis- to trans-isomerization was very fast in
aqueous solution and only detectable by flash laser photolysis.
By contrast, the thermal isomerization was found to be relatively
slow in dimethyl sulfoxide (DMSO), and PSRs up to 78% cis
could be observed in this solvent. Nevertheless, these photo-
switches performed well in aqueous solution when incorporated
in DNA.17 Preliminary experiments with our compounds show
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Figure 5. Effect of PhENAQ on neuronal firing. (a) Voltage-clamp recording from a hippocampal neuron treated with 50 μM PhENAQ. Voltage-gated
potassium current was elicited using a 200 ms depolarization from ꢀ60 to +40 mV, under 480 nm light irradiation or in the dark. (b) Reproducibility of
light-evoked membrane depolarization in neurons. Current clamp recordings were made from a hippocampal neuron treated with 50 μM PhENAQ.
Average trace (black) and standard deviation (gray) of four light cycles are shown. Apparent thermal relaxation rate was fitted with a monoexponential
decay equation (red): y = y0 + A exp(ꢀx/τ) with y0 = ꢀ46.78 ( 0.01 mV, A = 3.86 ( 0.02 mV, and τ = 237 ( 3 ms. (c) Optical regulation of action
potential firing. (d) Multielectrode array recording from an acute rat cerebellar slice. Top, raster plot of spiking; bottom, average firing rate calculated in
100 ms time bins. (e) Extracellular recording from a single cell using the multielectrode array. Color bars represent illumination with 480 nm (blue) or
periods of darkness (black).
red-shifted action spectrum and decreased thermal stability with
respect to AAQ and BzAQ.
residue lining the inner lumen of the channel, which can only take
effect when the azobenzene is cis-configured. Also noticeable is
the difference in the rate of thermal relaxation (t1/2 = 305 (
57 ms vs 2.6 ( 0.1 s). This could reflect either differences in the
dissociation from the channel protein as the rate-determining
step or different rates of the thermal isomerization in solution.
Detailed structural and kinetic investigations will be needed to
clarify these points.
The remaining azobenzenes shown in Figure 2 were tested on
HEK cells expressing various channels but failed to show func-
tional features that significantly go beyond DENAQ or PhENAQ.
Despite its seemingly small chemical modification, DENAQ-F4
proved too to toxic to be viable as a photochromic blocker.
AFM2-10 had virtually no effect on the conductance of potassium
channels, even at very high concentrations (500 μM). It appears
that subtle changes in the composition the molecules influence
not only the photophysical and thermal but also the pharma-
cological properties of our photochromic blockers.
The compound PhENAQ has similar spectral properties but
shows a key difference in terms of its blocking state. It is a red-
shifted azobenzene that preferentially blocks in its thermodyna-
mically less stable cis-form, that is, at 480 nm (Figure 4a). In this
case, electrophysiological recordings were performed in HEK
293 cells transiently expressing Shaker-IR in whole-cell voltage
clamp mode. Percent photoswitching, as determined by the dif-
ference between the steady-state current in the dark and in
480 nm light, divided by the current in the dark, was found to be
29.4 ( 4.8% (n = 3 cells). cis-Block occurred at all membrane
potentials tested (Figure 4b) and was fully reversible over many
cycles (Figure 4c). Once the light is turned off, PhENAQ quickly
reverts to the less blocking trans state (t1/2 = 2.6 ( 0.1 s, averaged
for n = 3 cells) (Figure 4d). Thus, PhENAQ has little effect when
added in the dark (or at longer wavelengths) but becomes an
efficient blocker when irradiated with blue light.
In comparison, DENAQ and PhENAQ show some key dif-
ferences, beyond their trans versus cis activity. Structurally, they
are only distinguished by the presence of an ethyl and phenyl
substituent, respectively. However, DENAQ does not appear to
affect Shaker-IR channels, which we routinely use in our inves-
tigations on photochromic Kv blockers, yet it cleanly blocks Kv3.1
channels. By contrast, PhENAQ blocks Shaker-IR and a range
of other Kv channels (not shown). Why it does so preferentially
in its cis-form remains an open question. It could be due to an
attractive interaction of the phenyl ring with an amino acid
Optical Regulation of Neuronal Excitability. cis-Blockers,
such as PhENAQ, have the inherent advantage that they have
little effect on ion channels in their thermodynamically more
stable, dark-adapted state, which should make them less toxic to
excitable cells. We therefore decided to focus on PhENAQ and
investigate its effect on neuronal firing patterns. In accordance
with its effect on Shaker-IR in HEK 293 cells, PhENAQ behaves
as a cis-blocker of voltage-gated potassium channels endogen-
ously expressed in hippocampal neurons (percent photoswitch-
ing = 24.5 ( 4.5%, n = 3 cells, Figure 5a). To look at optical
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modulation of membrane potential, we performed current clamp
experiments. When the membrane potential of the cell was close
to the resting potential, PhENAQ rapidly and reliably elicited
membrane depolarization upon 480 nm light irradiation (Δ =
6.0 ( 0.4 mV, n = 4 cells) without inducing cell firing (Figure 5b).
When switched back to the dark, repolarization occurred within a
second (t1/2 = 163 ( 2 ms, n = 4 cells). When current was
injected to bring the cell closer to threshold for firing, light-
induced depolarization triggered action potential firing, which
ceased rapidly when the light was switched off (Figure 5c).
The depolarization required to induce spiking depends not
only on the various ion channels expressed in a given type of cell
but also on the strength and variability of its synaptic input. To
test whether PhENAQ can modulate action potential firing of
cells that are not artificially depolarized, we recorded extracellu-
larly from cerebellar slices using a multielectrode array (MEA)
with three-dimensional electrodes. Cerebellar slices are marked
by high levels of spontaneous neuronal activity, probably origi-
nating from Purkinje neurons.18 As can be seen in Figure 5d,
neuronal firing was increased upon irradiation with 480 nm but
decreased markedly once the light was turned off. Although the
effects are not large, they are reversible and reproducible. They
are probably limited by the low solubility of PhENAQ in buffer
solution, which makes it difficult to deliver uniformly in brain
slices. Figure 5e shows an example of a single cerebellar neuron
whose activity was strongly photomodulated. As a control, we
checked that light itself has no effect on the activity of naïve
cerebellar slices (Supporting Information Figure 2).
’ METHODS
Synthesis. See the Supporting Information.
Spectroscopic Analysis. UV/Vis spectra were measured at room
temperature using a SmartSpec Plus photometer (Biorad). UV/Vis
spectra of thermally unstable compounds were measured on a Nano-
Drop 2000c (Thermo Scientific) instrument at concentrations of ap-
proximately 1 mM in DMSO solvent. Photoisomerization was achieved
by directly illuminating a 1 μL sample on the pedestal with 473 nm light
from a 200 μm, 0.22 NA fiber optic cable placed perpendicular to the
optical path such that the sample was at the focal point of the fiber tip.
150 mW was delivered to the sample from a 200 mW DPSS blue 473 nm
laser (MBL-III-473, Opto Engine).
Cell Culture. HEK 293 cells were grown in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS).
Cells were plated on12 mmdiameter poly-L-lysinecoatedglass coverslips
at a density of 20 000 cells/cm2. Transfection was performed using the
calcium phosphate method, as already described.9 We transfected cells
either with Shaker-IR (Inactivation Removed)25 or rat Kv3.1 cDNA,
using a bicistronic GFP-expression vector (pIRES). Cells were recorded
12ꢀ48 h after transfection. Primary cultures of neonatal rat hippocampal
neurons were performed using standard procedures, as previously
described.9 Recordings were performed 2ꢀ3 weeks after plating.
Cerebellar Slice Preparation. Sagittal cerebellar slices were
prepared from 13ꢀ17 day-old SpragueꢀDawley rats. Briefly, the animal
was decapitated under isoflurane anesthesia and the brain was quickly
removed. Sagittal slices (340 μm thick) were cut with a microtome
(Leica VT1000S; Leica Microsystems, Wetzlar, Germany) in an ice-cold
artificial cerebrospinal fluid (ACSF) and then placed in an incubating
chamber for 30 min at 34 °C. Thereafter, slices were kept at room
temperature. Bicarbonate-buffered ACSF was used as the slicing,
storage, and recording solution; composition in mM: NaCl 126, KCl
2.5, NaHCO3 26, NaH2PO4 1.25, CaCl2 2.5, MgSO4 1.3, and glucose 10,
saturated with O2/CO2 (95/5%).
Animal care and experimental protocols were approved by the
University of California Berkeley Animal Care and Use Committee.
Electrophysiology. Cells were incubated with 10 μM to 1 mM
(as indicated) photoswitch for 15 min in the cell incubator (37 °C,
7% CO2, dark). The photoswitch was diluted in extracellular solution
(contains in mM: NaCl 138, KCl 1.5, MgCl2 1.2, CaCl2 2.5, HEPES
free acid 5, and glucose 10, pH 7.4). Final DMSO content was always
e1% vol/vol. Cells were then washed twice with 500 μL of extracellular
solution and directly used for electrophysiological recordings. Whole-cell
recordings were performed using an PC-505B amplifier (Warner Instru-
ments, CT) linked to a personal computer equipped with pClamp8
(Molecular Devices). Patch pipettes had resistances between 3 and
4 MΩ and were filled with intracellular solution (contains in mM: NaCl
10, K+ gluconate 135, HEPES free acid 10, MgCl2 2, MgATP 2, EGTA 1,
pH 7.4). For neuronal recordings only, synaptic transmission was
blocked using 25 μM DNQX and 20 μM biccuculine, and voltage-gated
sodium channels were blocked using 1 μM tetrodotoxin in the extra-
cellular solution (in voltage-clamp mode only). To measure voltage-
gated K+ current from HEK cells or neurons, the holding membrane
potential was set to ꢀ60 mV and stepped to +40 mV (unless otherwise
indicated) for 200 ms at 0.5 or 1 Hz. In current clamp mode, injection of
current was used to depolarize the cell and induce action potential firing. For
illumination, we used a monochromator (Polychrome V, Till photonics)
controlled using Clampex and connected to the back of the microscope
using a UV/Vis quartz fiber. Light output measured using a hand-held
power meter (Newport 840-C) and through a 20ꢁ objective was
4ꢀ7 mW/mm2. Data were filtered at 2 kHz and acquired at a sampling
frequency of 10 kHz.
’ CONCLUSION
Following a general paradigm for tuning azobenzene photo-
switches, we have developed compounds that function as red-
shifted photochromic blockers of potassium channels, which turn
themselves off automatically in the dark. The lessons learned
during this study could be applied to other types of photo-
switches, including photochromic versions of the neurotransmit-
ter glutamate19,20 and covalently tethered blockers, agonists, and
antagonists of neural receptor proteins (so-called photochromic
tethered ligands, or PTLs).12,21ꢀ24
One of our most advanced compounds, DENAQ, is a blocker
of Kv3.1 channels that is active in its dark-adapted trans state. By
contrast, PhENAQ is a cis-blocker that becomes active upon
irradiation with blue light, which makes it an attractive molecule
for the optical regulation of neuronal excitability. A photochro-
mic cis-blocker of Shaker channels has been previously observed,
but it was neither red-shifted nor thermally instable and it proved
to be too toxic to be of any practical use.7 Although tonic blockers
have performed remarkably well in complex neural systems, cis-
blockers such as PhENAQ could have advantages where a mini-
mum of perturbation upon addition of the compound is desir-
able. Both DENAQ and PhENAQ revert thermally, that is, in the
dark, to their default trans states within hundreds of milliseconds
to seconds.
The use of these compounds in the photopharmacological
control of electrical activity with visible light in neural tissue such
as the retina is currently under investigation. Their effects on
other voltage-gated ion channels, such as Nav and Cav channels,
are also under study in our laboratories. Finally, the incorpora-
tion of red-shifted, thermally destabilized azobenzenes into other
soluble and tethered photochromic ligands is under active
investigation and will be reported in due course.
Multielectrode Array (MEA) Recordings. Experiments were
performed at room temperature in ACSF saturated with O2/CO2. Slices
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were incubated with 50 μM PhENAQ (DMSO concentration 0.5% vol/
vol) for 15 min at room temperature and then washed 5 min prior to
recording. Slices were then placed on a tridimensional microarray made
of 60 pyramid-shape microelectrodes (MEA60 200 3D GND, Ayanda
Biosystems SA, Lausanne, Switzerland). Recordings were acquired with
an MEA-1060 amplifier board (gain 1200, sampling frequency 20 kHz,
Butterworth second order highpass filter 300 Hz, Multi Channel Systems,
Reutlingen, Germany) positioned on the stage of an inverted micro-
scope (Olympus IX71). Principal component analysis of spike wave-
forms was used for sorting spikes generated by individual cells (Offline
Sorter; Plexon, Denton, TX). Light was delivered using the 100 W
halogen lamp of the microscope and a 480/40 bandpass filter, and was
focused on the slice using a 4ꢁ objective (Olympus UPLanFL N, NA
0.13). Light was computer-controlled using a filter-wheel controller
(Lambda 10-3, Sutter Instruments) and an ultrafast shutter (Uniblitz
VCM-D1, Vincent Associates). Light intensity measured at the back of
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’ ASSOCIATED CONTENT
S
Supporting Information. Additional figures and experi-
b
mental procedures, and general experimental details. This ma-
terial is available free of charge via the Internet at http://pubs.acs.
org.
’ AUTHOR INFORMATION
(16) Kamei, T., Kudo, M., Akiyama, H., Wada, M., Nagasawa, J.,
Funahashi, M., Tamaoki, N., and Uyeda, T. Q. P. (2007) Visible-Light
Photoresponsivity of a 4-(Dimethylamino)azobenzene Unit Incorpo-
rated into Single-Stranded DNA: Demonstration of a Large Spectral
Change Accompanying Isomerization in DMSO and Detection of Rapid
(Z)-to-(E) Isomerization in Aqueous Solution. Eur. J. Org. Chem. 2007
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Corresponding Author
*E-mail: dirk.trauner@lmu.de.
Author Contributions
These authors contributed equally to this work.
Funding Sources
(17) Kamei, T., Akiyama, H., Morii, H., Tamaoki, N., and Uyeda,
T. Q. (2009) Visible-light photocontrol of (E)/(Z) isomerization of the
4-(dimethylamino)azobenzene pseudo-nucleotide unit incorporated
into an oligonucleotide and DNA hybridization in aqueous media.
Nucleosides, Nucleotides Nucleic Acids 28 (1), 12–28.
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organotypic cerebellar slice cultures. Brain Res. 1218, 54–69.
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E. Y., and Trauner, D. (2007) Reversibly caged glutamate: a photo-
chromic agonist of ionotropic glutamate receptors. J. Am. Chem. Soc. 129
(2), 260–261.
Support for the work was provided by the Nanomedicine Devel-
opment Center for the Optical Control of Biological Function,
PN2EY018241 (D.T. and R.H.K.), the National Institutes of
Health (RO1 EY018957 and RO1MH088484, R.H.K.), and the
Deutsche Forschungsgemeinschaft (SFB 749, D.T.).
’ ACKNOWLEDGMENT
We thank Caleb M. Smith for his help with the MEA data
analysis.
(20) Abrams, Z. R., Warrier, A., Trauner, D., and Zhang, X. (2010) A
Signal Processing Analysis of Purkinje Cells in vitro. Front. Neural
Circuits 4, 13.
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R. H. (2004) Light-activated ion channels for remote control of neuronal
firing. Nat. Neurosci. 7 (12), 1381–1386.
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(2006) Light-induced depolarization of neurons using a modified Shaker
K(+) channel and a molecular photoswitch. J. Neurophysiol. 96 (5),
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Author Contributions
M.R.B., A.M., and D.T. designed the research. A.M. and D.T.
wrote the paper. M.A.K., M.S., and F.M.E.H. synthesized the
compounds. A.M. and T.F. carried out the biological in-
vestigations.
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