Optical Control of a Delayed Rectifier and a Two-Pore Potassium Channel with a Photoswitchable Bupivacaine

Article abstract of DOI:10.1021/acschemneuro.8b00279

Photoswitchable blockers of potassium channels can be used to optically control neuronal excitability and hold great promise for vision restoration. Here, we report a series of improved photoswitchable blockers that feature a new pharmacophore based on the local anesthetic bupivacaine. These azobupivacaines (ABs) enable optical control over the delayed rectifier channel Kv2.1. and target the two-pore domain potassium channel TREK-1. For the first time, we have identified a compound that blocks conductance in the dark and potentiates it upon illumination. Using light as a trigger, ABs efficiently and reversibly silence action potential firing of hippocampal neurons in acute mouse brain slices.

Full text of DOI:10.1021/acschemneuro.8b00279

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Letter
Optical Control of a Delayed Rectifier and a Two-Pore
Potassium Channel with a Photoswitchable Bupivacaine
Philipp Leippe, Nils Winter, Martin Sumser, and Dirk Trauner
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00279 • Publication Date (Web): 12 Jul 2018
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Optical Control of a Delayed Rectifier and a Two-Pore Potassium
Channels with a Photoswitchable Bupivacaine
†
,‡,#
†,#
†
*,†,
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⊥
Philipp Leippe , Nils Winter , Martin P. Sumser , Dirk Trauner
†Department of Chemistry and Center for Integrated Protein Science, LMU Munich, 81377 Munich, Germany.
Present address: Max Planck Institute for Medical Research, Department Chemical Biology, Jahnstr. 29, 69120 Heidelberg,
Germany.
New York University, Department of Chemistry, Silver Center for Arts and Science, 100 Washington Square East, New
‡
⊥
York, NY 10003, USA.
*
Correspondence should be addressed to D.T. (dirktrauner@nyu.edu)
ABSTRACT
Supporting Information Placeholder
(
photoswitchable tethered ligands, PTLs) or can operate on enꢀ
ABSTRACT: Photoswitchable blockers of potassium
dogenous, wildꢀtype proteins as freely diffusible photopharmacoꢀ
phores (photochromic ligands, PCLs). Both approaches have
made considerable advances in recent years, especially in the field
channels can be used to optically control neuronal ex-
citability and hold great promise for vision restoration.
Here, we report a series of improved photoswitchable
blockers that are furnished with a new pharmacophore
based on the local anesthetic bupivacaine. These azobu-
pivacaines (ABs) enable optical control over the delayed
5–7
of vision restoration.
Azobenzeneꢀcontaining PCLs based on the quaternary ammonium
ion (QA) tetraethylammonium (TEA) such as MAQ, AAQ and
DENAQ have enabled optical control over K channels (Fig. 1a).
In particular, DENAQ confers lightꢀsensitivity to degenerated
retinae from blind mice and is a potential treatment for degeneraꢀ
tive eye diseases such as retinitis pigmentosa and ageꢀrelated
macular degeneration. Despite their importance, one drawback
has been their low affinity to their targets, with useful concentraꢀ
5
8
7
v
rectifier channel K 2.1. and target the two-pore domain
v
potassium channel TREK-1. For the first time, we have
identified a compound that blocks conductance in the
dark and potentiates it upon illumination. Using light as
a trigger, ABs efficiently and reversibly silence action
potential firing of hippocampal neurons in acute mouse
brain slices.
5,7
9
tions in the high micromolar to single digit millimolar range.
This is presumably due to their permanent charge, which hampers
their ability to reach their binding site in the inner cavity ion
channel. While this issue may be alleviated by uptake through
auxiliary channels, such as P2X7, their comparatively low potenꢀ
cy prompted us to explore different pharmacophores. We beꢀ
came intrigued by the first description of TEAꢀinsensitive ‘leak’
channels in myelinated nerves in 1992 which identified bupivaꢀ
caine as an effective blocker, indicating a different mode of
1
0
KEYWORDS: Photochromic ligand, local anesthetics, photoꢀ
pharmacology, channel blocker, Kv2.1, TREKꢀ1
Light is an ideal input signal for the precise manipulation of bioꢀ
logical systems: it can be focused to defined areas, and can be
switched on and off rapidly. Therefore, it is a supreme tool in
neuroscience where events take place on a millisecond timescale
and in defined subpopulations of neurons. This explains the sucꢀ
cess of Optogenetics, which uses photoreceptors featuring naturalꢀ
ly occurring chromophores that can be recombinantly expressed
20
blockage. Therefore, we decided to investigate photoswitchable
versions of this widely used local anesthetic. Here, we describe
the design, synthesis and electrophysiological characterization of
a series of photoswitchable channel blockers derived from bupiꢀ
vacaine. Our resulting compounds not only target K channels,
such as K 2.1, but have also been shown to operate on the twoꢀ
v
v
1,2
in various tissues. A complementary approach, termed Photoꢀ
pore channel K 2.1, also known as TREKꢀ1.
Results and Discussion
2P
pharmacology, was described in 2004, wherein the photosensitiviꢀ3
ty was conveyed by a synthetic smallꢀmolecule photoswitch.
Design and synthesis of azobupivacaines AB1-3. One strategy
Azobenzenes are routinely employed as photoswitches due to
their remarkable photostability, small size, wellꢀdefined geometry
1
1
to render drugs lightꢀsensitive is termed ‘azoꢀextension’. In this
approach, furnishing drugs with a photoswitch is achieved by
identifying positions in their chemical structure where modificaꢀ
tions change biological activity but are not completely deleterious.
After modifying that position with a photoswitch, it is likely that
photoisomerization leads to lightꢀdependent change in biological
activity. Accordingly, we envisioned to install an azobenzene in
the paraꢀposition of the pipecolic amide of bupivacaine (Fig. 1b).
Isomerization of the azobenzene from its cisꢀ to its transꢀ
4
and ease of sythesis. In the dark, azobenzenes exist in their therꢀ
modynamically more stable transꢀconfiguration and they undergo
isomerization to the cisꢀconfiguration upon illumination. The
backꢀrelaxation can either occur thermally (i.e. in the dark), a
process whose kinetics are largely dependent on the azobenzenes
substituents, or it can be triggered by illumination with longerꢀ
wavelength light. These smallꢀmolecule photoswitches are then
either targeted to geneticallyꢀencoded bioconjugation motifs
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configuration by light should then change the PCLs efficacy
Next, the photoswitching properties of AB1-3 were evaluated
with UV/Vis spectroscopy. The optimal wavelengths for cisꢀtoꢀ
trans isomerization were determined in a wavelengthꢀscreen by
changing the illumination wavelength while looking at miniꢀ
mal/maximal extinction at the absorbance maximum near the πꢀπ*
transition as described previously. UV/Vis spectra of AB1-3
were then recorded under illumination of these optimal switching
wavelengths (Fig. 2 upper panels; AB1: λcis = 360 nm (light gray),
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and/or affinity to the ion channel, enabling optical control over
ion currents (Fig 1c). It should be noted that bupivacaine contains
an asymmetric carbon but is sold and used as a racemic mixture.
Therefore, our modular synthesis was carried out in the racemic
series to yield three azobupivacaines AB1-3 bearing different
substituents on the azobenzene for fineꢀtuning switching waveꢀ
lengths and kinetics, including a glycine moiety for AB2 that
facilitates further derivatization on the free amine,.
14
λtrans = 420 nm (violet); AB2: λcis = 380 nm (dark gray), λtrans
=
500 nm (green); AB3: λcis = 460 nm (blue); no illumination is
black in all cases). Both AB1 and AB2 exhibit a comparatively
slow thermal back relaxation to the transꢀisomer in the dark (Fig.
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lower panel). AB3 is redꢀshifted to longer wavelengths and
exhibits fast thermal relaxation in the dark. A fast thermal relaxaꢀ
tion can be beneficial in applications such as vision restoration,
where a quick turnꢀoff in the dark is required. In addition, longerꢀ
wavelength light allows deeper tissue penetration. In addition,
UV/Vis spectra of AB1ꢀ3 were recorded in aqueous PBS buffer
(Supporting Figure 4).
Figure 1. Logic and design of photoswitchable azobupivacaines
AB1-3. a) Photoswitchable blockers based on TEA. b) ‘Azoꢀ
extension’ strategy of azobupivacaines. c) Azobupivacaines AB1-
3
block channels in one configuration while allowing ion flow in
the other.
The synthesis of AB1-3 commenced with acyl chloride formation
of pipecolic acid (1) followed by amide bond formation with 4ꢀ
12
bromoꢀ2,6ꢀdimethylaniline to give pipecholyl xylidine 2. Direct
treatment of 2 with nꢀbutylbromide furnished tertiary amine 3 in
moderate overall yield (Scheme 1). Pdꢀcatalyzed amination of 3
using LiHMDS as the nitrogen source followed by direct deproꢀ
tection of the silyl groups gave aniline 4 as the common precursor
Figure 2. Top: UV/Vis spectra of AB1-3 (15 ꢀM in DMSO) in
the dark (black) and illuminated at the indicated wavelengths.
Bottom: Photoswitching is stable over many cycles. a) Black:
darkꢀadapted, violet: 420 nm, light gray: 360 nm; b) Black: darkꢀ
adapted, green: 500 nm, dark gray: 380 nm and c) Black: darkꢀ
adapted, blue: 460 nm.
1
3
for further derivatization. AB1 was then accessed by Baeyerꢀ
Mills reaction of 4 with nitrosobenzene. Treatment of 4 with tꢀ
BuONO followed by trapping of the resultant diazonium ion with
N,Nꢀdimethylaniline furnished AB3 in moderate yields. Aniline 6
was obtained from 4 after BaeyerꢀMills reaction with 4ꢀ
nitronitrosobenzene followed by reduction of the nitro group
using sodium sulfite. Amide bond formation of 6 with chloroaceꢀ
tyl chloride, followed by Finkelstein reaction and careful substituꢀ
tion with ammonia furnished AB2 in good overall yields.
ABs enable optical control of K 2.1
v
To evaluate the effect of ABs on K channels, HEK293T cells
v
were transiently transfected with the slowlyꢀdesensitizing, deꢀ
layedꢀrectifier K 2.1 channel. Prior to compound washꢀin, an IVꢀ
v
curve was recorded for normalization to account for cellꢀtoꢀcel+l
variances like size, conductance or expression. In particular, K
currents were evoked by depolarization from –80 to +40 mV in
wholeꢀcell voltageꢀclamp mode. Then, ABs were washed in and
illuminated with the appropriate wavelength to isomerize to the
trans-configuration (i.e. violet/green or dark) while recording two
IVꢀcurves. Maximum block was only achieved after the second
IV (trans-) which indicates that the channel has to be open to
1
) HCl, SOCl
2
1) LiHMDS
Pd(t-Bu P)
3 2
Pd(dba)
2
2) HCl
2)
Br
Br n-BuBr
CO
Br
H
N
O
O
O
H
N
K
2
3
2
H N
N
OH
N
H
N
H
DMF
PhMe, THF
(97%)
(16% over
PhMe
1
2
2 steps)
3
15
PhNO
AcOH
allow blockage by ABs. Next, two IVꢀcurves were recorded
N
NH
2
O
O
O
N
O
under the appropriate switching wavelengths to the cisꢀ
configuration to unblock the channel (Fig. 3a). The block was
more pronounced at high voltages which indicates a channel block
CH
2
Cl
2
N
N
N
N
N
H
N
H
(29%)
AB1
4
15
from the intracellular side (Fig. 3b). We tested AB1-3 at differꢀ
ent concentrations and determined AB2 as the most potent comꢀ
NO
2
1) t-BuONO
TFA
N
2) Me
2
NPh
N
ON
AcOH, CH
70%)
N
pound on K 2.1 channels, showing the maximum lightꢀdependent
v
2
Cl
2
MeCN
(65%)
N
H
(
AB3
block at 5 ꢀM (Fig. 3c and S3). As such, AB2 blocks 70.1±1.5%
+
R
of K current under green light, of which 58.8±4.7% can be reꢀ
1
)
O
N
H
N
O
N
leased under UVꢀillumination (Fig. 3 b,c). Interestingly, the subꢀ
stitution at the paraꢀposition of the azobenzene impacts potency
greatly since both AB1 and AB3 showed a decreased block (Fig.
3c). At a high concentration of 100 ꢀM AB1 blocked under violet
light with Imax = 0.22 ± 0.03 and unblocked under UVꢀlight with
Imax= 0.64 ± 0.03 (n = 4 cells). AB3 was only soluble up to 50 ꢀM
in extracellular buffer and at this concentration blocked currents
in the dark with Imax = 0.25 ± 0.06 while unblocking under blue
illumination with Imax = 0.67 ± 0.07 (n = 4 cells). Finally, to highꢀ
Cl
Cl
NH
2
N
N
H
DMAP, NEt3
N
O
N
2) NaI
3) NH3
(28% over
3 steps)
Na
2
S
N
H
5: R = NO
2
1,4-dioxane
H O
2
AB2
6: R = NH
2
(78%)
Scheme 1. Synthesis of AB1-3.
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light the reversibility and rapid speed of block and unblock with
AB2, we switched between UV/green illumination while holding
the cell at +20 mV (Fig. 3d).
100 ꢀM in neither the cisꢀ nor the transꢀ configuration was obꢀ
served (Fig. S2). Then, AB1-3 (Fig. 4a) were tested by looking at
the IVꢀrelationship from –80 to +40 mV. One IVꢀcurve for norꢀ
malization was recorded before washꢀin of compounds AB1-3.
The cell was then illuminated with light of the appropriate waveꢀ
length for the transꢀisomerization. (i.e. violet/green nm or dark).
Then the channel was unblocked by illumination with UV or blue
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light. Interestingly, and in contrast to the experiments with K 2.1,
AB1 emerged as the most efficacious compound for potentiating
TREKꢀ1 currents (Fig. 4b and S3). cisꢀAB1 potentiates TREKꢀ1
v
at 100 ꢀM with a maximum normalized current of Imax, norm.
=
1
.20ꢀ0.06 while transꢀAB1 blocks the channel efficiently with
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Imax, norm. = 0.39±0.07 (Fig. 4c). Two aspects of TREKꢀ1 blockade
by AB1-3 were different in comparison to K 2.1: Firstly, TREKꢀ1
v
blockage was maximal at the first recorded IVꢀstep and did not
require an earlier opening of the channel. Secondly, the percentꢀ
age of block did not increase at positive potentials. Taken togethꢀ
er, this points to a different mechanism of block on TREKꢀ1
compared to K 2.1.
Figure 3. Lightꢀgated K 2.1 currents in transiently transfected
v
v
HEK293T cells by AB1ꢀ3. a) After recording an initial IVꢀcurve
in 10 mV steps from –80 to +40 mV, AB2 (5 ꢀM) was washed in,
followed by illumination with green light (500 nm) to isomerize
+
AB2 to its trans-configuration, leading to a block of K current.
After isomerization to its cis-configuration by UVꢀlight (380 nm),
the blocking could be reversed. b) Representative singleꢀcell IVꢀ
curve before washꢀin, and in presence of AB2 under illumination
with 380 and 500 nm. c) Over multiple experiments, maximum
lightꢀgated currents were normalized to the maximum current
before washꢀin in the dark. AB1 (5 ꢀM) opened the channel under
UVꢀlight (360 nm) with Imax. = 0.86±0.04, while blocking under
violet light (420 nm) (Imax =0.60±0.03). AB2 (5 ꢀM) showed
currents of Imax = 0.89±0.05 under UVꢀlight (380 nm) and Imax.
=
0
.30±0.01 under green light (500 nm). AB3 showed no significant
block at 5 ꢀM with Imax. (460 nm) = 0.95±0.01 and Imax. (dark) =
0.89±0.01. d) To demonstrate the reversibility and fast kinetics of
block and unblock, the cell was held constantly at –20 mV while
switching with UV/green light (380/500 nm) in presence of AB2
(5 ꢀM).
ABs are photoswitchable blockers for the K2P channel TREK-
Figure 4. Lightꢀgated TREKꢀ1 currents in transiently transfected
HEK293T cells by AB1-3. a) After recording an initial IVꢀcurve
in 10 mV steps from –80 to +40 mV, AB1 (100 ꢀM) was washed
in, followed by illumination with violet light (420 nm) to isomerꢀ
ize to trans-AB1, leading to a block of K+ current. After isomeriꢀ
zation to cis-AB1 by UVꢀlight (360 nm), the block was not only
reversed but the channel was potentiated to larger currents than
before washꢀin. b) Representative singleꢀcell IVꢀcurve before
washꢀin, and in presence AB1 under illumination with UVꢀlight
1
+
TREKꢀ1 is a twoꢀporeꢀdomain K (K ) channel which is widely
2p
expressed in the central nervous system (CNS) and exhibits
16
unique physiological and pharmacological properties. Initially
described as ‘background’ or ‘leak’ channel, this channel was
17
cloned in 1996 and is an outward rectifier that is open at negaꢀ
tive membrane potentials. Therefore, expression of TREKꢀ1
drives the membrane potential closer to the potassium equilibrium
potential. TREKꢀ1 is activated by a variety of molecules such as
lipids and general anesthetics but is also responsive to nociceptive
(360 nm) and violet light (420 nm). c) Over multiple experiments,
1
8
maximum lightꢀgated currents were normalized to the maximum
current before washꢀin in the dark. AB1 (100 ꢀM) opened the
channel under UV light (360 nm) with Imax. = 1.29±0.06, while
blocking under violet light (420 nm, Imax =0.39±0.07). AB2 (100
ꢀM) showed currents of Imax = 1.01±0.10 under UV light (380
nm) and Imax. = 0.44±0.08 under green light (500 nm). AB3 (50
stimuli like membrane stretch. TREKꢀ1 is implicated in processꢀ
es ranging from neuroprotection, anesthesia and pain to mood
disorders such as depression and constitutes a fairly novel drug
1
9,20
target.
TREKꢀ1 is only weakly sensitive to quaternary ammoꢀ
21
niums like TEA. Therefore, optical control of TREKꢀ1 blockade
using the photoswitch MAQ could only be enabled at very high
local concentrations by tethering MAQ to a genetically introduced
ꢀ
M) blocked the current in in transꢀconfiguration (Imax.
=
2
2
0
.50±0.10) but could not be unblocked significantly under illumiꢀ
cysteine at its pore region. While this elegant approach could
unravel TREKꢀ1 contributions to hippocampal GABA signaling,
nation with blue light (460 nm, Imax. = 0.63±0.07). d) To demonꢀ
strate the reversibility and fast kinetics of block and unblock, the
cell was held constantly at +20 mV while switching with
UV/violet light (360/420 nm) in presence of AB1 (100 ꢀM).
B
it is limited by the requirement of genetic modification and by the
inherent drawbacks of cysteineꢀmaleimide chemistry such as
hydrolysis or its reactivity towards glutathione. Since bupivacaine
2
3
is a TREKꢀ1 blocker, we hypothesized that azobupivacaines
could photoswitch this channel. First, the inability of permanently
charged PCLs to block TREKꢀ1 was reassessed. Indeed, after
transfection of HEK293T cells with TREKꢀ1 no block by AAQ at
Optical control of action potential firing in acute hippocampal
slices by AB2
Having characterized our compounds in nonꢀexcitable transfected
cells, we tested the most potent compound AB2 (5 µM) for effects
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in horizontal brain slices of the mouse hippocampal CA1 region.
microscopy techniques widely used in life science research. In
order to enhance selectivity, we are currently working on equipꢀ
ping azobupivacaines with additional functionality, e.g. by tetherꢀ
ing these photoswitches to nanobodies or other proteinꢀbased
strong binders. Thereby, we hope to combine the advantages of
smallꢀmolecule photoswitches with the specificity of antibodies to
target potassium channels.
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Action potentials (APs) were evoked in wholeꢀcell currentꢀclamp
mode by injection of an aboveꢀthreshold current. Under illuminaꢀ
tion with green light (500 nm), the neuron could not sustain action
potential firing (Fig. 5a). This is due to the blockage of potassium
channels by transꢀAB2, resulting in inefficient repolarization.
Next, the neuron was illuminated with UV light (380 nm) to
isomerize to cisꢀAB2, relieving the channel blockage. In the
presence of cisꢀAB2, the neuron can fire repeatedly. To demonꢀ
strate the reversibility, the neuron was again illuminated with
METHODS
UV/Vis Spectroscopy. UV/Vis spectroscopy was performed
using a VARIAN Cary 50 Scan UV/Vis spectrometer. Compound
solution was placed in a standard quartz cuvette (d = 1 cm) illuꢀ
minated by a light fiber cable from above. For AB3 photoswitchꢀ
ing in PBS, illumination was performed using a SOLIS 445nm
HighꢀPowerꢀLED (Supporting Figure 4).
+
green light and was silenced. Similar as in transfected cells, K
currents could be blocked and unblocked rapidly by switching
between UV/green light (380/500 nm) (Fig. 5b).
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Cell Culture and whole-cell electrophysiology. HEK293T
(obtained from LeibnizꢀInstitute DSMZ: #305) cells were mainꢀ
tained in Dulbecco’s modified Eagle’s medium (DMEM) suppleꢀ
mented with 10% fetal calf serum (Biochrom, Merk Millipore,
Germany) in a 10% CO atmosphere at 37°C and were split at 80ꢀ
2
9
0% confluency. 40ꢀ60,000 cells were plated on acidꢀetched glass
coverslips in 500 µL full medium. Cells were transfected accordꢀ
ing to the manufacturer protocol (jetPRIME). TREK1 (50 ng) and
YFP (50 ng) or Kv2.1_GFP (50 ng)) (per coverslip: 50 uL) were
used for lipofection. Electrophysiology was performed 16ꢀ36h
postꢀtransfection. Electrophysiology was performed using a
standard electrophysiology setup equipped with a HEKA Patch
Clamp EPC10 USB amplifier and PatchMaster software (HEKA
Electronic). Micropipettes (Science Products) were generated
using a Narishige PCꢀ10 or PCꢀ100 vertical puller. The patch
pipette resistance varied between 5ꢀ9 Mꢁ. The bath solution
contained (in mM): NaCl (137), KCl (4), MgCl (1), CaCl (1.8),
2
2
HEPES (10), DꢀGlucose (10), pH to 7.4 with NaOH. The pipette
solution contained (in mM): KCl (12), Kꢀgluconate (140), HEPES
Figure 5. Optical control of action potenital firing in a hippocamꢀ
pal neuron in acute brain slices by AB2 (5 µM). a) transꢀAB2
(10), MgATP (4), Na GTP (0.4), NaCl (4), pH to 7.4 with KOH.
2
All analyzed cells were first visualized to contain YFP or Kv2.1ꢀ
GFP by irradiation at 480 nm using a Polychrome V (Till Photonꢀ
ics) monochromator. All patch clamp experiments were perꢀ
formed at room temperature. For photoswitching, light was apꢀ
plied via Polychrome V (Till Photonics) monochromator.
leads to silencing of the neuron due to blockage of K channels,
v
+
resulting in inefficient repolarization. b) K current was evoked
by depolarization to +60 mV and potentiated by switching
UV/green light (380/500 nm).
In conclusion, we report three novel photoswitchable channel
blockers that operate on K 2.1 at comparatively low concentraꢀ
v
tions. In addition, these compounds act as photochromic blockers
on the K2p channel TREKꢀ1. An opposing trend in efficacy deꢀ
pending on the azobenzene could be observed: TREKꢀ1 could not
accommodate large substituents on the azobenzene with the
smallest photoswitch AB1 exhibiting the largest potentiation of
Brain slice preparation. Hippocampal brain slices were prepared
from mice strain C57Bl6JRj (postnatal day 12ꢀ16) as described
27
previously. Briefly, mice were euthanized, the skull was quickly
removed and the brain placed in ice cold saline solution saturated
with carbogen (95/5 = O /CO ). The saline solution contained (in
+
2
2
K current. Furthermore, the cis-configuration was not only reꢀ
mM): 87 NaCl, 2.5 KCl, 1.25 NaH PO , 25 NaHCO , 0.5 CaCl , 7
+
2
4
3
2
lieving the blockage but potentiated the K currents towards larger
MgCl , 25 Dꢀglucose, 75 sucrose. Horizontal brain slices of 250
2
values. On K 2.1, the glycineꢀsubstituted AB2 proved to be the
v
mM thickness were prepared using a vibratome (Campden Inꢀ
struments). Prior to recording, the brain slice was placed in artifiꢀ
cial cerebrospinal fluid (aCSF) oxygenated with carbogen (95/5 =
most potent photoswitch.
Potassium channels are implicated in processes ranging from
cancer and metabolism to neurological and cardiovascular diseasꢀ
O /CO ). The aCSF contained (in mM): 125 NaCl, 2.5 KCl, 1.25
2
2
25
NaH PO , 1 MgCl , 2 CaCl , 26 NaHCO , and 20 Dꢀglucose.
es. However, this therapeutic potential has remained largely
2
4
2
2
3
Experiments were performed in continuous flow of aCSF in presꢀ
ence of AB2 (5 ꢀM).
untapped due to the lack of selective pharmacology, which i2n6
addition limited target validation to genetic knockout models.
While photoswitchable blockers like azobupivacaine inherit their
parent drugs’ lack of selectivity, the additional layer of control by
confining its activity in area and in time using light makes them a
useful tool. The advantage of photopharmacology lies in its simꢀ
plicity: it requires no genetic manipulation and photochromic
ligands can be applied like regular drugs. It only requires efficient
delivery and sculpting of light, which can be provided by the
ASSOCIATED CONTENT
Supporting Information
Detailed experimental procedures and characterization of phoꢀ
toswitches can be found in the supporting information, together
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with additional biological characterization and extended photoꢀ
(8)
Fortin, D. L.; Banghart, M. R.; Dunn, T. W.; Borges,
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chemical data.
K.; Wagenaar, D. A.; Gaudry, Q.; Karakossian, M. H.; Otis, T. S.;
Kristan, W. B.; Trauner, D.; et al. Photochemical Control of Enꢀ
dogenous Ion Channels and Cellular Excitability. Nat. Methods
2
008.
AUTHOR INFORMATION
(9)
Banghart, M. R.; Mourot, A.; Fortin, D. L.; Yao, J. Z.;
Corresponding Author
Kramer, R. H.; Trauner, D. Photochromic Blockers of Voltageꢀ
Gated Potassium Channels. Angew. Chem. Int. Ed. 2009, 48 (48),
9097–9101.
Dirk Trauner, dirktrauner@nyu.edu
(10)
Tochitsky, I.; Helft, Z.; Meseguer, V.; Fletcher, R. B.;
Author Contributions
Vessey, K. A.; Telias, M.; Denlinger, B.; Malis, J.; Fletcher, E. L.;
Kramer, R. H. How Azobenzene Photoswitches Restore Visual
Responses to the Blind Retina. Neuron 2016, 92 (1), 100–113.
P.L., N.W. and D.T. designed the study. N.W. performed the
chemical synthesis. P.L. and N.W. performed the UV/Vis experiꢀ
ments. P.L. performed the patch clamp experiments in transfected
cells. P.L. and M.P.S. performed patch clamp experiments in
brain slices. P.L. and D.T. wrote the manuscript with input from
all authors.
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Broichhagen, J.; Frank, J. A.; Trauner, D. A Roadmap
to Success in Photopharmacology. Acc. Chem. Res. 2015, 48 (7),
1947–1960.
(12)
bupivacaine and Analogues Thereof. WO9612700 (A1), May 2,
996.
13)
Frampton, G.; Zavareh, H. Process for Preparing Levoꢀ
#
P.L. and N.W.: equal contribution.
1
(
Hartwig, J.; Jorgensen, M.; Lee, S. Catalytic Method to
Funding Sources
Convert Aryl Compounds to Aryl Amines. WO03006420 (A1),
January 23, 2003.
D.T. was supported by an Advanced Grant from the European
Research Council (268795). P.L. and M.P.S. were supported by
the Deutsche Forschungsgemeinschaft within priority program
(14)
Barber, D. M.; Schönberger, M.; Burgstaller, J.; Levitz,
J.; Weaver, C. D.; Isacoff, E. Y.; Baier, H.; Trauner, D. Optical
Control of Neuronal Activity Using a LightꢀOperated GIRK
Channel Opener (LOGO). Chem. Sci. 2016, 7 (3), 2347–2352.
SPP1926.
N.W.
was
supported
by
the
Deutsche
Forschungsgemeinschaft within program TRR152.
(15)
French, R. J.; Shoukimas, J. J. Blockage of Squid Axon
Notes
Potassium Conductance by Internal TetraꢀNꢀAlkylammonium
Ions of Various Sizes. Biophys. J. 1981, 34 (2), 271–291.
The authors declare no competing financial interests.
(
16)
Focus on TREK1. Nat. Rev. Neurosci. 2007, 8 (4), 251–261.
17) Fink, M.; Duprat, F.; Lesage, F.; Reyes, R.; Romey, G.;
Honoré, E. The Neuronal Background K2P Channels:
ACKNOWLEDGEMENTS
(
P.L. gratefully acknowledges Dr. Johannes Broichhagen and Dr.
Timm Fehrentz for stimulating discussions and for proofreading.
P.L. gratefully acknowledges Prof. Dr. Kai Johnsson for support
and advice.
Heurteaux, C.; Lazdunski, M. Cloning, Functional Expression and
Brain Localization of a Novel Unconventional Outward Rectifier
K+ Channel. EMBO J. 1996, 15 (24), 6854–6862.
(18)
Vivier, D.; Bennis, K.; Lesage, F.; Ducki, S. Perspecꢀ
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