New Molecular Scaffolds for Fluorescent Voltage Indicators

Article abstract of DOI:10.1021/acschembio.8b00978

The ability to non-invasively monitor membrane potential dynamics in excitable cells like neurons and cardiomyocytes promises to revolutionize our understanding of the physiology and pathology of the brain and heart. Here, we report the design, synthesis, and application of a new class of fluorescent voltage indicators that make use of a fluorene-based molecular wire as a voltage-sensing domain to provide fast and sensitive measurements of membrane potential in both mammalian neurons and human-derived cardiomyocytes. We show that the best of the new probes, fluorene VoltageFluor 2 (fVF 2), readily reports on action potentials in mammalian neurons, detects perturbations to the cardiac action potential waveform in human induced pluripotent stem cell-derived cardiomyocytes, shows a substantial decrease in phototoxicity compared to existing molecular wire-based indicators, and can monitor cardiac action potentials for extended periods of time. Together, our results demonstrate the generalizability of a molecular wire approach to voltage sensing and highlight the utility of fVF 2 for interrogating membrane potential dynamics.

Full text of DOI:10.1021/acschembio.8b00978

Articles
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Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
New Molecular Scaffolds for Fluorescent Voltage Indicators
Steven C. Boggess,† Shivaani S. Gandhi,† Brian A. Siemons,‡,§ Nathaniel Huebsch,‡,§,#
Kevin E. Healy,‡,§ and Evan W. Miller*,†,∥,⊥
‡
§
∥
⊥
†Departments of Chemistry, Bioengineering, Materials Science & Engineering, and Molecular & Cell Biology and Helen Wills
Neuroscience Institute, University of California, Berkeley, California 94720, United States
S
* Supporting Information
ABSTRACT: The ability to non-invasively monitor membrane potential dynamics in
excitable cells like neurons and cardiomyocytes promises to revolutionize our understanding
of the physiology and pathology of the brain and heart. Here, we report the design,
synthesis, and application of a new class of fluorescent voltage indicators that make use of a
fluorene-based molecular wire as a voltage-sensing domain to provide fast and sensitive
measurements of membrane potential in both mammalian neurons and human-derived
cardiomyocytes. We show that the best of the new probes, fluorene VoltageFluor 2 (fVF 2),
readily reports on action potentials in mammalian neurons, detects perturbations to the
cardiac action potential waveform in human induced pluripotent stem cell-derived
cardiomyocytes, shows a substantial decrease in phototoxicity compared to existing
molecular wire-based indicators, and can monitor cardiac action potentials for extended
periods of time. Together, our results demonstrate the generalizability of a molecular wire
approach to voltage sensing and highlight the utility of fVF 2 for interrogating membrane
potential dynamics.
ptical methods for measuring biochemical and bio-
physical events in living cells provide a powerful
the cell of interest and an electrode. Electrophysiology is highly
invasive, low-throughput, and difficult to interpret in samples
like cardiac tissue or cardiomyocyte monolayers, where
electrical coupling between cells confounds single-cell
measurement. Optical recording of the membrane potential
using voltage-sensitive fluorescent indicators provides an
attractive alternative to probing Vm and AP dynamics in
multiple cells, in monolayers, or three-dimensional tissue.6
Recently, we initiated a program to develop a new class of
voltage-sensitive fluorescent indicators that utilize photo-
induced electron transfer (PeT) as a rapid trigger to sense
changes in Vm.7,8 These small molecule voltage-sensitive
fluorophores, or VoltageFluors (VF dyes), combine a
xanthene-based dye as a fluorescent reporter and a conjugated
molecular wire that localizes the indicator to the cell
membrane and facilitates PeT from an electron-rich aniline
donor to the fluorophore within the low-dielectric environ-
ment of the lipid bilayer. Phenylene-vinylene (PV)-based
conjugated molecular wires (Scheme 1, VF2.1.Cl)9,10 are
attractive because of their exceptionally low electron transfer
attenuation values in donor−bridge−acceptor (DBA) systems
(β = 0.04 Å−1).11,12 Within a PV wire framework, voltage
sensitivity can be improved by altering the redox potentials of
the fluorophore electron acceptor and the aniline electron
donor.13 Additionally, fluorophores such as rhodamine9 and
silicon-rhodamine10 can be substituted for fluorescein, after
some adjustment to the identity of the aniline donor. To date,
O
approach for monitoring cellular physiology in a non-invasive
and high-throughput manner. The success of such light-based
ventures depends critically on the ability to design and
construct molecules that change their optical properties, for
example, color, fluorescence intensity, or lifetime, in response
to changes in the cellular environment.1 Fluorescence
microscopy is one of the most commonly used modalities
because it is operationally simple, the instrumentation is widely
available, and an ever-growing library of small molecule
fluorescent indicators exists to probe the dynamics of a host
of cellular analytes, properties, and structures.2−5 Of particular
interest is the plasma membrane. Changes in the electro-
chemical potential across the lipid bilayer profoundly shape
cellular physiology. Excitable cells employ a consortium of ion
channels both to maintain tight control over their membrane
potential (Vm) and to initiate and propagate rapid changes in
Vm. Rapid changes in Vm such as an action potential (AP) drive
the unique physiology of excitable cells like neurons and
cardiomyocytes. In neurons, APs evoke the release of the
neurotransmitter into the synaptic cleft, and in heart tissue,
waves of APs coordinate contraction and maintain regular
rhythm. Disruption of the frequency, timing, and/or shape of
APs is linked to serious human diseases ranging from epilepsy
to long QT syndrome. Because of the importance of Vm to
both health and disease, robust methods for optically
monitoring the membrane potential remain a critical comple-
ment to more traditional approaches. The gold standard for
measuring Vm and APs in live cells is electrophysiology: direct
determination of Vm through the physical interaction between
Received: November 1, 2018
Accepted: January 24, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acschembio.8b00978
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Scheme 1. Synthesis of Fluorene VoltageFluor Dyes (fVF dyes)
Scheme 2. Synthesis of Electron-Rich Fluorene VoltageFluor Dyes
we have not explored alterations to the identity of the
molecular wire component of PeT-based voltage indicators.
Now, we present a new class of PeT-based voltage-sensitive
fluorescent indicators that use a 9,9-dimethyl-9H-fluorene
monomer in place of the canonical 1,4-divinylbenzene moiety
(Scheme 1). In other DBA scaffolds, 2,7-oligofluorene bridges
effectively facilitate electron transfer across large distances and
demonstrate low β values (0.09 Å−1) comparable to values for
PV wires (0.04 Å−1).14 Because β values depend both on the
identity of the wire and on the donor/acceptor pair,15,16 we
wanted to explore fluorene molecular wires as a platform for
optical voltage sensing, demonstrating the generalizability of a
PeT-based approach to voltage sensing. We now report the
design, synthesis, characterization, and application of a new
series of fluorene-based VoltageFluors, or fVF dyes.
The synthesis of fluorene-based voltage indicators starts with
Suzuki−Mirayura cross-coupling of bromo-iodo-fluorene 117
with either boronic ester 818 or phenylboronic acid, providing
monomeric fluorenes 2 and 3 as bright yellow solids (Scheme
1). Attachment to a sulfonated dichlorofluorescein was
achieved by Pd-catalyzed cross-coupling of a pinacol boronic
ester with the terminal aryl bromide to provide 4 and 5.
Suzuki−Mirayura cross-coupling yielded voltage indicator 6
(fVF 1) and indicator 7 (fVF 0), which lacks an aniline donor
(Scheme 1). For the wires with electron-rich donors [12 and
13 (Scheme 2)], transformation of the nitro group to an
aniline was performed with tin(II) chloride to yield wires 14
and 15. This was followed by reductive amination of
formaldehyde with NaCNBH3 to provide alkylated wires 16
New fVF dyes have a λmax centered around 520 nm and a
second major absorption band around 340 nm arising from the
fluorene molecular wire (Figure 1, Figure S1, and Table 1).
Each fVF dye has a maximum emission around 535 nm,
indicating little ground state interaction between the fluorene-
based molecular wire and xanthene chromophore. fVF 1−3
have fluorescence quantum yields (Φfl, 0.05−0.19) that are
lower than that of control indicator fVF 0 [0.77 (Table 1)].
To measure the voltage sensitivity of these indicators, we
used whole cell voltage-clamp electrophysiology in tandem
with epifluoresence microscopy. By applying voltage steps
ranging from 100 to −100 mV in 20 mV increments to
HEK293T cells stained with fluorene voltage indicators, we
observe that indicators fVF 1−3 possess moderate sensitivity
to changes in Vm. Similar to PV-based molecular wire voltage
indicators, fVF 1−3 become brighter in response to
depolarizing (more positive) membrane potentials. The two
most sensitive compounds, fVF 2 and 3, have sensitivities of 11
and 13% ΔF/F per 100 mV, respectively; however, fVF 2 has
an overall signal that was much brighter in cells, resulting in a
better signal-to-noise ratio (SNR) (Figure 1, Figure S2, and
Table 1). fVF 1 is also very bright in cells but has a low SNR
due to a low sensitivity (5% ΔF/F). Somewhat surprisingly, the
electron-deficient compound fVF 0 [7 (Scheme 1)] exhibits a
small amount of voltage sensitivity, −0.3% ΔF/F per 100 mV,
becoming less fluorescent in response to depolarizing
potentials (Table 1, Figure S2, and Table 1), a behavior that
is the opposite of that of every molecular wire indicator
synthesized in our laboratory. We chose to characterize fVF 2
in subsequent experiments due to its brightness and superior
SNR.
and 17. These were prepared via a method similar to that
depicted in Scheme 1 to yield electron-rich voltage indicators
20 (fVF 2) and 21 (fVF 3).
fVF 2 readily detects fast changes in membrane potential in
mammalian neurons. In cultured rat hippocampal neurons, fVF
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observe evoked spikes with a ΔF/F of −1% and a SNR of 15:1.
However, a 5-fold higher concentration was needed to make
these recordings (Figure S4 and Table S1). In addition to
reporting on neuronal activity, we detect no changes to
neuronal membrane properties or action potential kinetics
when comparing the electrophysiological parameters of
neurons in the absence or presence of fVF2 (Figure S6 and
Table S2).
We sought to use fVF 2 for optical measurements of cardiac
AP waveforms to provide a holistic assessment of drug
cardiotoxicity in vitro, a major goal of the Comprehensive in
vitro Proarrythmic Assay (CiPA) initiative.19−21 We cultured
hiPSC-CM monolayers and tested the ability of fVF 2 to
report cardiac AP waveforms in spontaneously beating
monolayers.21−25 fVF 2 clearly stains the sarcolemma of
hiPSC-CMs and faithfully reports ventricular-like AP wave-
forms, showing a large increase in fluorescence just before
contraction of the monolayer (Figure 2a−e). Using methods
previously described,26 we calculate the action potential
duration (APD) for each AP waveform in the fluorescence
trace at 70, 50, and 10% of the maximum depolarization
(APD30, APD50, and APD90, respectively).26−28 To correct
for APD variation arising from the difference in beat rate from
spontaneously beating monolayers, we used Fridericia’s
formula to provide a beat-rate corrected APD (cAPD).28,29
From our optical measurements, we calculated cAPD90 values
from 500 to 700 ms in spontaneously beating monolayers after
14 days in culture, consistent with previous reports for hiPSC-
CMs.27,30
To demonstrate the utility of fVF 2 for parsing the
pharmacological effects of drug treatment on cardiomyocytes,
we treated hiPSC cardiomyocytes with cisapride. Cisapride,
formally a useful gastroprokinetic agent, was withdrawn from
the U.S. market in 2000 due to its connection to torsades de
pointes (TdP) induced by acquired long QT syndrome caused
by blockage of Kv11.1.31 Cisapride is also one of 12 training
and calibration compounds used in the CiPA initiative.20,32
The observation of cardiomyocyte monolayers treated acutely
with cisapride results in several phenotypic alterations to
cardiac AP, which can be readily detected by fVF 2. At a
concentration of 300 nM, we saw three different manifestations
of the effect of prolonged cAPD caused by IKr blockade:33,34 an
extended phase 3 (Figure 2g), a tachycardia-like train of
drastically shortened APs (Figure 2h), and the appearance of
early after depolarizations (EADs) (Figure 2i). Kv11.1/hERG
channel blockade results in action potential prolongation
(Figure 2g), which has been connected to a higher risk of TdP
and higher arrhythmogenic potential. The rapid, subthreshold
spiking activity may be analogous to tachycardia,35 which was
accompanied by a loss of monolayer automaticity (Figure 2h).
The appearance of EADs (Figure 2i) corresponds to the
Figure 1. Characterization of fluorene VoltageFluor 2 (fVF 2). (a)
Live cell fluorescence microscopy image of fVF 2 in HEK cells. The
scale bar is 10 μm. (b) Normalized absorption and emission spectra of
fVF 2. Spectra were acquired in phosphate-buffered saline (pH 7.2)
and 0.1% sodium dodecyl sulfate. For an emission scan, excitation was
provided at 485 nm. (c) Voltage sensitivity of fVF 2 in patch-clamped
HEK cells. (d) Plot of ΔF/F vs membrane potential (in millivolts) for
fVF 2. The red line is the line of best fit. Error bars are standard errors
of the mean for eight independent determinations. (e) Live cell, wide-
field fluorescence images of rat hippocampal neurons stained with 500
nM fVF 2. The scale bar is 20 μm. (f) Representative ΔF/F plot of
evoked neuronal activity of a single cell recorded optically with fVF 2.
2 gives clear membrane staining (Figure 1e) and faithfully
records evoked action potentials with an average ΔF/F of 5.1%
and SNR of 21:1 [n = 54 spikes (Figure 1f and Figure S4)].
fVF 2 clearly resolves spontaneous activity in cultured rat
hippocampal neurons (Figure S5). Despite the lower nominal
voltage sensitivity of fVF 2 relative to a first-generation
VoltageFluor dye [VF2.1.Cl, 27% ΔF/F per 100 mV in HEK
cells,7 10% ΔF/F and a SNR of 43:1 in evoked action
potentials; n = 54 spikes (Figure S4)], the improved brightness
of fVF 2 relative to VF2.1.Cl [1.2 times brighter in neurons
(Table S1)] makes it useful for recording action potentials in
neurons. When we evaluate fVF 2 against an electrochromic
VSD with a similar voltage sensitivity, di-4-ANEPPS, we
Table 1. Properties of Fluorene VoltageFluor Dyes (fVF dyes)
a
a
a
a
b
b
entry
R1
R2
-H
-H
-H
absorbance (nm)
emission (nm)
QY
% ΔF/F
SNR
fVF 0 (7)
fVF 1 (6)
fVF 2 (20)
fVF 3 (21)
-H
519
519
520
520
535
534
535
535
0.77
0.05
0.07
0.19
−0.3 0.03
4.7 0.5
10.5 0.8
12.2 1.6
1.9:1
22:1
39:1
13:1
-N(Me)2
-N(Me)2
-N(Me)2
-OMe
a
b
Measured in phosphate-buffered saline and 0.1% sodium dodecyl sulfate (pH 7.2). Per 100 mV. Recorded in HEK 293T cells at an optical
sampling rate of 0.5 kHz.
C
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toxicity.35−37 Therefore, we were pleased that fVF 2 displays
lower phototoxicity in hiPSC-CMs relative to VF2.1.Cl, which
itself requires a light power an order of magnitude lower than
that of di-4-ANEPPS, thereby avoiding phototoxic effects.37
Others note phototoxic effects of di-4-ANEPPS can begin
within seconds of illumination.38 We stained hiPSC-CM
monolayers with either fVF 2 or VF2.1.Cl and continuously
illuminated them for 10 min and optically recorded membrane
potential dynamics every minute. Despite the initial photo-
bleach of fVF 2 (Figure 3a and Figure S2i), the shapes of
recorded action potentials (Figure 3c), SNR (Figure 3e), and
action potential duration (Figure 3f,g) remain relatively
constant (Figure 3c,d and Figure S7). In sharp contrast,
however, VF2.1.Cl had a dramatic, detrimental impact on
cardiomyocyte function. Although VF2.1.Cl initially has a high
SNR compared to that of fVF 2 (Figure 3b,e), the SNR drops
quickly after the first minute of illumination (Figure 3b,e), and
both action potential shape (Figure 3d) and duration (Figure
3f,g) undergo substantial and significant changes, as early as 2
min into illumination (Figure 3b,d,f,g). Even after 4 min, only
subthreshold activity was recorded with VF2.1.Cl and required
an automated analysis script to detect these events (Figure
S7h). After 5 min, monolayers imaged with VF2.1.Cl cease to
contract (Figure 3d and Figure S7). However, monolayers
imaged with fVF 2 continue to beat even after continuous
illumination for 10 min (Figure 3c and Figure S7). Increasing
the illumination intensity from 9 to 29 mW/mm2 when making
recordings with fVF 2 did not alter cAPD (Figure S7l).
Toxicity requires both dye and illumination, as regions of the
coverslip not exposed to illumination light beat properly, in the
case of fVF 2 (Figure S8). For VF2.1.Cl, we observe a small
increase in cAPD values, which was not statistically significant
(Figure S8). Together, these results suggest that fVF 2 has a
phototoxicity that is lower than that of VF2.1.Cl and can be
used to measure activity in cardiomyocyte monolayers for
prolonged periods of time. Previous reports note slight
differences in the structure of transmembrane probes can
explain differences in phototoxicity.38 Experiments are under-
way to probe the precise molecular mechanisms underlying the
faster photobleaching but reduced phototoxicity of fVF 2
compared to that of VF2.1.Cl.
To assess the ability of fVF 2 to measure changes to cardiac
electrophysiology in response to chronic drug treatment, we
calculated IC50 values for cisapride using in-well dose
escalation with optical recording of cAPD. Measurements
were taken after incubation with increasing cisapride
concentrations in each well from 0.1 to 300 nM. Our optical
measurements show an increase in cAPD90 (IC50 = 10.6 nM;
14 days in culture) to 300 nM cisapride (Figure 4e,f), which is
in the range of IC50 values previously measured for cisapride in
other in vitro studies (Table S3).32 We also observe an increase
in cAPD50 to 100 nM but a decrease at higher concentrations
of cisapride. Similarly, the measured cAPD30 seems to vary
little from that of the dimethyl sulfoxide vehicle control;
however, a decrease is detected at higher concentrations of
cisapride (Figure 4e,f). Together, these results demonstrate the
ability to fVF 2 to enable the rapid assembly of dose−response
data in hiSPCs using an all-optical approach.
Figure 2. Fluorene VoltageFluor 2 (fVF 2) reliably reports on cardiac
action potential (cAP) dynamics in human induced pluripotent stem
cell-derived cardiomyocytes (hiPSC-CMs). (a) Bright field. (b)
Membrane localization of fVF 2 (1 μM) in hiPSC-CM monolayers.
(c) Hoescht 33342 nuclear stain. (d) Merge of membrane and nuclear
stains. The scale bar is 20 μm. (e) Representative image used to
acquire functional AP data. The scale bar is 10 μm. (f−i)
Representative fluorescence traces acquired using 1 μM fVF 2. (f)
Baseline measurement of a spontaneously contracting monolayer.
Treatment with cisapride (300 nM) results in (g) prolonged APs, (h)
shorter and more frequent APs, and (i) early after depolarizations
(EADs). (j) Overlay of single APs from panels f−h to highlight
observed waveform changes from cisapride treatment. The baseline
trace in black shows a normal ventricular-like shape; the red trace
depicts an extended AP after treatment (from panel g), and the blue
trace depicts a shortened AP from a tachycardia-like phenotype (from
panel h).
observed cAPD prolongation and significantly increases the
risk of arrhythmia and TdP.
In summary, we present the design, synthesis, and
application of a new class of molecular wire-based fluorescent
indicators. We show, for the first time, that fluorene-based
molecular wires provide a platform for PeT-based voltage
sensing. In general, these fluorene-based indicators have lower
Monitoring the effect of potentially cardiotoxic drug relies
on the ability to make stable, long-term recordings from
cardiomyocytes. When using voltage-sensitive fluorescent
indicators, this often requires careful titration of illumination
intensity and indicator concentration to minimize photo-
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Figure 3. fVF 2 displays low phototoxicity in cardiomyocyte monolayers. Fluorescence intensity vs time for (a) fVF 2 and (b) VF2.1.Cl in
monolayers of hiSPC-CMs. The raw fluorescence intensity from an entire field of view over an entire 10 s recording session is plotted vs the total
illumination time (in minutes). Individual action potential (AP) traces for (c) fVF 2 and (d) VF2.1.Cl are indicated by red stars in panels a and b.
Plot of the mean (e) signal-to-noise ratio (SNR), (f) cAPD30, and (g) cAPD50 as a function of total illumination time for fVF 2 (black) and
VF2.1.Cl (blue). For panels e−g, mean values are determined from n = 3 independent trials, and error bars are standard error of the mean.
Statistical tests were two-tailed, unpaired t tests for each cAPD at the indicated time vs time zero. **p < 0.005. ***p < 0.001. **** p < 0.0001.
Figure 4. Fluorene VoltageFluor 2 (fVF 2) reveals changes to cardiac action potentials (APs) upon treatment with cisapride. (a) Action potential
duration (APD) values calculated from the maximum derivative of the depolarization to the repolarization at 70, 50, and 10% of the maximum
depolarization value are corrected for beat rate and reported as cAPD30, cAPD50, and cAPD90, respectively. Treatment of monolayers cultured for
14 days with doses of cisapride from 0.1 to 300 nM (red) results in (b) little change in cAPD30, (c) a moderate increase in cAPD50, and (d) a
clear increase in cAPD90. Black points indicate data for dimethyl sulfoxide-treated control samples. Plots indicate mean values the standard error
of the mean for n = 4 independent experiments.
nominal voltage sensitivities (ΔF/F per 100 mV of 5−13% in
HEK cells, compared to approximately 27% for VF2.1.Cl) to
changes in membrane potential but are brighter than their
phenylene-vinylene counterparts. fVF 2 exhibits adequate
sensitivity and excellent brightness in cells for reporting AP
waveforms in neurons and cardiomyocytes with high SNRs.
More importantly, fVF 2 displays a substantially reduced
phototoxicity in cardiomyocytes relative to that of VF2.1.Cl,
allowing for prolonged, continuous measurement of cardio-
myocyte activity. Fluorene-based molecular wires may provide
an attractive, general solution to the phototoxicity often
associated with voltage-sensitive fluorescent indicators.
ASSOCIATED CONTENT
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S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.8b00978.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: evanwmiller@berkeley.edu.
ORCID
Kevin E. Healy: 0000-0002-8524-3671
Evan W. Miller: 0000-0002-6556-7679
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Present Address
#N.H.: Department of Biomedical Engineering, Washington
University in St. Louis, St. Louis, Missouri 63130.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge financial support of this
research from the following organizations: National Institutes
of Health, Grants R35GM119855 (E.W.M.), T32GM066698
(S.C.B.), and R01HL130417 (K.E.H.); and the California
Institute for Regenerative Medicine (Grant DISC2-100090 to
K.E.H.).
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