ExoSensor 517: A dual-analyte fluorescent chemosensor for visualizing neurotransmitter exocytosis

Article abstract of DOI:10.1021/cn400128s

A dual-analyte fluorescent chemosensor (ExoSensor 517) for the direct visualization of neurotransmitters released upon exocytosis is presented. The sensor exploits the high concentration of neurotransmitters (e.g., glutamate, norepinephrine, and dopamine) and the pH gradient between the vesicle and synaptic cleft. The cooperative recognition elements require both binding and a change in environmental pH to afford a fluorescence response which makes ExoSensor 517 one of the first integrated molecular logic gates to be used for biological applications.

Full text of DOI:10.1021/cn400128s

Letter
pubs.acs.org/chemneuro
ExoSensor 517: A Dual-Analyte Fluorescent Chemosensor for
Visualizing Neurotransmitter Exocytosis
Jessica L. Klockow, Kenneth S. Hettie, and Timothy E. Glass*
Department of Chemistry, University of MissouriColumbia, 601 South College Avenue, Columbia, Missouri 65211, United States
S
* Supporting Information
ABSTRACT: A dual-analyte fluorescent chemosensor (ExoSensor 517) for the
direct visualization of neurotransmitters released upon exocytosis is presented.
The sensor exploits the high concentration of neurotransmitters (e.g., glutamate,
norepinephrine, and dopamine) and the pH gradient between the vesicle and
synaptic cleft. The cooperative recognition elements require both binding and a
change in environmental pH to afford a fluorescence response which makes
ExoSensor 517 one of the first integrated molecular logic gates to be used for
biological applications.
KEYWORDS: chemosensor, neurotransmitter, logic gate, exocytosis, fluorescence
eurotransmitters are critical to the regulation of the
central and peripheral nervous systems and command a
irreversible affinity for glutamate with limited dynamic range
and overall small changes in fluorescence. To avoid the use of
protein-based fluorophores, a pH-sensitive fluorescent false
neurotransmitter (FFN) has been developed to monitor
exocytosis. This fluorescent tracer is loaded into vesicles
expressing VMAT and fluoresces upon exocytosis similar to the
synapto-pHluorins.17
We recently developed NeuroSensor 521 (NS521) as a
fluorescent chemosensor for selective labeling and imaging of
the neurotransmitters norepinephrine and dopamine inside
secretory vesicles (Figure 1).18 The sensor enters the vesicle
N
number of functions such as learning, memory, sleep, and
movement.1,2 Discerning the machinery involved in vesicular
fusion, the spatiotemporal mechanisms of synaptic release, and
the chemical activity of neurotransmitters is vital to under-
standing both normal and atypical cellular processes. The
ability to effectively monitor exocytotic operations bolsters
research in neuroscience, serving as a useful tool in the study of
neurophysiology and neuropsychiatric disorders. Methods to
evaluate exocytosis include fluorescence imaging,3−7 capillary
electrophoresis,8−11 microelectrochemistry,12,13 and mass spec-
trometry.14 Nonoptical techniques are limited by poor
throughput and a lack of spatial resolution.15 Conversely,
fluorescence methods offer a sensitive means to elucidate the
spatial distribution of neuronal vesicles and chemical
messengers.
Fluorescence imaging of secretion was studied early on by
loading chromaffin cells with acridine orange and observing a
loss in fluorescence upon exocytosis.4 More recently, exocytosis
has been visualized using the genetically encoded synapto-
pHluorins, wherein a pH-sensitive GFP construct is expressed
on the inner membrane of secretory vesicles. The engineered
vesicles fluoresce upon exocytosis due to a change in pH from
the acidic synaptic vesicle (∼5) to the neutral synaptic cleft
(∼7.4).5 These methods solely monitor the process of vesicle
membrane fusion during an exocytotic event but do not directly
image active neurotransmitters released upon exocytosis. In
recent years, a genetically encoded CFP/YFP FRET biosensor
was developed to monitor glutamate release, spillover, and
reuptake by fluorescence.16 However, these protein-based
biosensors require genetic manipulation and display high,
Figure 1. Structures of NS521 and ES517.
and binds to the primary amine of the catecholamine, creating a
positively-charged iminium ion. The charged complex cannot
translocate across the vesicular membrane and becomes
trapped, accumulating inside the vesicle. Formation of the
iminium ion also induces a bathochromic shift in absorbance
that can be selectively excited at 488 nm allowing the
neurotransmitter to be imaged directly, giving the signature
punctate fluorescence. To image exocytosis of these neuro-
Received: June 26, 2013
Accepted: August 8, 2013
© XXXX American Chemical Society
A
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ACS Chemical Neuroscience
Letter
transmitters, we sought to create a pH-sensitive sensor based
on the NS521 model.
causes the bound sensor to become trapped within the vesicle,
encouraging sensor accumulation. Exocytosis releases the
bound sensor complex into the synaptic cleft and the change
in environmental pH switches the fluorescence “on” for only
the released sensor in its bound state.
To create a sensor that would respond in this way, the diethyl
amino group of NS521 was exchanged for an electron-poor
sulfamide that can be deprotonated at neutral pH to become
electron-rich (Scheme 1). The sensor fluoresces via an
Herein, we report an approach that utilizes a novel dual-
analyte chemosensor, ExoSensor 517 (ES517), that selectively
labels primary amine neurotransmitters found at high
concentrations within vesicles (e.g., glutamate, norepinephrine,
and dopamine) and allows for direct visualization of only active
neurotransmitters released upon exocytosis by capitalizing on
the pH gradient between the vesicle and the synaptic cleft.
ES517 incorporates two distinct recognition elements into a
single molecular receptor and requires both analyte binding and
pH change to afford strong fluorescence at 517 nm. The logic
gate design strategy allows for direct imaging of released
neurotransmitters while minimizing interference with native
neurotransmitter trafficking due to the reversible recognition
motif. To our knowledge, this is the first integrated molecular
logic gate for biological applications.
Scheme 1. Neurotransmitter Binding and Deprotonation of
a
ES517
As depicted in Figure 2, the dual-analyte sensor can enter the
vesicle and selectively bind to primary amine neurotransmitters
at high concentrations, yet remain fluorescently “off” due to the
acidic environment. Formation of a positively-charged complex
a
Quantum yields were measured in triplicate in buffer (50 mM bis-tris
propane, 120 mM NaCl, 1% DMSO) alone (A and C) and with
saturating glutamate (300 mM, B and D) at pH 5.0 (A and B) and 7.4
(C and D). Excited at 488 nm. Calculated by using fluorescein as a
fluorescence standard (Φfl = 0.850).
intramolecular charge transfer (ICT) state in which the
sulfamide acts as an electron donor and the aldehyde acts as
an electron acceptor. Both analyte binding and sulfamide
deprotonation enhance the charge transfer across the
fluorophore. Since binding to the analyte and deprotonation
of the sensor both produce large bathochromic shifts, exciting
the sensor at the highest wavelength of absorption will allow
visualization of only the active neurotransmitters in the synapse.
Any unbound sensor molecules would not be visualized as they
absorb at much lower wavelengths.
In the cytosol, ES517 (A) will exist largely in the
deprotonated form (C) due to the neutral pH and relatively
low concentration of amines. Both forms A and C would have
weak fluorescence as they lack the iminium ion as a strong
acceptor. When ES517 enters the vesicle, it binds to the
neurotransmitter due to its high concentration producing the
iminium ion B. Form B would have marginal electron transfer
and weak fluorescence since the protonated sulfamide is a weak
donor. Upon exocytosis, the bound complex enters the synaptic
cleft, becomes deprotonated (structure D), and produces a
marked fluorescence increase due to the enhanced ICT.
This mechanism of action is similar to that of known
molecular logic gates;19,20 however, this approach is distinct in
that it integrates the switch, fluorophore, and receptor into one
unit for the purposes of biological chemical sensing. To obtain
the maximum fluorescence increase upon exocytosis, the sensor
pKa must be between the pH of the vesicle (5) and the synaptic
cleft (7.4). The sulfamide functional group was chosen as
Figure 2. Sensing mechanism of ES517. (a) Sensor enters vesicle and
selectively labels the neurotransmitter (NT). The bound complex
accumulates and fluorescence is “off” due to low pH (5). (b) Influx of
Ca2+ triggers exocytosis. The increase in environmental pH in the
synaptic cleft (7.4) switches the fluorescence “on” for only the bound
complex.
B
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ACS Chemical Neuroscience
Letter
literature precedent indicated the pKa would be approximately
6.21
ES517 was prepared in four steps (Scheme 2). Compound 1
was synthesized from commercially available starting materi-
Scheme 2. Synthesis of ES517
als22 and underwent a Suzuki coupling to achieve the phenyl-
substituted compound 2. Demethylation and triflation of the
resulting phenol produced compound 4. The sulfamide
substituent was appended using Buchwald-Hartwig coupling
conditions to produce ES517.
ES517 was studied via UV/vis and fluorescence spectrosco-
py. Initially, binding studies were performed by titrating ES517
with glutamate and exciting at 488 nm, a region that is
sufficiently red to prohibit absorbance and subsequent emission
from the unbound sensor (Figure 3). A 12-fold fluorescence
enhancement was obtained upon binding to glutamate though
with a low binding constant (Ka = 8.6 M−1). While low, this
binding constant is actually preferred for reversible binding to
the high millimolar concentration of glutamate in a vesicle
(∼300 mM).23−25
To test the pKa of the bound sensor, ES517 was saturated
with glutamate and the spectroscopic properties measured over
a wide pH range (Figure 4). The protonated form of the bound
sensor (Figure 2, structure B) absorbed at 368 nm. Upon
deprotonation, two bands were observed at 428 and 458 nm.
The 428 nm band was assigned to the unbound deprotonated
form of the sensor (structure C, compare with Figure S1 in the
Supporting Information). The 458 nm band therefore
represents the bound, deprotonated sensor (structure D).
The plot of pH vs intensity was fit to a pH isotherm, and the
pKa determined to be 6.3. The fluorescence quantum yield
(Φfl) for ES517 was determined in each state using glutamate
as the analyte (shown in Scheme 1). The quantum yields were
similar, with the bound states being slightly lower than the
unbound states. Since the sensor is selectively excited at a
wavelength at which only form D absorbs, these quantum yields
are sufficient for imaging purposes.
Figure 3. (a) Absorbance and (b) fluorescence titration of ES517 (20
μM) in buffer (50 mM bis-tris propane, 50 mM Na2S2O3, 1% DMSO,
pH 7.4) adding 20−800 μL aliquots of 1.5 M glutamate. λex = 488 nm.
Inset is the fit to a binding isotherm.
was saturated with glutamate, and the resulting iminium ion
had a pKa of ∼9. This result indicates that the imine formed
upon binding to the neurotransmitter will be protonated within
the physiological pH ranges described.
To estimate the response of the sensor−glutamate complex
to exocytosis, its fluorescence at pH 7.4 was compared to that
at pH 5.0 and a 12-fold difference was calculated. This implies
that the sensor−glutamate complex will produce a significant
fluorescence enhancement upon exocytosis.
The key spectroscopic properties of ES517 binding to
glutamate are tabulated in Table 1 along with several other
neurotransmitters. As with the non-pH-sensitive NS521, all
primary amine neurotransmitters bound relatively weakly with a
preference for aromatic neurotransmitters. By contrast, the
non-aromatic neurotransmitters produced the largest change in
fluorescence upon binding at pH 7.4 due to the absence of PET
quenching effects from the electron-rich aromatic groups.
Importantly, all nonaromatic neurotransmitters produced large
fluorescence increases upon pH change from 5.0 to 7.4. The
catecholamines gave smaller, but still significant fluorescence
increases and serotonin was unique in that it gave little
fluorescence change (Figure 5).
In summary, ExoSensor 517, a dual-analyte fluorescent
chemosensor, was developed for the visualization of neuro-
transmitters released upon exocytosis. The logic-based design
strategy exploits high vesicular neurotransmitter concentrations
and the pH gradient between the vesicle and synaptic cleft. The
glutamate-bound sensor afforded a 12-fold fluorescence
enhancement to conditions mimicking synaptic release, and
A pH titration of ES517 alone gave a pKa of 6.3, indicating
that formation of the iminium ion does not alter the sulfamide
pKa (Figure S1, Supporting Information). To verify that the
spectroscopic changes observed were not due to deprotonation
of the iminium ion, a control test using compound 2 was
performed (Figure S7, Supporting Information). Compound 2
C
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ACS Chemical Neuroscience
Letter
Figure 5. Fluorescence enhancements of ES517 saturated with various
amine analytes under pH conditions simulating exocytosis.
ASSOCIATED CONTENT
* Supporting Information
Spectroscopic parameters, experimental procedures, and NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Funding
This work was supported by the National Science Foundation
(CHE-1112194).
■
Figure 4. (a) Absorbance and (b) fluorescence pH titration of ES517
(20 μM) with 300 mM glutamate in buffer (50 mM bis-tris propane,
120 mM NaCl, 1% DMSO) adjusting the pH from 4.4 to 7.7. λex = 488
nm. Inset is the fit to a pH isotherm.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Kevin Gillis for helpful discussions regarding the
design of the sensor.
■
Table 1. Binding and Spectroscopic Properties of ES517
with Various Primary Amine Neurotransmitters
a
b
c
neurotransmitter
Ka (M−1
)
Isat/I0
IpH7.4/IpH 5
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d
■
glutamate
GABA
8.6
8.3
9.2
49
12
27
25
14
10
0.5
12
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