Sensing acetylcholine and anticholinesterase compounds

Article abstract of DOI:10.1002/anie.201307754

Acetylcholine is a key neurotransmitter, and anticholinesterase agents are essential compounds used as medical drugs, pesticides, and chemical warfare agents. A semisynthetic fluorescence-based probe for the direct, real-time detection of acetylcholine and anticholinesterase compounds is introduced. The probe possesses good sensitivity, tunable detection range, and can be selectively targeted to cell surfaces, thereby making it an attractive tool for applications in analytical chemistry and quantitative biology.

Full text of DOI:10.1002/anie.201307754

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DOI: 10.1002/anie.201307754
Fluorescent Probes
Sensing Acetylcholine and Anticholinesterase Compounds**
Alberto Schena and Kai Johnsson*
Abstract: Acetylcholine is a key neurotransmitter, and anti-
cholinesterase agents are essential compounds used as medical
drugs, pesticides, and chemical warfare agents. A semisynthetic
fluorescence-based probe for the direct, real-time detection of
acetylcholine and anticholinesterase compounds is introduced.
The probe possesses good sensitivity, tunable detection range,
and can be selectively targeted to cell surfaces, thereby making
it an attractive tool for applications in analytical chemistry and
quantitative biology.
A
cetylcholinesterase (AChE) catalyzes the hydrolysis of the
neurotransmitter acetylcholine (ACh) to choline and acetate.
It is mainly found at the neuromuscular junction and in the
animal synapse, where it terminates the signaling triggered by
ACh release.[1] Despite the importance of ACh in biology, no
molecular probes exist for its real-time quantification in cell
culture experiments or in vivo. Existing tools either permit
only indirect detection of ACh or show low signal-to-noise
ratio and impact cellular signaling.[2,3] Moreover, a direct
quantification of anticholinesterase compounds (ACs), that is,
compounds that target AChE such as drugs for AChE-related
pathologies,[4] pesticides, and chemical weapons,[5] is of great
practical importance. Here we introduce the semisynthetic
protein ACh-SNIFIT, a fluorescent probe for the direct, real-
time quantification of ACh and ACs within the extracellular
matrix. ACh-SNIFIT possesses good signal-to-noise ratio, fast
kinetics and can be targeted to selected cells, thereby making
it well suited for applications in analytical chemistry and
neurobiology.
Figure 1. a) ACh-SNIFIT is a fusion protein of AChE, CLIP tag, and
SNAP tag in which CLIP is labeled with Cy3 and SNAP with a molecule
containing a FRET acceptor and a ligand for AChE. Binding of analyte (A)
to AChE displaces the intramolecular ligand (L) and affects the FRET
efficiency. b) Molecules utilized in this study: benzylguanine (BG) deriva-
tives 1 and 2 permit the labeling of SNAP fusion proteins; benzylcytosine
derivative BC-Cy3 permits the labeling of CLIP.
ACh-SNIFIT is a Fçrster resonance energy transfer
(FRET)-based semisynthetic probe designed according to
the SNIFIT concept[6–9] in which the competition between
a tethered ligand and free analyte results in a change in FRET
efficiency within the probe (Figure 1). Our SNIFIT sensor is
constituted of a fusion protein of AChE, CLIP tag, and
SNAP tag. Functional ACh-SNIFIT is then formed upon site-
specific labeling with synthetic molecules: CLIP is site-
specifically labeled with a FRET donor and SNAP with
a molecule (Figure 1) containing a FRET acceptor and
a ligand for AChE. In the absence of ACs, the tethered
ligand is bound to AChE and the two fluorophores are in
close proximity. At sufficiently high concentration of ACh or
an AC the intramolecular ligand is displaced from AChE,
thereby resulting in opening of ACh-SNIFIT and detectable
decrease in FRET ratio (Figure 1).
To maximize the change in FRET efficiency, CLIP and
SNAP tags were fused to the C terminus of AChE and thus
close to its active site, ensuring proximity of the two
fluorophores in the closed state of the probe. The linkers
between the three building blocks were optimized (Figure 1)
by directly fusing CLIP to the C terminus of AChE and by
inserting a long linker between the two tags, ensuring a large
distance between the fluorophores in the open state. Active-
site residue Ser231 was mutated to Ala to obtain a catalytically
inactive probe. The synthetic FRET acceptor was sandwiched
between the intramolecular ligand and BG, reacting with
SNAP tag (Figure 1).
[*] A. Schena, Prof. K. Johnsson
Ecole Polytechnique Fꢀdꢀrale de Lausanne
Institute of Chemical Sciences and Engineering (ISIC)
Institute of Bioengineering
National Centre of Competence in Research (NCCR) in
Chemical Biology
1015 Lausanne (Switzerland)
E-mail: kai.johnsson@epfl.ch
[**] This work was supported by the Swiss National Science Foundation.
ˇ
We thank R. Griss, Dr. Reymond, Dr. Lukinavicius, and Dr. Hovius
for discussions.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201307754.
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Table 1: Affinity and inhibitory activity of ACs towards ACh-SNIFIT-D;
inhibition assays were performed on apo-SNIFIT with wild-type AChE.
We chose as tethered ligand a derivative of decametho-
nium (Deca, Figure 1), a muscle relaxant drug reported to
inhibit AChE with an inhibitory constant of 9.6 mm.[11]
AC
c50 [m]
IC50 [m]
We first characterized the soluble probe by labeling
recombinant apo-SNIFIT with BC-Cy3 and BG-Cy5-Deca
(Figure 1) to obtain ACh-SNIFIT-D with tethered Deca.
Titration of the probe with increasing concentrations of Deca
resulted in a decrease in emission intensity of the acceptor
fluorophore and increase of the emission intensity of the
donor fluorophore (Figure 2), in accordance with the design
acetylcholine
decamethonium
tacrine
(10.0Æ0.4)x10À3
(3.2Æ0.1)x10À5
(1.5Æ0.3)x10À6
(6.9Æ0.6)x10À9
–
(8.0Æ0.1)x10À6
(1.1Æ0.04)x10À6
(5.9Æ0.9)x10À9
edrophonium-C13
FRET-based probes utilizing muscarinic acetylcholine recep-
tors have been introduced, but these probes show low ratio
changes (< 10%), are saturated at micromolar concentrations
of ACh, and require excitation with blue light.[3] Approaches
that measure downstream effects such as changes in calcium
concentrations only provide an indirect measure of changes in
ACh concentrations.[2] We therefore decided to target ACh-
SNIFIT to the outer cell membrane of living cells for a direct
quantification of ACh and ACs on cell surfaces. We generated
doxycycline-inducible cell lines from human embryonic
kidney cells (HEK293)[10] expressing the probe protein
anchored on the outer cell surface through a growth factor
receptor transmembrane domain[6] and labeled cells express-
ing apo-SNIFIT with BC-Cy3 and BG-Cy5-Deca (Figure 3).
Analysis by fluorescence confocal microscopy demonstrated
correct labeling and localization of the probe on the cell
surface (Figure 3). Since both BC-Cy3 and BG-Cy5-Deca are
membrane-impermeable, only those probes properly
exported to the cell surface will be labeled.[6]
To demonstrate the use of ACh-SNIFIT for quantification
of ACs on cell surfaces we perfused cells displaying ACh-
SNIFIT with ACs and analyzed the response of individual
cells by plotting the ratio of the fluorescence intensities of the
Cy5 and Cy3 channels of a wide-field fluorescence micro-
scope. By perfusing cells with different Deca concentrations
we were able to measure the c50 value for Deca of ACh-
SNIFIT-D on cell surfaces. The measured c50 of 48 mm is
comparable to the c50 value obtained with soluble probe
(32 mm; Table 1). We determined an overall FRET-ratio
change of 38% (Figure 3). These data demonstrate that
ACh-SNIFIT-D permits the direct quantification of ACs on
living cells.
Figure 2. a) Fluorescence emission spectra of ACh-SNIFIT-D in the
presence and absence of Deca (lEXC =500 nm). b) In vitro titrations of
soluble ACh-SNIFIT-D with ACs.
To demonstrate that the response range of SNIFITs can
be tuned by tethering ligands with different affinity for the
receptor protein, we labeled ACh-SNIFIT with the edropho-
nium derivative BG-Cy5-Edro (Figure 1), resulting in ACh-
SNIFIT-E. Edrophonium (Edro) is a reversible cholinesterase
inhibitor with a Ki = 0.62 mm[11] and the tetherable derivative
edrophonium-C13 showed a stronger affinity towards AChE
than Deca in vitro (Figure 2 and Table 1). As expected, the
measured c50 value of ACh-SNIFIT-E for Deca (580 mm) is
higher than the corresponding value measured for ACh-
SNIFIT-D (48 mm; Figure 3). Thus, ACh-SNIFIT-D responds
to Deca concentrations in the range of 5–500 mm, while ACh-
SNIFIT-E responds to Deca concentrations in the range of
100–10000 mm, demonstrating how readily the properties of
ACh-SNIFIT can be fine-tuned.
principles. The observed maximum change in FRETratio was
65%. We then titrated ACh-SNIFIT-D with other ACs and
used the c50 value, defined as the analyte concentration that
resulted in 50% of the maximum ratio change, as a parameter
to evaluate the affinity of ACh-SNIFIT for the analyte
(Figure 2). The c50 values observed for different ACs corre-
lated with the corresponding IC50 values measured in
enzymatic assays using apo-SNIFIT comprising wild-type
AChE (Table 1 and Figure S2 in the Supporting Information).
ACh-SNIFIT-D thus also permits a high-throughput-compat-
ible characterization of the relative affinities of ACs.
AChE is a secreted protein and ACh plays its role when
released in the synaptic cleft. Thus, we investigated the use of
our probe for a direct, noninvasive quantification of concen-
trations of ACh or ACs on surfaces of living cells. Methods for
fluorescence imaging of ACh action on cell surfaces have
been demonstrated,[2,3] but they possess several shortcomings.
For a probe to be applied for live cell imaging a fast
response to the analyte concentration is highly desirable; thus
we estimated the kinetics of the opening and closing of ACh-
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Figure 3. a) Anchored ACh-SNIFIT model. b) Confocal images of HEK cells expressing ACh-SNIFIT and labeled with BC-Cy3 and BG-Cy5-Deca.
Scale bars: 10 mm. c) Perfusions of decamethonium on cells expressing ACh-SNIFIT and d) quantification of sensor response. ACh-SNIFIT-E (red
&
*
) with the stronger tethered ligand edrophonium responds at higher analyte concentrations than ACh-SNIFIT-D (black ).
SNIFIT on the cell surface. Opening of the SNIFIT probe is
mainly governed by the koff of the tethered ligand from the
receptor protein, while the closing of the probe is related to
the koff of the analyte.[7] We perfused Deca and tacrine (Tac),
a cholinesterase inhibitor previously used to treat Alzheim-
erꢀs disease[4] (in vitro c50 = 1.5 mm), on live cells expressing
ACh-SNIFIT-D. The probe readily responded to the addition
of the inhibitors with opening half-life (topen), which is defined
as the time to reach half-maximum ratio change, for the two
analytes of 1.5 s and 1.8 s, respectively (Table 2). Note that
ACh-SNIFIT responds on a second timescale and that it can
be used to determine the kinetics of the interaction of ACs
with AChE.
Finally, we investigated the use of our probe for visual-
izing ACh concentrations on cell surfaces. Initial experiments
demonstrated that when perfusing cells with concentrations
of ACh as high as 100 mm, ACh-SNIFIT-E gave more
reproducible results than ACh-SNIFIT-D (data not shown).
However, even at 100 mm ACh we observed only a partial
opening of the sensor (Figure 4). To increase the sensitivity of
ACh-SNIFIT-E towards ACh we mutated Trp310 to
Ala, as this residue has been reported to specifically
contribute to the interaction with edrophonium
Table 2: Kinetics of opening and closing of ACh-SNIFIT with different tethered
ligand and analytes perfused.
derivatives.[12] The resulting probe ACh-SNIFITWA
-
ACh-SNIFIT-D
ACh-SNIFIT-D ACh-SNIFIT-E
ACh-SNIFITWA-E
E responded in perfusion experiments to ACh in the
range 1–100 mm with c50 of 20 mm and 52% FRET-
ratio change (Figure 4). The ACh concentration
reached upon presynaptic release in the synaptic
cleft is not precisely known, but in cholinergic
synaptic vesicles is estimated to be 100 mm,[13] thus
Perfused Deca (100 mm) Tac (30 mm)
Deca (100 mm) ACh (50 mM)
topen
tclose
(1.5Æ0.2) s
(5Æ1) s
(1.8Æ0.2) s
(39Æ3) s
(3.6Æ0.3) s
(4Æ1) s
(2.4Æ0.4) s
(4Æ1) s
these rates are similar to the speed with which the bath
solution inside our perfusion chamber is exchanged;[7] the real
above the c50 value of ACh-SNIFITWA-E. The kinetics of
sensor opening and closing (Table 2) most likely is too slow to
follow concentration changes during synaptic release. How-
ever, nicotinic ACh receptors in the central nervous system
are extrasynaptic, far from the region of ACh release, and
ACh action is significantly slower than the kinetics of synaptic
release.[13] We have demonstrated that the c50 value of ACh-
SNIFIT can be easily fine-tuned and we believe this probe
represents a good starting point for imaging of physiological
relevant concentration changes of ACh. Furthermore, FRET
sensors with comparable ratio changes have already been
shown to be suitable for applications in vivo.[2]
probe opening could thus be faster. The closing half-life (tclose
)
was measured by washing out the inhibitor. Deca showed
a tclose around 8-fold smaller than tacrine (5 s and 39 s,
respectively). This suggests a smaller koff value of tacrine
compared to Deca, a result compatible with the lower c50
value measured for tacrine in vitro (Table 1). To demonstrate
that opening and closing kinetics can be controlled by using
different tethered inhibitors, we perfused Deca on cell-
anchored ACh-SNIFIT-E. As expected, using a stronger
tethered ligand increased topen, while tclose values remained
unchanged (Table 2). These experiments demonstrate that
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for the biochemical experiments are given in the Supporting
Information. apo-ACh-SNIFIT, as a soluble protein or anchored to
the outer cell membrane, has been cloned using standard cloning
techniques and transfected in HEK293 cells. Cells were grown at
378C in DMEM (Gibco) complemented with 10% fetal bovine serum
(Gibco). Labeling reactions, in vitro titrations, and perfusion experi-
ments were performed in HBSS (Gibco) at 258C.
Received: September 3, 2013
Revised: October 24, 2013
Published online: December 13, 2013
Keywords: acetylcholine · acetylcholinesterase · biosensors ·
.
FRET · imaging
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SNIFIT cell lines.
ˇ
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Experimental Section
Detailed experimental procedures for the synthesis of BG-Cy5-Deca
and BG-Cy5-Edro, for protein expression and characterization, and
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