Fluorescent epibatidine agonists for neuronal and muscle-type nicotinic acetylcholine receptors

Article abstract of DOI:10.1002/anie.200604807

Singled out: Specific high-affinity binding to and activation of nicotinic acetylcholine receptors (nAChRs) by fluorescent epibatidine (EPB) agonists is detected by fluorescence microscopy and electrophysiology (see picture). The optical and pharmacologic

Full text of DOI:10.1002/anie.200604807

Angewandte
Chemie
DOI: 10.1002/anie.200604807
Receptor Agonists
Fluorescent Epibatidine Agonists for Neuronal and Muscle-Type
Nicotinic Acetylcholine Receptors**
Jörg Grandl, Elias Sakr, Florence Kotzyba-Hibert, Florian Krieger, Sonia Bertrand,
Daniel Bertrand, Horst Vogel,* Maurice Goeldner,* and Ruud Hovius
Nicotinic acetylcholine receptors (nAChRs) are archetypical
ligand-gated ion channels mediating synaptic transmission at
the neuromuscular junction, autonomic ganglia, and in the
of single AChRs because of the lack of ligands that are both
brightly fluorescent and receptor agonists.
Fluorescent nAChR-activating molecules are thus
required with minimal changes in receptor specificity and
[
1,2]
central nervous system.
Selective binding of the natural
[
9,10]
neurotransmitter acetylcholine (ACh) is the key event for
receptor activation which, after transmembrane conforma-
tional changes of the protein, leads to transient opening of an
ion channel, thus triggering an action potential. Structural
changes in ligand-gated ion channels upon activation have
been observed, but the molecular mechanism of the trans-
affinities.
Former attempts to label the natural, nonfluor-
escent agonist ACh, or analogues thereof, with fluorophores,
[
9,11]
[10]
such as dansyl,
pyrene,
or N-(7-nitrobenz-2-oxa-1,3-
[
12]
diazol-4-yl) (NBD), yielded either fluorescent antagonists
or poorly fluorescent agonists of moderate affinities,
[
11,12]
which precludes their application to single-molecule/single-
channel studies in living cells. However, these ACh deriva-
tives allowed investigation of, for example, ligand binding and
allosteric effects using purified nAChR from the electric
[
3,4]
membrane signaling reactions is far from being elucidated.
Ligand binding to nAChR is typically investigated either
by incubating the sample for some time with radioactive or
fluorescent ligands, thereby driving the receptor to its
[
13]
organ of Torpedo. Recently, fluorescent anabaseine ago-
nists on the neuronal a7 subtype have been described to
display increased fluorescence upon binding to ACh-binding
[
5,6]
desensitized state that can be probed,
or indirectly by
[
6]
measuring channel currents upon receptor activation.
[
14]
Important new insights into how agonist binding to nAChR
induces opening of its ion channel, and thus leads to
transmembrane signaling, are expected to be gained from
direct observation of the formation of single ligand–receptor
complexes by simultaneous optical and electrophysiological
proteins. Additional important questions on the function of
AChRs might be answered by investigating single agonist–
receptor complexes: 1) How are different (activated/non-
activated) AChRs distributed in neuronal cells and how is this
distribution influenced by cellular and synaptic develop-
[
6]
[15,16]
measurements. Although single receptors have been imaged
on the surface of living cells in a few cases by fluorescence
ment?
2) Does partitioning in microdomains or mono-
mer–oligomer formation play a role in AChR-mediated
[
7,8]
[16,17]
microscopy,
it was still impossible to resolve the activation
signaling?
3) Do AChRs interact with other cellular
proteins and thereby influence other central cellular process-
[
18]
[
+]
es besides triggering action potentials?
[
*] Dr. J. Grandl, Prof. H. Vogel, Dr. R. Hovius
Herein, we report the synthesis and characterization of
novel fluorescent agonists for nAChRs, based on labeling of
the frog alkaloid epibatidine (EPB, 1, Scheme 1), a potent
Laboratoire de Chimie Physique des Polym›res et Membranes
Ecole Polytechniques FØdØrale de Lausanne (EPFL)
1015 Lausanne (Switzerland)
[
19,20]
Fax: (+41)216-936-190
E-mail: Horst.Vogel@epfl.ch
agonist of various nAChRs.
These fluorescent EPBs bind
to and activate neuronal and muscle-type nAChRs, and
feature subtype selectivity. We demonstrate that the excep-
tional optical and pharmacological properties of the fluores-
cent agonists offer new possibilities to study AChRs on a
single-cell and single-molecule level.
[
+]
Dr. E. Sakr, Dr. F. Kotzyba-Hibert, Dr. F. Krieger, Prof. M. Goeldner
Laboratoire de Chimie Bioorganique UMR 7175 LC1 CNRS
FacultØ de Pharmacie
UniversitØ Louis Pasteur Strasbourg
BP 24, 67401 Illkirch Cedex (France)
Fax: (+33)390-244-306
E-mail: Goeldner@bioorga.u-strasbg.fr
The synthetic ligands contain EPB to recognize nAChR,
and a fluorescent moiety linked by a spacer to the 3’-position
of the pyridine ring of EPB (Scheme 1). All synthesized
compounds were purified by HPLC and characterized by
mass spectrometry and NMR spectroscopy (see the Support-
ing Information). The optical properties of the fluorescent
EPBs were identical to those reported for the original
fluorescent groups (see Table 1 of the Supporting Informa-
tion).
The receptor affinity and efficacy of the fluorescent EPBs
were evaluated from radioligand binding and electrophysiol-
ogy measurements to gain insight into subtype specificity for
the major neuronal and muscle-type nAChRs. Compared to
EPB, the affinity of its fluorescent derivatives was reduced by
S. Bertrand, Prof. D. Bertrand
DØpartement des Neurosciences
Centre MØdical Universitaire
1
221 Gen›ve (Swizerland)
] Equally contributing authors
**] We thank P.-J. Corringer and T. Grutter for plasmids encoding a7-
HT and muscle-type nAChR subunits. This work was supported by
+
[
[
5
3
the Swiss National Science Foundation (H.V. and D.B.), the
Association Française contre les Myopathies (AFM), the Centre
National de la Recherche Scientifique, and the UniversitØ Louis
Pasteur Strasbourg (M.G.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2007, 46, 3505 –3508
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
while, as expected, no activation of a7
and a7-5HT3 receptors could be
detected at concentrations up to 1–
1
0 mm (Table 2). Interestingly, on the
muscle nAChR gain of function
mutant dS268K the fluorescent EPBs
featured 50% effective concentration
(
EC ) values comparable to that of
50
EPB itself. Remarkably, the Cy5 and
both coumarin derivatives were full
agonists with a twofold lower EC50
value compared to that of EPB,
whereas EPB-Cy3 and EPB-Atto610
were partial agonists (Table 2 and
Supporting Information). Although
enantiomers of EPB itself are not
[
21]
discriminated,
substituted EPB
Scheme 1. Fluorophore conjugation to EPB (1). 7-Boc-3’-aminoepibatidine (2; Boc: tert-butoxy-
derivatives might display, depending
on their structure, stereoselective
receptor recognition and activation
that account for the partial agonist
effect.
carbonyl) was first modified with an aminoacetyl linker and then reacted with an N-hydroxysuccin-
imide-activated fluorophore X to yield after deprotection a fluorescent EPB (3); X: coumarin (EPB-
Cou, 4), 6-chlorocoumarin (EPB-Cl-Cou, 5), Cy3 (EPB-Cy3, 6), Cy5 (EPB-Cy5, 7), Atto610 (EPB-
Atto610, 8). For details, see the Supporting Information.
one to two orders of magnitude for neuronal receptors and
Table 2: Activation of nAChR subtypes by EPB ligand conjugates
evaluated from electrophysiology.
three orders of magnitude for muscle-type receptors
Table 1). As expected for EPB derivatives, the affinities for
[a]
(
[
b]
[b]
[c]
[d]
Ligand
a4b2
a3b4
a7
a bgd
2
rat
rat
human
Table 1: Inhibition constants K in nm of EPB ligands for nAChR subtypes
evaluated from radioligand competition binding assays.
I
3
EPB (1)
23Æ7
59%
31%
n.d.
19Æ6
n.d.
85%
n.d.
(2.0Æ0.5)”10
53Æ4
EPB-Cou (4)
EPB-Cl-Cou (5)
EPB-Atto610 (8)
0%
0%
0%
26Æ5
[
a]
[b]
a7-5HT3R
[b]
Ligand
a4b2
a bgd Torpedo
2
29Æ4
rat brain
chicken/mouse
127Æ30
(50%)
115Æ24
(52%)
30Æ10
[
c]
[c]
[c]
EPB (1)
0.37Æ0.19
75Æ13
51.6Æ9.5
3
EPB-Cy3 (6)
EPB-Cy5 (7)
n.d.
n.d.
0%
0%
EPB-Cou (4)
EPB-Cl-Cou (5)
EPB-Cy3 (6)
EPB-Cy5 (7)
8.5
4.75
44
n.d.
16”10
3
3[c]
25”10
(10.4Æ8.9)”10
6
3[c]
ꢀ2%
25%
>1”10
(93Æ16)”10
[
c]
73Æ34
n.d.
n.d.
[a] EC50 values in nm stem from concentration–current curves. If no
complete curve has been measured, the channel current evoked by 1 mm
of ligand is listed as %-value of the current evoked by 100 mm ACh. Partial
agonism is indicated in parentheses as percentage of maximal activation
evoked by 100 mm ACh. n.d.: not determined. [b] Rat a4b2 and a3b4
receptors were expressed in oocytes. [c] EPB and EPB-Cou were
investigated on rat a7 receptor expressed in oocytes, the remaining
3
[
a] Inhibition of binding of [ H]cytisine to rat-brain membranes by the
indicated ligands; a4b2 is the predominant nAChR in rat brain.
1
25
[
b] Protection by the ligands for [ I]-a-bungarotoxin binding to a7-
5
HT R in membranes of HEK293 cells or a bgd nAChR in membranes of
3
2
the electric organ of Torpedo. n.d.: not determined. [c] Data are mean Æ
standard deviation from two or three experiments.
ligands on a7-5HT R chimeric receptors in HEK293 cells. [d] Human
3
fetal muscle nAChR gain of function mutant dS268K was expressed in
HEK293 cells.
[
19]
a7-5HT chimeric receptors were very low, but interest-
3
ingly, the fluorescent molecules displayed an unprecedented
selectivity for a4b2 over a7. EPB-Cou and EPB-Cl-Cou
showed better affinities than EPB-Cy3 and EPB-Cy5, which
might be a result of the larger size and/or presence of several
charges on the cyanine dyes.
The ability of the fluorescent compounds to activate the
nAChR was investigated by patch-clamp experiments. Com-
pared to the currents evoked by 100 mm ACh, the neuronal-
type receptors a4b2 and a3b4 showed 31 to 85% activation by
Taken together, the successful design and synthesis of
highly fluorescent, efficient agonists for both muscle and two
major neuronal nAChR subtypes have been achieved.
Fluorophore conjugation of EPB decreased similarly both
the affinity and efficacy for neuronal receptors. However, for
the muscle receptor efficacy was hardly affected. This might
be because of structural and functional differences between
subtypes of nAChR.
1
mm of the coumarin derivatives 4 and 5, whereas 1 mm EPB-
Cy5 evoked weak responses at a3b4 and a4b2 receptor
subtypes (Table 2 and Supporting Information). Conjugation
of the fluorescent groups to EPB decreased the efficacy for
the neuronal nAChRs by two to three orders of magnitude,
The results of the following two key experiments demon-
strate the great potential of the fluorescent EPBs to inves-
tigate agonist binding and single-molecule imaging on indi-
vidual living cells. First, HEK293 cells transiently expressing
3
506
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Angewandte
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À1 À1
À1
the human wild-type adult muscle nAChR, imaged by
fluorescence microscopy, showed an increasing fluorescence
intensity upon perfusion with a solution containing 50 nm
EPB-Cy5 until a steady level was reached; then, upon rinsing
with buffer, the fluorescence intensity decreased gradually to
the initial background level (Figure 1A; movie 1 of the
kon = (2.48 Æ 0.06) mm
s
and koff = (0.26 Æ 0.03) s , respec-
tively, which resulted in a dissociation constant K = (103 Æ
d
53) nm for the complex of the ligand with the basal state of the
adult wild-type muscle nAChR.
Next, we evaluated EPB-Cy5 for single-molecule fluores-
cence imaging of muscle nAChR in the plasma membrane of
HEK293 cells (Figure 2A shows a micrograph of such a cell).
Figure 2. Single-molecule microscopy. A) Transmission micrograph of
an HEK293 cell expressing adult wild-type muscle nAChR. B) After
adding 5 nm EPB-Cy5 to the cell shown in (A), the AChRs bind the
fluorescent agonist and appear as single fluorescent spots, such as the
one shown in the white box. C) Trajectory (red line) over 52 frames
(
ꢁ4.3 s) of the single fluorescent receptor boxed in (B) diffusing in
the cell membrane.
After perfusion with 5 nm Epi-Cy5, we observed on the upper
surface of the cells bright fluorescent spots (Figure 2B)
blinking and photobleaching in one step, which are typical
properties of single molecules. By reason of their absence on
nontransfected-cells, the spots represent nAChRs on which
only one of the two binding sites is occupied by a fluorescent
agonist. On average, trajectories of 28 frames could be
acquired before the EPB-Cy5 molecules photobleached or
dissociated from the receptor (movie 2 in the Supporting
Information). Single-molecule trajectories, constructed by
connecting the corresponding spots in consecutive frames
Figure 1. Binding of fluorescent EPB to the surface of a living HEK293
cell expressing the adult wild-type muscle nAChR. A) Time course of
the fluorescence intensity: addition of 50 nm EPB-Cy5 (arrow 1) leads
to an increase of fluorescence; upon washing with buffer (arrow 2) the
signal decreases to the initial value. Four images were acquired per
second. The rate constants, evaluated from monoexponential fits
[
8]
using a custom-designed single-particle-tracking algorithm,
revealed the lateral diffusion of the nAChR within the cell
membrane (Figure 2C).
In summary, we have shown the synthesis and character-
ization of EPBs comprising five structurally different fluo-
rophores. These novel compounds are either partial or full
agonists on muscle and on neuronal nAChR subtypes
featuring (sub)micromolar receptor affinities and efficacies,
and receptor-subtype specificity. The combination of excep-
tional optical properties—high quantum yield and photo-
stability covering the visible spectrum—and high receptor
affinities makes the fluorescent EPBs ideally suited for
application to ligand-binding assays for screening purposes,
and to elucidating the different facets of functioning of the
nAChRs in vitro, in heterologous cells, or even native tissues,
with single-molecule sensitivity.
(
g) for three independent experiments, were for the fluorescence
À1
intensity accumulation k =(0.38Æ0.05) s and depletion
acc
À1
kdepl =(0.256Æ0.032) s . B) Experiments at different image acquisition
rates showed a weak linear dependence of k on the image
depl
À1
acquisition rate. Evaluation yielded k =(0.298Æ0.012) s ; at 4 Hz
off
À1
kbleach =(0.022Æ0.006) s . The dissociation constant of EPB-Cy5 was
K =(103Æ53) nm. For details, see the Supporting Information.
d
Supporting Information). As fluorescence was only observed
on transfected cells, identified by fluorescence of co-trans-
fected green-fluorescent protein (GFP), it reflects the specific
binding of the ligand to AChRs on the cell surface. Repetition
of the experiments on the same cell with varying image
acquisition rates showed only minor influence on the time
course of fluorescence accumulation or depletion, which
demonstrates that photobleaching did not significantly con-
tribute to the decreasing fluorescence signal during washing
Experimental Section
Details of the synthesis (Scheme 1) of fluorescent EPB ligands and all
other experiments are provided in the Supporting Information and
also in references [22–24]for radioligand competition and protection
binding experiments, in references [25–27]for expression and electro-
(
Figure 1B). The rate constants evaluated for binding of the
fluorescent agonist to the receptor and its dissociation were
Angew. Chem. Int. Ed. 2007, 46, 3505 –3508
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3507

Communications
physiological characterization of the AChRs, and in reference [28]for
recording fluorescence images and evaluating single-molecule traces
on cell membranes.
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Keywords: alkaloids · fluorescence · ion channels · receptors ·
single-molecule studies
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Angew. Chem. Int. Ed. 2007, 46, 3505 –3508