Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

Article abstract of DOI:10.1038/nchem.1234

Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately 'blind' neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3β4 and α4β2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.

Full text of DOI:10.1038/nchem.1234

ARTICLES
PUBLISHED ONLINE: 10 JANUARY 2012 | DOI: 10.1038/NCHEM.1234
Optochemical control of genetically engineered
neuronal nicotinic acetylcholine receptors
1
2
1
2
3
†
Ivan Tochitsky †, Matthew R. Banghart , Alexandre Mourot , Jennifer Z. Yao , Benjamin Gaub ,
Richard H. Kramer1,3 and Dirk Trauner2,4
*
*
Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the
systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-
sensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be
photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic
acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design,
we produced heteromeric a3b4 and a4b2 nAChRs that can be activated or inhibited with deep-violet light, but respond
normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the
physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric
ligand-gated ion channels.
cetylcholine (ACh) and its receptors have always been at the the millisecond precision and subcellular spatial resolution that
forefront of new developments in physiology. With its iso- only light can provide. This has been achieved already with
A
lation as ‘Vagusstoff’ in 1921, Otto Loewi established that a voltage-gated ion channels12,14 and glutamate receptors13,15. We
small diffusible molecule could mediate nervous activity and now demonstrate that our approach towards photosensitizing
shaped the concept of a neurotransmitter1. Nicotinic acetylcholine nature’s molecular machines can be applied to pentameric ligand-
receptors (nAChRs) were among the first ion channels investigated, gated ion channels as well, and introduce the genetically engineered,
long before the advent of molecular cloning and heterologous light-controlled nAChR (LinAChR).
expression2. Numerous techniques in biophysics and chemical
biology, including patch clamp electrophysiology3, cryoelectron Results
microscopy4 and the expansion of the genetic code5, were developed PTL design. The development of LinAChR first required the design
using nAChRs, and the powerful methods of molecular cloning were and synthesis of appropriate PTLs. Our PTLs typically consist of a
applied to nAChRs early on6–8. These studies established that maleimide as the cysteine reactive group, an azobenzene
nAChRs are pentameric ligand-gated cation channels expressed photoswitch and a ligand head group that resembles known receptor
throughout the mammalian nervous system and at the neuromuscu- agonists or antagonists. Three compounds known to interact
lar junction1. The neuronal nAChR subtypes are composed of with nAChRs guided the choice of the ligand: the photoaffinity label
a2–a10 and b2–b4 subunits and can assemble both as heteromeric AC-5 (1), the agonist homocholine phenyl ether (HoChPE) (2) and
(for example, (a4)2(b2)3) and homomeric (for example, (a7)5) pen- the antagonist MG-624 (3). The presence of an aromatic ring
tamers1. Whereas the function of muscle nAChRs is well estab- separated by seven atoms from the ACh moiety in AC-5 (Fig. 1a),
lished, the physiological roles of neuronal nAChRs are still being which acts as a full agonist at muscle nAChRs16, suggests that the
unravelled. For instance, they are implicated strongly in the patho- steric bulk of an azobenzene photoswitch may be tolerated in PTLs
physiology of several psychiatric disorders, as well as in nicotine that act as tethered agonists. Although the much shorter molecule
addiction and Alzheimer’s disease1,9. However, progress in this HoChPE is an agonist of nAChRs17,18, the stilbene derivative
regard has been held back by the lack of subtype-selective nAChR MG-624 is a potent antagonist of neuronal receptors19. Therefore,
pharmacology and the difficulties associated with selectively target- on the basis of the steric similarity of stilbenes and azobenzenes, we
ing nAChRs in different parts of the brain.
anticipated that corresponding PTLs could function as antagonists.
These issues can be addressed using an approach called ‘opto- Based on these model compounds, we synthesized trans- and
chemical genetics’10,11. In essence, this is an effort to photosensitize cis-maleimide–azobenzene–acylcholine (MAACh) (4a and 4b,
innately ‘blind’ receptors using synthetic photoswitches. These can respectively) (Fig. 1a), a putative photoswitchable agonist for
be attached covalently as photoswitchable tethered ligands (PTLs), nAChR. We also synthesized trans- and cis-maleimide–azobenzene–
which require a reactive functional group for bioconjugation (typi- homocholine (MAHoCh) (5a and 5b, respectively) (Fig. 1b),
cally the sulfhydryl group of a cysteine12,13). By introducing the wherein the choline moiety was replaced with homocholine and the
cysteine through genetic manipulation, these receptors can be spacer was removed to resemble MG-624. MAACh and MAHoCh
targeted in a subtype-selective manner based on the subunit that were prepared by multistep total synthesis, as detailed in
contains the mutation14. Furthermore, the mutant receptors can Supplementary Fig. S1. By taking absorbance measurements of the
be expressed in specific cell types in the brain and controlled with ligands in solution, we determined that both molecules can be
1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA, 2Department of Chemistry, University of California,
Berkeley, California 94720, USA, 3Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720, USA, 4Department of Chemistry,
Ludwig-Maximilians-Universita¨t, Mu¨nchen and Center for Integrated Protein Science, 81377 Munich, Germany; †These authors contributed equally to this
work. e-mail: rhkramer@berkeley.edu; dirk.trauner@lmu.de
*
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1234
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a
b
N
N+
O
O
N+
O
N+
N
N
2
H
O
HoChPE
1
AC-5
O
H
N
N
N+
O
O
O
N
N
O
O
N+
3
N
H
N
H
MG-624
O
4a
O
trans-MAACh
H
N
N
O
N
N
O
500nm
or kBT
O
N+
380nm
5a
trans-MAHoCh
N
N
O
O
N+
N
H
N
H
500nm
or kBT
O
380nm
4b
NH
O
O
cis-MAACh
N
N
N
O
N+
O
5b
cis-MAHoCh
O
NH
O
N
O
c
d
β
β
β
β
380nm
380nm
α
β
α
β
α
β
α
β
500nm
or kBT
500nm
or kBT
α
β
α
β
α
β
α
β
Figure 1 | Photoswitchable ligand design for the optical control of neuronal nAChRs. a,b, Structures of AC-5 and MAACh (a) as well as HoChPE, MG-624
and MAHoCh (b). The PTL molecules photoisomerize from trans to cis on illumination with 380 nm light and revert to trans under 500 nm light or in the
dark. c, A tethered agonist is converted from its trans- into its cis-configuration under 380 nm light, and thus activates heteropentameric nAChRs. Illumination
with 500 nm light or thermal relaxation deactivates the receptor. d, A tethered antagonist is converted from its trans- into its cis-configuration under 380 nm
light, which blocks heteromeric nAChRs from binding to ACh (black circles). Illumination with 500 nm light or thermal relaxation unblocks the receptor,
which allows it to be activated by the neurotransmitter. kB ¼ Boltzmann constant.
converted maximally into their cis-isomer by illumination trans-configuration (at 500 nm or in the dark), allowing it to be
with 380 nm light and can then be reset to the trans-isomer by activated by free ACh (Fig. 1d).
500 nm illumination or thermal relaxation in the dark
(Supplementary Fig. S2).
Attachment-site screening. These functional requirements can be
met by the proper choice of cysteine attachment sites, which we
decided to place on the b2 or b4 subunits of heteropentameric
neuronal nAChRs. Using the X-ray structure of an acetylcholine-
binding protein (AChBP) in complex with carbamylcholine (PDB
ID:1uv6)20 and the model of AC-5 docked into the Torpedo
nAChR, we identified an antiparallel beta sheet in the b-subunit
that faces the ligand-binding site as a potential region for PTL
attachment16. Based on the calculated structures of our PTLs and
distance measurements in the protein structures, we decided to
Preferably, a photosensitive receptor would be inactive in the
dark, where azobenzene photoswitches generally reside in a trans-
configuration (Fig. 1c). Illumination with 380 nm light would
produce the cis-configuration of the azobenzene and activate the
receptor. Deactivation could then be achieved through conversion
of the photoswitch back to its inactive trans-configuration by illumi-
nation with 500 nm or by thermal relaxation in the dark. Similarly, a
tethered antagonist should block the receptor in its cis-configuration
(that is, at 380 nm), but should leave the receptor unimpaired in its
106
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1234
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a
a
c
380/500nm
100 μM ACh
b
113
115
117
1.0
0.8
0.6
0.4
0.2
0
63
32
34
25s
1
10
100 1,000 10,000
61
[ACh] (μM)
d
b
c
380/500nm
100 μM ACh
d
1.0
0.8
0.6
0.4
0.2
0
25s
0.1
1
10
100 1,000 10,000
[ACh] (μM)
Figure 2 | Choice of cysteine attachment sites for photoswitchable ligand
conjugation. a,b, Homology model of the a4b2 nAChR with ACh (green and
red spheres, respectively) docked in the binding site and the engineered
cysteine mutants shown in red and numbered in the enlarged view of the
receptor (b). Only one a/b (a, yellow; b, grey) interface is shown for clarity.
c,d, Docking results with unconstrained cis-MAACh (violet) (c) and trans-
MAACh (green) (d) in the a4b2 homology model. Six of the most
energetically favourable conformations are shown.
Figure 3 | Photoactivation of nAChRs with a tethered agonist. a,b,
Photoactivation of the a3b4E61 (a) and a4b2E61C (b) mutant receptors by
tethered MAACh in Xenopus oocytes. Illumination at 380 nm (violet lines)
triggers photoactivatable current and at 500 nm (green lines) shuts it off.
c,d, ACh dose–response curves for the a3b4 (c) and the a4b2 (d) WT
(black lines), E61C mutant (blue lines) and E61C mutant with tethered
MAACh in the dark (red lines). Data are mean+s.e.m., n ¼ 5.
current in the cis- but not in the trans-configuration: E61C,
install cysteines in positions S32, R34, E61, T63, R113, N115 and R113C and S117C. In the structure of carbamylcholine-bound
S117 of the b4 nAChR subunit. Homologous residues with AChBP (ref. 20) the distances from the alpha carbons of each of
identical numbering were chosen for b2. These residues are the three positions and the carbonyl carbon of carbamylcholine
shown mapped onto a homology model of the a4b2 receptor21 were very similar (10.43 Å, 11.28 Å and 12.11 Å for the E61C,
(PDB ID:1ole) (Figs. 2a,b), based on the structure of AChBP R113C and S117C mutants, respectively). The remaining residues
(ref. 22). A sequence alignment that relates these positions on were farther away (19.03 Å, 14.28 Å, 16.39 Å and 16.54 Å for
various complementary subunits to AChBP is also provided S32C, R34C, T63C and N115C, respectively).
(Supplementary Fig. S3).
Our choice of cysteines was validated further by molecular Photoactivation of engineered nAChRs. For a comprehensive
docking studies, which were carried out using the same homology electrophysiological analysis, these mutant b4 subunits were
model (Fig. 2c). Unconstrained cis-MAACh and trans-MAACh expressed as a3b4 heteropentamers, which have
a more
were docked into the a4b2 model and the rotatable bonds in the established presence in the nervous system25. After labelling the
ligand allowed to move freely. We found that the maleimide a3b4E61C receptor with MAACh, illumination with 380 nm light
moiety of MAACh comes very close to several of the engineered produced an inward current that could be reversed with 500 nm
cysteine mutants shown in Fig. 2b in the cis-form of the azobenzene light (Fig. 3a). The amplitude of the photoactivatable current was
photoswitch, in particular to cysteines in positions 61, 63 and 117 6.8+1.3% (mean+standard error of the mean (s.e.m.), n ¼ 10) of
(Fig. 2c), but extends past most of them in its trans-form the saturating cholinergic current and the receptor still responded
(Fig. 2d). Molecular modelling studies with MAHoCh were not normally to perfusion of ACh in the dark. Neither the E61C
undertaken because of the lack of ligand similarity in the antagon- mutation nor MAACh conjugation greatly changed the half-
ist-bound AChBP structures23 and the absence of a homology model maximum effective concentration (EC50) of the receptor for ACh
of an antagonist-bound heteromeric neuronal nAChR.
(Fig. 3c, Supplementary Table S1). MAACh labelling at the other two
To test our predictions experimentally, we first introduced the residues, b4R113C and b4S117C, also produced photoactivatable
seven independent cysteine mutations into the b4 subunit. Then, currents of similar magnitude (Supplementary Fig. S4).
we screened a4b4 mutant nAChRs expressed heterologously in
Next, we transposed the E61C mutant from the b4 to the b2
Xenopus oocytes, because this particular heteropentamer desensi- subunit and tested whether MAACh conjugation at the b2E61C
tizes very slowly compared to nAChRs composed of other neuronal position could photoactivate the neuronal a4b2 receptor. Indeed,
subunits24. The oocyte system was chosen for its robust expression the MAACh-labelled a4b2E61C receptor also produced an
of heteromeric nAChRs and the lack of endogenous receptors. By inward current using 380 nm light, which could be terminated
contrast, non-neuronal mammalian cell lines often express with 500 nm light (Fig. 3b). The amplitude of the photoactivatable
nAChRs poorly and many neuronal mammalian cell lines possess current was 16.1+2.1% (mean+s.e.m., n ¼ 15) of the saturating
endogenous nAChRs that would complicate the screening process. cholinergic current. The E61C mutation increased the EC50 of the
Using two-electrode voltage-clamp recordings, we screened these a4b2 receptor for ACh. Labelling with MAACh did not change
a4b4 mutants for cis-agonism by treating oocytes with MAACh in this EC50 further (Fig. 3d, Supplementary Table S1).
the dark and then looking for the induction of a current on illumi-
The prominence of the E61C mutant motivated us to carry out
nation with 380 nm light. We identified three cysteine mutations on another molecular docking study, wherein the maleimide moiety
the b4 subunit where MAACh could evoke a photoactivatable of MAACh was ‘virtually tethered’ to be in the proximity of the
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1234
ARTICLES
a
a
c
300 μM ACh
b
1.0
0.8
0.6
0.4
0.2
0
10
100 1,000 10,000
2s
[ACh] (μM)
300 μM ACh
b
d
1.0
0.8
0.6
0.4
Figure 4 | Molecular model of a nAChR with a virtually tethered
photoswitchable agonist. a,b, Docking results with constrained cis-MAACh
(violet) and trans-MAACh (green) in the a4b2 homology model. The
maleimide is constrained to be near the sulfhydryl group of C61. Ribbon (a)
and surface (b) views of the minimum energy conformations are shown. The
positional constraint (sphere) is bright yellow.
0.2
0
10
100 1,000 10,000
3s
sulfhydryl group of the E61C residue (Fig. 4a,b). Allowing the
remainder of the PTL to move freely resulted in docking of the
ligand head group into the ligand-binding site in all conformations.
By contrast, none of the tethered trans-MAACh conformations
reached the ligand-binding site (Fig. 4a,b, Supplementary Fig. S5).
These molecular docking studies corroborate our finding that the
agonist is able to reach the ligand-binding site and activate the
receptor only in its cis-form, and not in its trans-configuration.
[ACh] (μM)
Figure 5 | Photoinhibition of nAChRs with a tethered antagonist. a,b,
Photoinhibition of the cholinergic current of the a3b4E61C (a) and
a4b2E61C (b) mutant receptors by tethered MAHoCh. The current shown
is in response to 300 mM ACh under 380 nm light (violet lines) and
500 nm light (green lines). c,d, ACh dose–response curves for the
a3b4E61C (c) mutant and the a4b2E61C (d) mutant with tethered
MAHoCh under 500 nm light (green lines) and 380 nm light (violet lines).
Data are mean+s.e.m., n ¼ 5.
Photoinhibition of engineered nAChRs. We then proceeded to test
the effect of MAHoCh attached to the three cis-agonist active
cysteine mutants in a3b4 heteromeric nAChRs. After treating thermal relaxation in the dark26. Thus, the MAACh-labelled
oocytes with the PTL in the dark, we determined that a4b2E61C receptor can be activated by 380 nm light and the
illumination with 380 nm and 500 nm light did not produce photoactivatable current decays over time in the dark (Fig. 6a). To
photoactivatable currents with any of the three mutants investigate whether tethering the PTL to a receptor affects the rate
(Supplementary Fig. S6). Instead, illumination with 380 nm light of cis-isomer thermal relaxation, we compared the half-life of
reduced dramatically the response of the a3b4E61C receptor to MAACh in solution, measured by changes in absorption at
ACh, and that with 500 nm light restored the initial response to 380 nm, to the calculated half-life of the decay of the
ACh (Fig. 5a). This indicates that tethered MAHoCh acts as a photoactivatable current (Fig. 6b). The half-life of MAACh in
cis-antagonist of the a3b4E61C receptor. ACh-evoked currents solution was 28 seconds, close to the previously reported value of
were not modulated substantially by illumination at the 26 seconds for a urea–azobenzene derivative27. In contrast, the
a3b4R113C and a3b4S117C receptors (data not shown). To thermal decay of the photoactivatable current could only be fitted
characterize the light-dependent block of the a3b4E61C receptor to a biexponential decay curve, with a fast half-life of 4.7 seconds
at different concentrations of ACh, we compared the amount of and a slow half-life of 94 seconds, which indicates that ligand
current observed under 380 nm and 500 nm illumination for the binding increases the stability of cis-MAACh. The MAHoCh-
same cells. The percentage of current blocked under 380 nm light labelled a4b2E61C receptor can be blocked by 380 nm light and
ranged from 80+5% at 30 mM ACh to 68+4% at a saturating the amount of block also decays slowly in the dark (Fig. 6c). We
10 mM ACh (mean+s.e.m., n ¼ 5) (Fig. 5c). Tethering MAHoCh measured the half-life of cis-MAHoCh in solution to be 74.4
at the a4b2E61C receptor produced a similar photoinhibition of minutes (Fig. 6d). The long half-life of cis-MAHoCh can be
the cholinergic current (Fig. 5b). The percentage of current attributed to the alkoxy substituent on the azobenzene, which
blocked under 380 nm light at the a4b2 receptor ranged from does not destabilize the cis-configuration to the same extent as the
75+10% at 100 mM ACh to 81+7% at a saturating 3 mM ACh urea substituent in MAACh26. The thermal relaxation rate of
(mean+s.e.m., n ¼ 5) (Fig. 5d).
tethered cis-MAHoCh appears to be of the same order of
In control experiments, we did not observe any photoactivation magnitude as that of cis-MAHoCh in solution.
after treating oocytes that expressed either a3b4E61C or a4b2E61C
nAChRs with AcAACh (6a,b), a non-reactive analogue of MAACh
(Supplementary Fig. S7). Furthermore, treatment of wild-type (WT)
receptors with either MAACh or MAHoCh did not impart any
photosensitivity (Supplementary Fig. S6). These results confirm
that covalent modification at the genetically introduced cysteine
residue is required for the optical control of the LinAChR function
and suggest that off-target actions of these ligands at endogenous
neuronal receptors should be minimal.
Discussion
In a classic set of studies carried out in the late 1970s and early
1980s, Erlanger and co-workers demonstrated that azobenzene
photoswitches could provide photosensitivity to endogenous
muscle nAChRs28,29. These pentameric ligand-gated ion channels
naturally possess a disulfide bond at the tip of the so-called
C-loops in their a-subunits1, which is in close proximity to the
ligand-binding site. This disulfide could be reduced to provide a
reactive thiol for the covalent attachment of a PTL, although such
Thermal relaxation of the photoswitchable ligands. Azobenzene treatment also reduced the affinity of the receptor for agonists by
PTLs can be converted from their cis-configuration into their up to 100-fold30. The PTL, called QBr, consisted of a benzylic
trans-configuration by 500 nm illumination, but they also bromide (the electrophile), an azobenzene photoswitch and a
spontaneously convert into the more stable trans-isomer through benzylic trimethylammonium moiety. Once attached, QBr activated
108
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a
b
1.0
0.8
0.6
0.4
Photoactivatable current
cis-MAACh in solution
0.2
0
100s
0
100
200
300
Time (s)
c
d
1.0
0.8
0.6
0.4
0.2
0
Photoinhibition
cis-MAHoCh in solution
10 min
0
50
100
150
200
250
Time (min)
Figure 6 | Thermal relaxation of the photoswitchable agonist and antagonist. a, Normalized photoactivatable current decay of the a4b2E61C receptor in
the dark with tethered MAACh. Decay of the photoactivatable current over time under 380 nm illumination (violet line) and in the dark (black line) is shown.
b, Thermal relaxation of cis-MAACh in phosphate-buffered saline (open squares) fitted with a monoexponential decay function (black line) (half-life
(t1/2) ¼ 28 seconds). Decay of the photoactivatable current (mean (blue line)+s.e.m. (grey line), n ¼ 4) fitted using a biexponential decay function (red line,
t
1/2(1) ¼ 4.7 seconds, t1/2(2) ¼ 94 seconds). Both measurements are normalized. c, Decay of the photoinhibition of cholinergic current of the a4b2E61C
receptor in the dark with tethered MAHoCh measured in Xenopus oocytes. ACh (100 mM) was perfused under 500 nm light (green line), 380 nm (violet)
light (violet line) and in the dark (black line). d, Thermal relaxation of cis-MAHoCh in phosphate-buffered saline (open squares) fitted with a
monoexponential decay function (black line) (t1/2 ¼ 74.4 minutes). Decay of the photoinhibition of cholinergic current of the a4b2E61C receptor with
MAHoCh (blue line) is shown (mean+s.e.m., n ¼ 4).
the channel in its trans-form, that is at 500 nm or in the dark, but functional PTLs and choose appropriate sites for their attachment.
could be switched to an inactive cis-form by irradiation with The X-ray structure of carbamylcholine bound to AChBP and the
330 nm light. The resulting light-activated nAChR could be used homology models of the neuronal receptors20,21 proved to be most
to stimulate reversibly Electrophorus electroplaques, frog muscles relevant for our design and computational evaluation of PTLs,
and rat myoballs with light28,29,31,32. At the time these studies were and were particularly helpful for identifying residues suited to the
carried out, however, receptors could not be manipulated genetically attachment of the photoswitchable agonist MAACh. Our molecular
and expressed heterologously, so the impact of synthetic photo- docking studies correctly identified positions 61 and 117 as suitable
switches on neuroscience research remained limited.
sites of attachment. However, the predicted site 63 turned out to be
As a result of the enormous progress of molecular biology since unsuitable, which highlights the importance of functional screening
the 1980s, it is now possible to express heterologously mutant recep- as a complement to in silico models.
tors in Xenopus oocytes, mammalian cells, neurons and even trans-
The antagonist action of tethered MAHoCh is more difficult to
genic animals12,13,33. Once expressed in a cell, our engineered explain based on the available X-ray structures, but it is consistent
LinAChR (E61C) can be conjugated with either an agonist or an with the known pharmacology of nAChRs (see Fig. 1). It is also con-
antagonist, which provides a powerful bi-directional control of sistent with the ‘foot-in-the-door’ mechanism of nAChR antagon-
receptor activity. Both variants of LinAChR behave like normal ism, wherein an antagonist binds, but prevents the complete
nAChRs in the dark, but on irradiation with 380 nm they can be conformational contraction of the ligand-binding site required for
turned on in the absence of ACh or off in the presence of the neu- receptor activation39. Apparently, the elongation of HoChPE with
rotransmitter. The red-shifted PTL action spectra (compared to that a diazene unit bearing an aryl group is sufficient to prevent the
of QBr) should also minimize potential phototoxic effects. In C-loop of the receptor a-subunit from closing in sufficiently to acti-
principle, their spectral sensitivity could be moved further towards vate the receptor. This also makes sense given the structural simi-
lower energies with appropriate substitution of the azobenzene larity of the azobenzene MAHoCh with the stilbene MG-624, and
photoswitch34,35
.
timethylammonium derivatives thereof, which also function as
Another field that has undergone remarkable progress in recent antagonists19. Our data demonstrate that once an attachment site
years is the structural biology of pentameric ligand-gated ion chan- is chosen, it is possible to convert a tethered photoswitchable
nels (Cys-loop receptors)36. Several X-ray structures of homopenta- agonist into a tethered photoswitchable antagonist by altering the
meric ligand-gated ion channels are now available37,38, as are high- structure of the tether and the ligand head group. This matches pre-
resolution structures of AChBPs bound to a variety of agonists and vious observations by Sullivan and Cohen, who established that
antagonists20,22. Without these, it would be difficult to design elongation of a covalently attached agonist by a single methylene
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109

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1234
ARTICLES
minimization and scoring of hits based on a discretized ChemScore function48,49
.
group could turn it into an antagonist40. Conversely, it might be
possible to use the same tethered ligand and two different sites of
attachment to achieve either photoagonism or photoantagonism40.
The diversity of neuronal nAChRs and their structural simi-
larities complicates selective targeting of specific nAChR subtypes
pharmacologically. This study reports the reversible photoactivation
and inhibition of common neuronal a4b2 and a3b4 nAChRs using
light following the genetically targeted conjugation of PTLs. The
findings should enable the rational design of other light-regulated
pentameric ligand-gated ion channels, homopentameric a7
nAChRs and neuromuscular nAChRs. The lessons learned in this
study should also facilitate the design of photosensitive GABAA
(g-aminobutyric acid), GlyRs, 5HT3 or GluCl receptors. Based on
the recent X-ray structure of GluCl and our expertise with photo-
switchable tethered glutamate derivatives13, for instance, it should
be possible to develop a hyperpolarizing light-gated chloride
channel41. It is also conceivable that photoswitchable tethered ago-
nists and antagonists could be extended to muscarinic ACh recep-
tors, which have not yet been characterized in atomic detail, but
for which extensive pharmacology exists42.
In future physiological investigations, LinAChRs will be
expressed in dissociated neurons, intact neuronal tissues and live
animals. We expect them to function well in these systems,
perhaps even better than in Xenopus oocytes, the opacity of which
makes it impossible to photoregulate receptors on the entire
surface of the cell. In any case, the amount of photoactivation
should be sufficient to replicate the major type of cholinergic trans-
mission in the mammalian brain. Neuronal nAChRs are mostly
extrasynaptic and respond to low micromolar concentrations of
ACh diluted after synaptic release (that is, volume transmission)43.
Expression of the cysteine-containing subunit alone should
produce functional LinAChRs in neurons that possess endogenous
nicotinic receptors, which would then allow optical manipulation
of the receptor function in vitro and in vivo44. Alternatively, native
nAChR subunits can be replaced by cysteine mutants using a
knock-in strategy45, which would preserve the endogenous pattern
and level of receptor expression. We intend to pursue these strat-
egies to study the physiological and pathological roles of hetero-
meric nAChRs in the brain and periphery and will report the
results of these investigations in due course.
Ligands were docked flexibly to a rigid receptor using standard precision and the top
hits were examined. Docking of the constrained ligands contained an additional
positional constraint that restricted the alkenyl carbons of the maleimide moiety to a
shell with an inner radius of 1 Å and an outer radius of 3.5 Å centred at the
sulfhydryl group of the C61 cysteine residue.
Photoswitch conjugation. All photoswitch compounds were dissolved in
dimethylsulfoxide (DMSO) to make a stock solution (10 mM) and diluted in oocyte
Ringer’s solution (see above) to 25 mM for conjugation. For all recordings, the
DMSO concentration was ,0.25% (volume/volume). Oocytes were incubated in the
photoswitch compound solution for 20 minutes in the dark at room temperature
before electrophysiological recordings. Photoswitch conjugation was followed by a
five minute wash in oocyte Ringer’s solution.
Electrophysiology. All electrophysiology experiments were conducted at room
temperature. Two-electrode voltage-clamp experiments were performed using an
OC-725C amplifier (Warner Instruments), DigiData 1200 interface and pClamp 8.0
software. The oocytes were placed in a perfusion chamber for the recordings. All the
recordings were performed in oocyte Ringer’s solution (see above). For each
experiment, the oocyte membrane potential was held at 280 mV. An eight-line
perfusion system with a VC-8 valve controller (Warner Instruments) was used for
perfusion of ACh into the chamber. Data were sampled at 1 kHz and filtered at
10 Hz. Cells were illuminated using a Lambda-LS illuminator that contained a
125 W xenon arc lamp (Sutter Instruments) equipped with narrow (+10 nm)
band-pass filters. The incident light intensity was 20 mW cm22 for both 380 nm
and 500 nm light, measured at the aperture using a handheld optical power
meter (1918-C, Newport).
Data analysis. Dose–response curves were created using equation (1):
ꢂ
ꢃ
ꢀ
ꢁ
log
(
x
(
−x ×p
)
)
0
y = A1 + A2 − A1 / 1 + 10
(1)
where p ¼ the Hill coefficient, 10logx ¼ EC50, A1 is the bottom asymptote and A2 is
0
the top asymptote of the dose-response curve.
Absorption spectra. Ultraviolet–visible spectra of the photoswitch compounds were
obtained using the SmartSpec Plus Spectrophotometer (BioRad Laboratories) in PBS
at pH 7.4. The change in absorbance of the compounds at 380 nm with time after
photoconversion to the cis-configuration was used to determine the kinetics of
thermal relaxation. The light source used for photoconversion of the ligands was the
same as that used in electrophysiological recordings.
Received 17 August 2011; accepted 21 November 2011;
published online 10 January 2012
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Acknowledgements
Support for the work was provided by the Nanomedicine Development Center for the
Optical Control of Biological Function PN2EY018241 (D.T., R.H.K.), The European
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Author contributions
M.R.B., A.M., R.H.K. and D.T. designed the research. M.R.B. and J.Z.Y. synthesized the
ligands. I.T., A.M. and B.G. performed the research and analysed the data. I.T., M.R.B. and
D.T. co-wrote the paper.
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The authors declare no competing financial interests. Supplementary information and
chemical compound information accompany this paper at www.nature.com/
naturechemistry. Reprints and permission information is available online at http://www.
nature.com/reprints. Correspondence and requests for materials should be addressed to
R.H.K. and D.T.
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