.
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360 nm light for deactivation can be kept at
a minimum as cis-PF2 remains stable under
physiological conditions (Figure S3). In gen-
eral, activation and deactivation occurred at
timescales that can be expected for a metabo-
tropic receptor.[26] As azobenzene photoswitch-
ing is known to occur on a sub-millisecond
timescale[21] and MOR ligands dissociate rap-
idly,[19] it can be assumed that the rate of
activation and deactivation is limited by the
kinetic parameters of the signaling cascade that
links the GPCR with the ion channel.
Recently, we described photochromic teth-
ered ligands (PTLs) that are covalently at-
tached to Family C GPCRs, namely, metabo-
tropic glutamate receptors.[26] This system,
termed LimGluR, was used to effectively con-
trol neuronal excitability with light. Our new
results described herein should enable the
extension of this concept to Family A GPCRs.
These “LiGPCRs” could provide a valuable
alternative to genetically encodable light-sensi-
tive GPCRs (Opto-XRs), which are based on
a fusion of rhodopsin with other Family A
GPCRs.[25]
Figure 3. Light-dependent activation of MOR by PF2. HEK293t cells were transiently
transfected with the human MOR and both GIRK1 and GIRK2 channels. A) Illuminating
a 25 mm solution of PF2 with 360 nm light keeps the system in an inactive state.
Switching to 480 nm quickly activates the receptor. B) Rapid, repeated photoactivation
of GIRK by action of MOR and PF2. C) Comparison of photocurrents (consecutive
stimulation with a 10 mm solution of the native agonist LE (n=11, one error bar
indicates ꢀSEM). D) Photoswitching can be repeated over many cycles without
a noticeable decrease in photocurrent.
In conclusion, we have developed a method
to optically control MORs with a photochromic
(Figure 3).[26] To this end, human MOR was transiently
transfected into HEK293t cells together with the G-protein-
coupled inward rectifier channels GIRK1 and GIRK2.[17]
These channels are common effectors in Gi/o signaling and
native signaling partner of MORs in the locus coeruleus.[18,27]
Upon testing several concentrations, we found that 25 mm PF2
gave optimal results for photoswitching. As shown in Fig-
ure 3A, trans-PF2, which predominates in the dark or under
irradiation with 420–480 nm light, acts as an effective agonist
of MOR. By contrast, cis-PF2, which predominates at 360 nm,
is much less active. As a consequence, switching from 360 nm
to blue light immediately triggers a potassium influx through
the GIRK channels (high external potassium concentrations
were applied). This process can be terminated by switching
back to 360 nm light, which abrogates MOR activation. The
action spectrum of PF2 between 400 and 500 nm is shown in
the Supporting Information (Figure S1). GIRK currents cease
as bg-G-proteins re-associate with inactive Gai/o subunits
bound to GDP. Interestingly, GIRK currents evoked through
the action of trans-PF2 are maintained at a high level.
Desensitization of the system, as is the case of other GPCRs
and agonists, is not observed.[17]
ligand, PF2. In a sense, our PCL imparts the logic of
rhodopsin onto a light-insensitive Family A GPCR. Rhodop-
sin is a member of this transmembrane protein class that uses
retinal as a covalently bound photoswitchable inverse agonist.
Our work outlines a potentially general chemical strategy to
turn other members of Family A GPCRs, such as dopamine
receptors or adrenergic receptors, into photoreceptors, laying
the foundation for a photopharmacology of these targets. PF2
itself could be used to investigate the role of ORs in various
parts of the brain and the periphery in a precise spatiotem-
poral fashion. Its application in neuroscience as well as its
potential use as a “photoanalgesic”, perhaps as a variant with
a red-shifted action spectrum, is currently under investigation
and will be disclosed in due course.
Received: November 5, 2013
Revised: December 5, 2013
Published online: February 12, 2014
Keywords: azobenzenes · fentanyl · opioid receptors ·
photopharmacology · photoswitches
.
The repeated photoactivation of MOR with PF2 is shown
in Figure 3B–D. Comparison between photocurrents of three
consecutive switching cycles and subsequent activation of the
same cell with a saturating concentration of LE revealed that
PF2 elicits, on average, 80% of the current elicited by the
native ligand (Figures 3C,D and Figure S2). Photoactivation
could be repeated a number of times, demonstrating the
stability of the system (Figure 3D). Since PF2 acts as a trans
agonist, the pharmacologically active isomer is also the
thermodynamically more stable one. However, exposure to
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[4] Y. S. Lee, J. Nyberg, S. Moye, R. S. Agnes, P. Davis, S. W. Ma, J.
[7] S. Granier, A. Manglik, A. C. Kruse, T. S. Kobilka, F. S. Thian,
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 3264 –3267