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Light in combination with PTLs also provides high temporal re-
solution, which is key, since synaptic signaling through iGluRs
is a fast, millisecond process. The binding and unbinding of
PTLs can be controlled with short light pulses within tens of
microseconds if high light intensities are used (Fig. 3E). Light-
gated iGluRs therefore enable us to mimic synaptic activation,
e.g. to generate excitatory postsynaptic currents with different
temporal profiles. Another exciting possibility is to use PTLs
to study the biophysical gating mechanism of iGluRs, for ex-
ample, to probe, how the ligand occupancy controls receptor
desensitization (Reiner, Isacoff, 2014b). Using MAG ligands, we
found that the fast ligand-induced desensitization (tempora-
ry inactivation) of kainate receptors depends on the receptor
occupancy, i.e. the number of occupied binding sites. Besides
the high time resolution, this approach has the advantage that
specific receptor subunits can be addressed, e.g. it allowed us
to study how the GluK2 subunits contribute to the gating of
GluK2/GluK5 heteromers (Reiner et al., 2012; Reiner, Isacoff,
2014b), which are the prevalent GluK2-containing complexes
in most brain regions.
Optical control of other targets
The high specificity and time resolution provided by PTLs
makes it possible to control specific iGluR subtypes in real time,
to differentiate between pre- and postsynaptic effects, and to
study the mechanisms through which iGluRs regulate synap-
tic strength and plasticity. PTLs have also been successfully
used to control a number of other cell surface receptors. For
example, mGluRs, which are GPCRs with important neuromo-
dulatory functions, can be controlled with MAG ligands similar
to those used for iGluRs. Photoactivation has been established
for mGluR2, mGluR3 and mGluR6 and can be used to optically
control native second messenger signaling cascades that regu-
late presynaptic neurotransmitter release (Levitz et al., 2013).
Next to photoactivation, MAG ligands have been used for pho-
toinhibition. The latter is achieved by tethering the ligand at
positions, where it acts as competitive antagonist or allosteri-
cally blocks receptor activation (Levitz et al., 2013), a feature
that is very useful to transiently suppress signaling through
specific receptor subtypes. PTLs have also been developed for
a number of other targets, including neurotransmitter-gated
ion channels, such as acetylcholine receptors (Bartels et al.,
1971; Tochitsky et al., 2012) and GABA
A
receptors (Lin et al.,
2014). A similar strategy has been used to photo-inhibit vari-
ous potassium channels (Banghart et al., 2004; Sandoz, Levitz,
2013). In this case the tethered photoswitch is functionalized
with a pore blocker instead of a ligand and is installed close to
the ion channel pore. Photoswitching then leads to block and
unblock of the ion permeation pathway – an approach that has
been applied to voltage-gated, Ca
2+
-gated and leak potassium
channels. Another variation is to use photoswitchable ligands
without tethering them to any specific receptor (Fehrentz et
al., 2011; Kramer et al., 2013). Here, the specificity of the so-
luble ligands is only determined by the ligand headgroup and
no genetically-encoded component is involved. In summary,
photoswitchable tethered ligands (PTLs) provide an important
and versatile addition to the available optogenetic toolset, as
they offer exciting possibilities to control and study the role of
specific receptors in genetically-selected cells with high spatial
and temporal precision.
Acknowledgements
The article summarizes work from the lab of Prof. Dr. Ehud Y.
Isacoff at the University of California, Berkeley. I want to thank
all present and past members of the lab involved in these efforts,
my colleagues for discussion, and Prof. Isacoff for continuing
support. The work was performed in corporation with the lab
of Prof. Dr. Dirk Trauner at the Ludwig-Maximilians-Universität
München, whose contributions are gratefully acknowledged. A
large part of the work was funded by the NIH Nanomedicine
Development Center for Optical Control of Biological Function
(to E.Y.I. 2PN2EY018241). Part of my postdoctoral research pro-
ject was funded by the Deutsche Forschungsgemeinschaft (DFG
RE 3101/1-1).
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