Optical Control of Endogenous Proteins
with a Photoswitchable Conditional Subunit
Reveals a Role for TREK1 in GABABSignaling
Guillaume Sandoz,1,2Joshua Levitz,1,3Richard H. Kramer,1,3and Ehud Y. Isacoff1,3,4,*
1Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, 271 Life Sciences Addition, University of California,
Berkeley, Berkeley, CA 94720, USA
2Institut de Pharmacologie Mole ´culaire et Cellulaire, CNRS, and Universite ´ de Nice Sophia-Antipolis, Sophia-Antipolis,
06560 Valbonne, France
3Biophysics Graduate group, University of California, Berkeley, Berkeley, CA 94720, USA
4Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
Selective ligands are lacking for many neuronal
signaling proteins. Photoswitched tethered ligands
(PTLs) have enabled fast and reversible control of
specific proteins containing a PTL anchoring site
and have been used to remote control overex-
pressed proteins. We report here a scheme for
optical remote control of native proteins using a
‘‘photoswitchable conditional subunit’’ (PCS), which
contains the PTL anchoring site as well as a mutation
that prevents it from reaching the plasma membrane.
In cells lacking native subunits for the protein, the
PCS remains nonfunctional internally. However, in
cells expressing native subunits, the native subunit
and PCS coassemble, traffic to the plasma mem-
brane, and place the native protein under optical
control provided by the coassembled PCS. We apply
this approach to the TREK1 potassium channel,
which lacks selective, reversible blockers. We find
that TREK1, typically considered to be a leak
channel, contributes to the hippocampal GABAB
While the production of pharmacological reagents targeted to
membrane signaling proteins has been a major objective for
both academic laboratories and the pharmaceutical industry,
many important membrane proteins are still without specific
blockers. Moreover, where specific blockers exist, they often
have high affinity and are selective only at low concentrations,
so that the onset of their effect upon exposure takes a long
time to develop and they bind so tightly that they are difficult to
remove. The development of photoswitched tethered ligands
(PTLs) that are targeted to an introduced cysteine near ligand
binding sites of membrane proteins opened the door to the
reversible control of membrane signaling, by using two wave-
lengths to photoisomerize the tether between one state that
permits ligand binding and a second state, which prevents
binding (Szobota and Isacoff, 2010). Because specificity derives
from the unique geometric relationship between the ligand
binding site and the engineered anchoring site, rather than
from tight binding, photoisomerization to the nonbinding state
rapidly removes the ligand. Moreover, the high effective concen-
tration of the ligand near its binding site in the permissive state
leads to rapid binding upon photoisomerization, itself a very
rapid transition (Szobota and Isacoff, 2010). Together, these
properties enable highly selective optical control of binding
and unbinding on the millisecond timescale and micron space-
scale (Szobota and Isacoff, 2010).
So far, optical control with PTLs has been applied to ion chan-
nels and receptors that are overexpressed in cells. Because the
introduction of the anchoring site can usually be done with
minimal perturbation to protein function (Szobota and Isacoff,
2010), it should be possible to introduce the mutation into the
native protein via genetic knockin. Still, generation of a knockin
animal is laborious and expensive, making sense only when
one is directly interested in the signaling by the targeted protein,
in typical pharmacological experiments. To address this, we
developed a scheme for targeting optical control via a PTL to
native proteins without the requirement for genetic knockin.
Our approach is to express a ‘‘photoswitchable conditional
subunit’’(PCS)thatcontains aPTLanchoring siteandamutation
that retains the subunit inside the cell. This engineered subunit
will not function in cells where native subunits are missing.
However, in cells that express the native subunits that are
required to form the functional protein complex, the native and
engineered subunits will assemble inside the cell and the
complex will be trafficked to the plasma membrane, thereby
placing the native protein under optical control provided by the
coassembled engineered subunit.
GABABreceptors set the inhibitory tone, provide the critical
feedforward and feedback inhibition that shapes the spread of
neural activity, regulates the filtering properties of neural circuits,
and prevents hyperexcitation and seizure (Kohl and Paulsen,
Neuron 74, 1005–1014, June 21, 2012 ª2012 Elsevier Inc. 1005
2010; Semyanov et al., 2004). In the hippocampus, the postsyn-
aptic GABABresponse was long thought to be mediated exclu-
sively through Kir3 potassium channels (Lu ¨scher et al., 1997;
Padgett and Slesinger, 2010; Ulrich and Bettler, 2007), but the
genetic knockout of Kir3 subunits has suggested that another
channel might also contribute to GABAB inhibition (Koyrakh
et al., 2005). The identity of this additional channel has not
been revealed and its function in tissue from wild-type animals
remains to be determined. Using the PCS approach we show
that TREK1, a 2P-potassium channel typically thought of as
a leak channel, is an additional target of GABABreceptors in
One interesting class of channels to consider for participation in
hippocampal GABABsignaling is the large family of 2P-potas-
sium channels. These channels are typically thought of as leak
channels, whose function is to set the resting potential (Noe ¨l
these channels has remained elusive due to a lack of specific
blockers. One of the 2P-potassium channels, TREK2, was found
recently to be involved in the GABABcontrol of spatial learning in
the entorhinal cortex (Deng et al., 2009). However, the entorhinal
GABABcurrent deactivates more than ten times more slowly
than the hippocampal GABABcurrent, suggesting that TREK2
is not the missing hippocampal channel. In the absence of
specific pharmacological blockers of most 2P-potassium chan-
nels, and because knockout of specific genes can lead to
compensatory expression of related genes, we searched for an
alternative approach for selective pharmacology. We turned to
the strategy of PTLs, which obtain their target selectivity not
from the specificity of the ligand but from their selective attach-
ment to the protein of interest and the precise geometric relation
of the attachment site to the ligand binding site (Banghart et al.,
2004; Fehrentz et al., 2011; Szobota and Isacoff, 2010; Volgraf
et al., 2006). Because the PTLs are photoisomerized between
two conformations by distinct wavelengths of light and because
only one of the conformations permits the ligand to bind, they
can activate or block the target protein rapidly and reversibly.
Thus, in principle, photoblock should provide a clear assay for
when the channel is activated.
PTL for the TREK1 Channel
We developed a light-blocked version of the 2P-Potassium
(M) that tethers the molecule to a genetically engineered
cysteine, a photoisomerizable azobenzene (A) linker, and a
pore-blocking quatenary ammonium group (Q) (Figure 1A, top).
In its relaxed state, MAQ is in the trans configuration (Figure 1A
and Figure 1B, left). It is rapidly photoisomerized to the cis con-
figuration by 380 nm light and rapidly photoisomerized back to
the trans form by 500 nm light (Figure 1B). MAQ was previously
employed to photocontrol the voltage-gated Shaker potassium
channel (Banghart et al., 2004).
We introduced single cysteine mutations as attachment sites
pore loops (P1 and P2) of TREK1 (Figure 1A) and expressed the
channel in HEK293 cells. MAQ was applied in the external solu-
tion to each of these cysteine-substituted mutants and photo-
switching was tested by measuring the modulation of the current
when illumination was switched back and forth from 500 nm to
380 nm. We first examined cysteine substitutions at residue
N122 in P1 and K231 in P2 of TREK1, since these are homolo-
gous to the optimal site for photoblock by MAQ in the Shaker
channel (Shaker 422) (Banghart et al., 2004). While both sites
showed photomodulation, they had a different dependence on
light, i.e., on the configuration of the MAQ photoswitch.
TREK1(K231C-MAQ) produced photoblock in the trans state
(500 nm illumination), as found in Shaker (Banghart et al.,
2004), but TREK1(N122C-MAQ) produced photoblock in the
cis state (380 nm illumination) (Table 1). The opposite photo-
switching at the two attachment positions indicates P1 and P2
differ structurally and that P2 more closely resembles the P
loop of Shaker. This is interesting in view of the levels of
homology of the conserved C-terminal portion of the P regions,
where TREK1’s P1 and P2 have 17% and 23% identity (55%
long loop precedes TREK’s P1 (Figure S1 available online).
Photomodulation was also seen at two other MAQ attachment
sites in P1 (Table 1). The strongest photomodulation was at
S121C (Table1), which displayed 64%± 3%(n =14)block under
380 nm light and was unblocked by isomerization to trans under
illumination at 500 nm (Figure 1C). Since MAQ thermally relaxes
into the trans state, TREK1(S121C-MAQ) has the advantage that
the channel is unblocked and can function normally in the dark.
Figure 1. Light-Gated TREK1
(A) (Top) MAQ consists of a maleimide (M), which tethers the photoswitch to
a cysteine introduced into the outer portion of the first P loop of the channel
(bottom), a photoisomerable azobenzene (A) linker, and a quaternary ammo-
nium (Q) pore blocker. (Bottom) Cartoon showing the membrane topology of
TREK1 channel and the different position tested.
(B) Schematic representation of light-gated TREK1. MAQ is covalently
attached to cysteine (S121C). MAQ blocks the pore in the cis configuration
(380 nm light). Exposure to 500 nm light places MAQ in the trans state where
the pore is unblocked.
(C and D) Whole-cell recording from HEK293T cell expressing TREK1(S121C)
and labeled with MAQ. Current was elicited by voltage-ramps (from ?100mV
to 50mV, 1 s in duration) (C). Alternating illumination at 500 nm (green) and
380 nm (magenta) reversibly blocks and unblocks constant outward current,
as seen at different holding potentials (D).
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