Optogenetic photochemical control of designer K?channels
in mammalian neurons
Doris L. Fortin,1Timothy W. Dunn,1Alexis Fedorchak,1Duane Allen,2Rachel Montpetit,1
Matthew R. Banghart,3Dirk Trauner,4John P. Adelman,2and Richard H. Kramer1
1Department of Molecular and Cell Biology, University of California, Berkeley, California;2The Vollum Institute, Oregon
Health & Science University, Portland, Oregon;3Department of Chemistry, University of California, Berkeley, California;
and4Department of Chemistry, University of Munich, Munich, Germany
Submitted 21 March 2011; accepted in final form 25 April 2011
Fortin DL, Dunn TW, Fedorchak A, Allen D, Montpetit R,
Banghart MR, Trauner D, Adelman JP, Kramer RH. Optogenetic
photochemical control of designer K?channels in mammalian neu-
rons. J Neurophysiol 106: 488–496, 2011. First published April 27,
2011; doi:10.1152/jn.00251.2011.—Currently available optogenetic
tools, including microbial light-activated ion channels and transport-
ers, are transforming systems neuroscience by enabling precise remote
control of neuronal firing, but they tell us little about the role of
indigenous ion channels in controlling neuronal function. Here, we
employ a chemical-genetic strategy to engineer light sensitivity into
several mammalian K?channels that have different gating and mod-
ulation properties. These channels provide the means for photoregu-
lating diverse electrophysiological functions. Photosensitivity is con-
ferred on a channel by a tethered ligand photoswitch that contains a
cysteine-reactive maleimide (M), a photoisomerizable azobenzene
(A), and a quaternary ammonium (Q), a K?channel pore blocker.
Using mutagenesis, we identify the optimal extracellular cysteine
attachment site where MAQ conjugation results in pore blockade
when the azobenzene moiety is in the trans but not cis configuration.
With this strategy, we have conferred photosensitivity on channels
containing Kv1.3 subunits (which control axonal action potential
repolarization), Kv3.1 subunits (which contribute to rapid-firing prop-
erties of brain neurons), Kv7.2 subunits (which underlie “M-current”),
and SK2 subunits (which are Ca2?-activated K?channels that con-
tribute to synaptic responses). These light-regulated channels may be
overexpressed in genetically targeted neurons or substituted for native
channels with gene knockin technology to enable precise optophar-
macological manipulation of channel function.
potassium; excitability; light-activated channel
K?CHANNELS CONSTITUTE a superfamily of integral membrane
proteins that are crucial for many physiological processes. K?
channels regulate cellular excitability by controlling resting
potential, action potential duration, the magnitude and duration
of afterhyperpolarizations, and the propensity for repetitive
firing (Brown 1990). K?channels regulate neurotransmitter
release from presynaptic terminals (Dodson and Forsythe
2004) and integration in postsynaptic dendrites (Johnston et al.
2003; Takagi 2000). In nonexcitable cells, including lympho-
cytes, smooth muscle, and glia, K?channels participate in the
control of cell volume, proliferation, and cell migration. The
importance of K?channels in normal cellular physiology is
underscored by the finding that many human-inherited dis-
eases, including cardiac arrhythmias and epilepsy, are caused
by mutations in K?channels (Abbott 2006; Kullmann 2002;
Mulley et al. 2003; Sanguinetti and Spector 1997).
Neurons coexpress many different types of K?channels,
and most are heteromeric, containing two or more different
types of subunits. This complexity makes it difficult to attribute
unambiguously a particular physiological function to a partic-
ular type of K?channel. In some cases, small molecules,
peptide toxins, or antibodies that block a particular type of K?
channel with high specificity have been discovered, facilitating
evaluation of channel function. However, high specificity im-
plies high affinity, and such blockers are often irreversible.
Channel function can also be deduced by evaluating deficits
that result from genetic knockout of a specific K?channel
gene. Although this approach has provided many interesting
results, developmental alterations and compensatory up- or
downregulation in the expression of other channels and recep-
tors are complicating factors in interpreting the phenotype of
knockout mice (Hoffman 2008).
Here, we describe a chemical-genetic strategy that allows
optopharmacological control of mammalian K?channels. The
chemical component of the system is a synthetic photoswitch
molecule called MAQ. We previously used MAQ to bestow
light sensitivity onto a modified Drosophila Shaker K?chan-
nel (Banghart et al. 2004). This channel, which we named
“SPARK,” contains an engineered cysteine that serves as the
MAQ attachment site, along with mutations that enhance
voltage-dependent activation and remove fast inactivation,
causing the channels to be constitutively active at typical
neuronal resting potentials. Exogenous expression of SPARK
in mammalian neurons silences action potential firing. Subse-
quent attachment of MAQ bestows light sensitivity by an
artificial light-sensitive gate that blocks or unblocks the chan-
nels under different wavelengths of light, restoring or resilenc-
ing firing. Like many other optogenetic tools (Deisseroth et al.
2006; Kramer et al. 2009; Miesenböck 2009), the SPARK
conductance was superimposed on the native ionic conduc-
tances of neurons, enabling an experimenter to take charge of
the cell and regulate activity by overriding its intrinsic elec-
The four varieties of photosensitive K?channels described
in this paper can be exogenously expressed in neurons of
interest to enable optical control over different electrophysio-
logical properties. These channels will allow an investigator to
manipulate the function of the targeted channel and conse-
quently specific properties of cells with light. With the excep-
Address for reprint requests and other correspondence: R. H. Kramer, Dept.
of Molecular and Cell Biology, Univ. of California, Berkeley, 121 Life
Sciences Addition, Berkeley, CA 94720-3200 (e-mail: rhkramer@berkeley.
J Neurophysiol 106: 488–496, 2011.
First published April 27, 2011; doi:10.1152/jn.00251.2011.
4880022-3077/11 Copyright © 2011 the American Physiological Societywww.jn.org
tion of a cysteine substitution in an extracellular loop (the S5-P
loop) to enable photoswitch attachment, the channels described
in this paper are identical to wild-type proteins, with no
apparent phenotypic differences. This is consistent with struc-
ture-function studies showing that mutations in this region do
not affect channel gating or permeation. Hence, it will be
possible to generate knockin mice in which each of these
minimally altered proteins substitutes for its native protein
counterpart. Subsequent exposure to MAQ will allow specific
and precise optopharmacological knockout of these channels,
providing a powerful means for evaluating their functional
roles in cells and networks.
MATERIALS AND METHODS
Cell culture, plasmids, and transfection. HEK-293T cells were
grown in DMEM containing 10% FBS. Cells were plated at 10–20 ?
103cells/cm2on poly-L-lysine-coated glass coverslips and transfected
using the calcium phosphate method. Recordings were performed
24–48 h after transfection. Hippocampal neurons were prepared from
neonatal rats, plated at 50 ? 103cells/cm2on poly-L-lysine-coated
coverslips, and grown in MEM containing 5% FBS, 20 mM glucose,
B-27 (Invitrogen), glutamine, and MITO? Serum Extender (BD
Biosciences) and transfected 7 days after plating. Recordings were
performed 14–25 days after plating.
Adeno-associated virus injection. Three- to eight-week-old C57BL/6J
mice were injected with adeno-associated virus (AAV) encoding a
tetracycline transactivator (tTA)-sensitive bidirectional promoter driv-
ing small-conductance Ca2?-activated K?channel type 2 (SK2)
Q339C in one direction and green fluorescent protein (GFP) in the
other. A helper virus was coinjected to produce the tTA protein. Both
viruses (2-?l total volume) were injected stereotaxically at coordi-
nates corresponding to the hippocampus as per the Paxinos mouse
atlas using a Quintessential Stereotaxic Injector (Stoelting, Wood
Dale, IL). Animal care and experimental protocols were approved by
the University of California, Berkeley (UC Berkeley), Animal Care
and Use Committee.
Hippocampal slice preparation. Hippocampal slices were prepared
from mice at least 10 days postinjection. Animals were anesthetized
with an intraperitoneal injection of ketamine-xylazine cocktail before
being perfused with ice-cold artificial cerebrospinal fluid (aCSF; in
mM: 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2.0 CaCl2, 1.0
MgCl2, 12 glucose) equilibrated with 95% O2-5% CO2. Hippocam-
puses were removed and transferred into a slicing chamber containing
sucrose-aCSF (in mM: 75 sucrose, 87 NaCl, 2.5 KCl, 21.4 NaHCO3,
1.25 NaH2PO4, 0.5 CaCl2, 7 MgCl2, 1.3 ascorbic acid, 20 glucose)
equilibrated with 95% O2-5% CO2. Transverse hippocampal slices
(300 ?m) were cut with a Vibratome (VT1000S; Leica Instrument,
Leitz, Nussloch, Germany) and transferred into a holding chamber
containing aCSF and equilibrated with 95% O2-5% CO2. Slices were
incubated at 34°C for 35 min before chemical modification protocol.
Photoswitch treatment. Cultured cells were incubated at 37°C in
the dark for 5 min with 1 mM DTT in cysteine-free DMEM, rinsed,
and then incubated for 15 min with 25–100 ?M MAQ in cysteine-free
DMEM. Hippocampal slices were incubated in 2 mM tris(2-carboxy-
ethyl)phosphine (TCEP) for 10 min in aCSF equilibrated with 95%
O2-5% CO2and then washed 3? with 10-ml aCSF. This was followed
by 30-min incubation in 50 ?M MAQ before slices were washed 3?
with 10-ml aCSF.
Electrophysiological recordings and photocontrol of K?currents.
For cultured cells, recordings were made in the whole cell patch-
clamp configuration in extracellular solution (in mM: 138 NaCl, 1.5
KCl, 1.2 MgCl2, 5 HEPES, 1.5 CaCl2, 10 glucose; pH 7.4) using
borosilicate pipettes (4–7 M?) with intracellular solution (in mM: 10
NaCl, 135 K-gluconate, 10 HEPES, 2 MgCl2,2 MgATP, 1 EGTA; pH
7.4). For recording of cells transfected with Kv7.2, the intracellular
solution contained (in mM) 112 K-gluconate, 10 KCl, 20 KOH, 20
HEPES, 10 EGTA, 5 Na-ATP, 0.25 Na-cAMP; pH 7.2. For cells
transfected with SK2 channels, we recorded in the inside-out patch
configuration using pipettes with 1- to 2-M? resistance. Pipette
solution contained (in mM) 150 NaCl, 10 KCl, 10 HEPES, 1 MgCl2,
3 CaCl2; pH 7.4. Bath solution contained (in mM) 160 KCl, 0.5
MgCl2, 1 EGTA, 10 HEPES and 1.5 ?M free CaCl2; pH 7.4. Free
calcium concentration was calculated using MAXCHELATOR
For cultured cells, illumination was provided using a xenon lamp
(175 W) with band-pass filters (379 ? 17 and 500 ? 8 nm). At the
back of the objective, light output was 6 mW/cm2for 380-nm light
and 2 mW/cm2for 500-nm light. When measured through a ?20
objective and normalized to the focal area at the specimen plane, light
output was 1.6 and 0.4 mW/mm2for the 380- and 500-nm light,
CA1 pyramidal cells in hippocampal slices were visualized with
modified DOT imaging contrast optics (Axioskop FS2; Carl Zeiss,
Thornwood, NY) and a charge-coupled device (CCD) camera, and
recordings were made in the whole cell patch-clamp configuration.
Patch electrodes (2?3 M?) were filled with a solution containing (in
mM) 135 K-gluconate, 8 NaCl, 1 MgCl2, 10 HEPES, 4 MgATP, 0.3
Na2GTP, and 10 phosphocreatine; pH 7.26. Electrophysiological
records were filtered at 5 kHz and sampled at 20 kHz. The input
resistance was determined from an ?30-pA (500-ms) hyperpolarizing
current-injection pulse following each event. All recordings were
performed at room temperature, and we evaluated only those cells
with a stable series resistance (80% compensated) and a resting
membrane potential between ?75 and ?55 mV. For recording small
conductance Ca2?-activated K?(IsK) tail currents, cells were depo-
larized from ?55 to ?20 mV for 100 ms followed by a return to ?55
mV (Hammond et al. 2006).
For current-clamp recordings, we only used cells with stable input
resistance and resting potential (?70 to ?50 mV), and we rejected
recordings where the series resistance changed by ?20%. Glass
micropipettes filled with saline were positioned in the stratum radia-
tum to stimulate presynaptic axons via a stimulus isolation unit (A-M
Systems, Sequim, WA). For excitatory postsynaptic potentials (EPSP)
measurements, a bias current was applied to maintain the membrane
potential at ?60 mV. SR-95531 (2 ?M) and CGP-55845 (1 ?M) were
added to reduce GABAAand GABABcontributions, respectively.
For neurons in brain slices, fluorescence illumination was gener-
ated by an X-Cite 120 light source, and exposures were ?2 s in
duration. Slices were alternately exposed to 500-nm light to induce
block of SK2 channels and then exposed to 380-nm light to unblock
the channels, at least three consecutive times.
Design of light-regulated K?channels. The photoswitch
used for regulating K?channels (Fig. 1A) contains a maleim-
ide (M) that tethers the molecule to a genetically engineered
cysteine, a photosensitive azobenzene linker (A), and a pore-
blocking quaternary ammonium group (Q). In darkness, MAQ
is in the low energy trans configuration, but 380-nm light
photoisomerizes the molecule to the cis configuration. The cis
form of MAQ spontaneously relaxes to the trans form over
several minutes, but in 450- to 520-nm light relaxation is
accelerated. Given sufficient light intensity, relaxation can
occur within milliseconds after light exposure. Hence, after
conjugating MAQ to the channel, the blocker can be toggled in
and out of the pore by photoswitching the azobenzene with
500- and 380-nm light, respectively, allowing regulation of ion
conduction (Fig. 1B). Photocontrol of channel activity can be
489OPTICAL CONTROL OF MAMMALIAN K?CHANNELS
J Neurophysiol • VOL 106 • JULY 2011 • www.jn.org
achieved with great temporal and spatial precision, enabling
acute and reversible blockade of K?channel function.
Light-sensitive Kv1.3. Members of the mammalian Kv1
family of voltage-gated K?channels are highly homologous to
Drosophila Shaker K?channels, so we began by engineering
a photosensitive version of Kv1.3. In neurons, Kv1 family
channels are targeted to axons (Gu et al. 2003) where they
contribute to membrane repolarization during the falling phase
of the action potential (Fadool et al. 2004). Kv1.3-containing
channels are particularly important for maintaining tonic firing
during sustained depolarization (Kupper et al. 2002) and reg-
ulate the differentiation of neuronal progenitors into neurons,
making the channels an intriguing drug target for neurodegen-
erative disease (Peng and Huss 2010; Wang et al. 2010). Kv1.3
also plays important physiological and pathological roles in
many nonneuronal cell types, including T cell lymphocytes,
where it has become an important target for potential thera-
peutic modulation of the immune system (Beeton et al. 2006),
and platelets, where it is the only voltage-gated K?channel
expressed (McCloskey et al. 2010).
Taking advantage of the extensive similarity between the
amino acid sequence of Shaker and Kv1.3 (Fig. 1C), we
introduced a cysteine in the extracellular loop (P374C) at the
position equivalent to that in SPARK to bestow photosensitiv-
ity onto Kv1.3. Because the affinity of Kv1 family channels for
the pore-blocker TEA is low, we introduced an additional point
mutation (H401Y) that increases the affinity for TEA to the
micromolar range (Kavanaugh et al. 1991). The resulting
channel was expressed in HEK-293T cells, and photosensiti-
zation was tested by recording whole cell currents after 15 min
of MAQ treatment. The mutated Kv1.3 channels indeed did
become sensitive to light, with 500-nm light blocking ?50% of
the voltage-activated current and 380-nm light relieving chan-
nel blockade (Fig. 2, A and C). Currents through photosensi-
tized Kv1.3 could be repeatedly blocked by 500-nm light and
unblocked by 380-nm light (Fig. 2A). For cells expressing
Fig. 1. Optopharmacological strategy to control the activity of K?channels. A: the synthetic photoswitch MAQ consists of a cysteine-reactive maleimide,
a photoisomerizable azobenzene linker, and a pore-blocking quaternary ammonium (QA; blue). MAQ undergoes trans-to-cis isomerization on illumination
with 380-nm light. Exposure to 500-nm light, or prolonged time in darkness, returns the molecule to the trans configuration. B: MAQ covalently attaches
to a genetically engineered cysteine located at the appropriate distance from the pore of a K?channel. C: sequence alignment of different K?channels.
We engineered cysteines at position equivalent to E422C in Kv1.3 (P374C), Kv3.1 (E380C), Kv7.2 (KCNQ; E257C), and small-conductance
Ca2?-activated K?channel type 2 (SK2; Q339C). S5 and S6 denote the 5th and 6th transmembrane domains. P denotes the K?selectivity filter.
490OPTICAL CONTROL OF MAMMALIAN K?CHANNELS
J Neurophysiol • VOL 106 • JULY 2011 • www.jn.org
wild-type Kv1.3 channels, application of MAQ resulted in no
detectable photosensitization. Pretreatment of cells expressing
the photosensitive Kv1.3 with another cysteine-reactive re-
agent, MTSET, prevented subsequent photosensitization by
MAQ (Fig. 2, B and C), confirming that MAQ photosensitiza-
tion is mediated by cysteine conjugation.
Light-sensitive Kv7.2. Heteromultimeric channels composed
of primarily Kv7.2 and Kv7.3 subunits underlie the M-current,
a slow voltage-gated current in neurons and cardiac muscle that
is activated at membrane potentials below action potential
threshold (Brown and Passmore 2009). M-current is named as
such because it is inhibited by muscarinic receptors, providing
a potent mechanism for neurotransmitter modulation of excit-
ability. Mutations in Kv7.2 cause channelopathies in humans,
including inherited juvenile epilepsy and peripheral nerve hy-
perexcitability (Maljevic et al. 2008).
To generate light-regulated Kv7.2-containing channels, we
again used MAQ in conjunction with a cysteine-scanning
approach. Kv7.2 E257C channels showed the greatest degree
of photosensitization, with 500-nm light blocking ?34% of the
current elicited during at step from ?70 to ?40 mV (Fig. 3A)
and a similar reduction in the tail current on repolarization to
?70 mV (Fig. 3B). Introduction of a cysteine at position K255
and G256 completely eliminated photosensitization, whereas
cysteine substitution at N258 resulted in no current on the cell
surface. Interestingly, cysteine substitution at D259 appears to
result in “reverse” photoswitching, with 380-nm light block-
ing the channel, although the effects of light were not as
pronounced as with E257C (Fig. 3C). These data underscore
the need for precise positioning of the MAQ attachment site
to achieve maximal photosensitization of the engineered
Fig. 2. Engineering a photosensitive Kv1.3. A: introduction of P374C and H401Y and subsequent treatment with MAQ photosensitizes Kv1.3. Whole cell
recording from a MAQ-treated HEK-293T cell expressing Kv1.3 P374C and H401Y. The cell was held at ?60 mV and stepped to ?20 mV every 5 s. The
resulting current was measured and plotted over time and wavelength changes. Illumination with 500-nm light blocks the channel, reducing ion flow, whereas
illumination with 380-nm light unblocks the channel. B: when the genetically engineered cysteine is protected by treatment with cysteine-reactive reagent MTSET
before treatment with MAQ, there is no photosensitization and no regulation of current with light. Kv1.3 channels exhibit cumulative inactivation with very slow
recovery (Kupper et al. 2002) that may account for the small decline in currents observed in A and B. C: fraction of current regulated with light in cells expressing
Kv1.3 P374C and H401Y treated with MAQ (48.2 ? 16.2%) and pretreated with MTSET to protect the cysteine and prevent attachment of MAQ (0.05 ? 5.5%).
The fraction of current photoregulated is defined as the difference between the current under 380- and 500-nm light divided by the amount of current in 380 nm.
Data represent average ? SD (n ? 6–13).
Fig. 3. Photosensitization of Kv7.2 channels. A: engineering of a cysteine in Kv7.2 (E257C) and subsequent treatment with MAQ enables photoregulation of
the channel. Whole cell recording from a cell expressing Kv7.2 E257C treated with MAQ. Current was elicited by stepping to ?40 mV from a holding voltage
of ?70 mV. Illumination with 500-nm light blocks the channel, whereas the 380-nm light unblocks the channel. B: tail current through Kv7.2 E257C is also
photoregulated after treatment with MAQ. Voltage protocol to elicit tail current is the same as in A. C: fraction of current regulated in cells expressing Kv7.2
K255C (?1.4 ? 6.2%), G256C (?0.7 ? 1.1%), E257C (33.8 ? 8.3%), and G259C (?14.2 ? 18.3%). There was no detectable Kv7.2 current in cells expressing
the mutant N258C (N/A). Currents were elicited as in A. The position of the engineered cysteine is crucial for photosensitization. Data represent averages ? SD
(n ? 3–9).
491OPTICAL CONTROL OF MAMMALIAN K?CHANNELS
J Neurophysiol • VOL 106 • JULY 2011 • www.jn.org
Light-sensitive Kv3.1. Kv3.1 subunits are widely expressed,
particularly in neurons that fire action potentials at high fre-
quencies, including those in the auditory brainstem and cere-
bellum (Gan and Kaczmarek 1998). Kv3.1-containing channels
deactivate very rapidly after a depolarizing pulse, an important
feature thought to enable action potentials to occur in rapid
succession. Optical manipulation of Kv3.1 channels would aid
in exploring the function of these channels in neuron and other
cell types where they are expressed. K?current through
homomeric Kv3.1 channels containing a cysteine at amino acid
380 (E380C) and treated with MAQ were photosensitive at all
voltages, but the fraction of current photoregulated was largest
at membrane potential more negative than 0 mV (Fig. 4A).
Voltage-gated currents elicited by stepping to ?10 mV were
reduced by ?70% after switching from 380- to 500-nm light
(Fig. 4B). As expected, wild-type channels lacking a geneti-
cally engineered cysteine were insensitive to light even after
treatment with MAQ (Fig. 4B). Currents through photosensi-
tive Kv3.1 are repetitively blocked by 500-nm light with little
decrement in the fraction of current regulated and no apparent
photobleaching of the photoswitch (Fig. 4, C and D).
In fast-spiking neurons, Kv3.1 subunits can heteromultim-
erize with Kv3.4 subunits, resulting in a channel that repolar-
izes spikes more rapidly (Baranauskas et al. 2003). Since MAQ
should interact with its target channel like any standard qua-
ternary ammonium compound, that is by interacting with a
single binding site (25–27), we predicted that introducing a
cysteine at position D420 in Kv3.4, the equivalent position to
E380 in Kv3.1, would enable MAQ to photosensitize not only
homomeric Kv3.4 channels, but also heteromeric Kv3.4/Kv3.1
channels (Fig. 4D). Indeed, the photosensitivity was the same
whether the channels comprised 4 identical cysteine-containing
subunits (i.e., homomeric mutant channels) or a mixture of
cysteine-containing and wild-type subunits (i.e., heteromeric
channels with only 1 subunit type mutated). Hence, photosen-
sitization is all-or-none, rather than varying incrementally with
the number of tethered MAQ molecules, consistent with the
channel possessing a single TEA binding site that mediates
photosensitive pore blockade (Blaustein et al. 2000; Hegin-
botham and MacKinnon 1992; Hille 1967).
To confirm that light-sensitive Kv3.1 can alter activity of
neurons, we transfected cultured hippocampal neurons with
Kv3.1 E380C. Current-clamp recordings obtained after MAQ
treatment from transfected neurons coexpressing GFP showed
that light exposure had a significant effect on repetitive firing
elicited by depolarizing current injection. For example, the
neuron in Fig. 5A fired for almost twice as long when the
channels were unblocked in 380-nm light than when they were
blocked in 500-nm light, and six out of seven transfected
neurons showed similar results (Fig. 5B). Hence, hippocampal
neurons expressing Kv3.1 E380C channels fire more action
potentials when the channels are in the unblocked state. Indeed,
action potentials repolarized more quickly with a larger after-
hyperpolarization when the channels were unblocked (Fig.
5C), presumably enabling faster recovery of voltage-gated Na?
channels from inactivation, which promotes repetitive firing.
Fig. 4. Photosensitization of Kv3 family channels.
A: steady-state current voltage curves from a volt-
age-clamped HEK-293 cell expressing Kv3.1
E380C. Currents were elicited by stepping to the
indicated voltage from a holding potential of ?70
mV and measured in 380-nm (open squares; violet)
or 500-nm (closed squares; green) light after treat-
ment with MAQ. B: fraction of current photo-
switched after MAQ treatment of cells expressing
Kv3.1 E380C (67.0 ? 6.5%) or wild-type Kv3.1
(9.2 ? 3.5%). Currents were elicited by stepping
from a resting potential of ?70 to ?10 mV. Data
represent average ? SD (n ? 3–9). C: representa-
tive currents elicited by stepping from a holding
potential of ?70 to ?10 mV in alternating 500-
(green), 380- (violet), and 500-nm (green) light.
Currents through photosensitive Kv3.1 are repeti-
tively blocked by 500-nm light with little decrement
in the fraction of current regulated and no apparent
photobleaching of the photoswitch. D: currents
were elicited and measured every 2 s as in C and
plotted over time and wavelength changes. Illumi-
nation with 500-nm light (green) blocks the chan-
nel, whereas illumination with 380-nm light (violet)
unblocks the channel. E: channels expressed in
HeLa cells composed of up to 2 (heteromers) or 4
photosensitized to a similar extent by MAQ treat-
ment (homomeric Kv3.4, 5.540 ? 3.6%; homo-
meric Kv3.4 D420C, 57.2 ? 15.9%; heteromeric
Kv3.4 D420C ? Kv3.1, 49.8 ? 6.0%; hetero-
meric Kv3.1 E380C, 75.6 ? 24.2%; heteromeric
Kv3.1 E380C ? Kv3.4, 59.1 ? 12.6%. Data
represent average ? SD, n ? 4–5 for each
492 OPTICAL CONTROL OF MAMMALIAN K?CHANNELS
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Photosensitization of SK2. SK channels are small conduc-
tance Ca2?-activated K?channels. In contrast to large-con-
ductance potassium (BK) channels for which gating is sensitive
to both Ca2?and voltage, SK channels are sensitive only to
Ca2?, which activates the channels by binding to auxiliary
calmodulin subunits (Maylie et al. 2004). There are four
members of the SK channel family, and three of them, SK1,
SK2, and SK3, are expressed in central nervous system neu-
rons (Stocker et al. 2000). In CA1 hippocampal neurons, SK2
channels modulate EPSPs and contribute to long-term poten-
tiation (LTP) of synaptic transmission (Lin et al. 2008; Ngo-
Anh et al. 2005).
Based on amino acid sequence alignment with the other
photosensitive K?channels engineered for these studies, we
chose position Q339 as the cysteine attachment site for SK2
channels (Fig. 1C). Inside-out membrane patches were ob-
tained from cells expressing SK2 Q339C and treated with
MAQ. Channels were activated by applying a solution con-
taining 1.5 ?M free Ca2?. The current elicited first in darkness
was increased ?2.5-fold in 380-nm light, consistent with relief
of channel block as the MAQ was converted from the trans to
the cis configuration. The Ca2?-elicited current was repeatedly
attenuated and restored by illuminating with 500 and 380 nm,
respectively (Fig. 6A).
Light-regulated SK2 current could also be observed under
whole cell patch-clamp (Fig. 6B). For these experiments, we
used a patch pipette that contained 1 ?M free Ca2?and applied
a voltage ramp from ?80 to ?80 mV. The Ca2?-elicited
current was decreased over the entire voltage range by switch-
ing from 380- to 500-nm light. We then completely blocked the
channels with the specific SK channel inhibitor apamin (Fig.
6B). This allowed us to estimate that ?60% of the SK2-
mediated current is suppressed in 500-nm light, consistent with
our using inside-out patch results (Fig. 6B).
To test whether light-regulated SK channels can be gener-
ated in situ, we used a virus to express SK2 Q339C subunits in
hippocampal CA1 neurons. AAV carrying the genes encoding
SK2 Q339C and GFP was introduced via transcranial stereo-
taxic injection into 3- to 5-wk-old mice. No behavioral changes
were noted in mice post-AAV injection. Hippocampal slices
were obtained ?10 days postinjection and treated with MAQ.
Cells expressing SK2 Q339C were identified based on coex-
pression of GFP. Resting membrane properties of fluorescent
SK2 Q339C-expressing cells were similar to those of nonex-
pressing cells (membrane potential ? ?63 ? 2 mV for control;
?62 ? 1 mV for fluorescent; input resistance (Rin) ? 201 ? 20
M? for control; 208 ? 25 M? for fluorescent; access resistance
(Ra) ? 21 ? 2 M? for control; 22 ? 2 M? for fluorescent; n ?
12–14 for each). Voltage-clamp recordings of SK2 Q339C-ex-
pressing neurons showed photosensitive SK channel activity. In
380-nm light, when MAQ is in its cis configuration and SK2
Q339C channels are relieved from block, we observed a slowly
relaxing outward tail current after a depolarizing pulse to ?20
mV, characteristic of IsK(Bond et al. 2004). This tail current
Fig. 6. Photosensitization of SK2 channels. A: inside-out patch recording from
a HEK-293T cell expressing SK2 Q339C and treated with MAQ. Ca2?-
activated currents were induced with a solution containing 1.5 ?M free Ca2?
buffered with EGTA. Ca2?-activated current is larger in 380- than 500-nm
light. B: whole cell recording showing light-sensitive currents during a depo-
larizing voltage ramp (?80 to ?80 mV). The pipette contained 1 ?M Ca2?to
ensure maintained activation of SK2 channels. Currents were maximal in
380-nm light and reduced in 500-nm light. Apamin (0.5 ?M) further inhibited
the current, indicating that blockade by trans MAQ was ?60% complete.
Fig. 5. Photosensitization of Kv3.1 channels in neurons enable control of
repetitive firing. A: photoregulation enables control of repetitive firing in a
neuron expressing Kv3.1 E380C after MAQ treatment. The number of action
potentials fired in response to sustained current injection (iinj) is higher under
380-nm (violet) than 500-nm (green) light. Light exposure did not affect
resting membrane potential. B: photoregulation of Kv3.1 E380C enables
control of action potential firing in transfected neurons. Six of seven cells
expressing Kv3.1 E380C and treated with MAQ fire more action potentials in
380-nm light in response to a depolarizing current injection. C: photoregulation
of Kv3.1 E380C changes the shape of the action potential in transfected
neurons treated with MAQ. Single action potentials were generated by short
(10 ms) current injection. The afterhyperpolarization is more pronounced
under 380-nm (violet) than in 500-nm (green) light.
493OPTICAL CONTROL OF MAMMALIAN K?CHANNELS
J Neurophysiol • VOL 106 • JULY 2011 • www.jn.org
was reduced after illumination with 500-nm light (Fig. 7, A and
C). The amount of photoregulation varied considerably in
different cells; exposure to 500-nm light reduced the amplitude
of IsKmeasured at 100 ms by ?30% on average (n ? 12),
presumably because the photoswitch-ready channels were ex-
pressed against a background of native, light-insensitive chan-
nels. The IsKrecorded from nonfluorescent (and therefore not
expressing SK2 Q339C) CA1 neurons from the same slice was
not altered by exposure to 380- or 500-nm light (Fig. 7, B and
C). Hence, MAQ selectively photosensitizes cysteine-substi-
tuted SK2 channels, and light can alter currents carried by
these channels in an acute brain slice preparation.
We next asked whether photoregulating SK2 channels could
affect synaptic communication in a hippocampal slice. SK2-
containing channels are found in dendritic spines of CA1
neurons (Ngo-Anh et al. 2005). Electrical stimulation of pre-
synaptic Shaffer collateral axons causes glutamate release and
postsynaptic activation of Ca2?-permeant NMDA receptors in
the spines. The resulting Ca2?influx activates SK channels,
providing a repolarizing conductance that reduces the gluta-
mate receptor-mediated depolarization (Ngo-Anh et al. 2005).
Consistent with this, pharmacological blockade of synaptic SK
channels with apamin causes an increase in EPSP amplitude.
EPSP amplitude was regulated by light in some SK2
Q339C-expressing neurons, but the magnitude of the effect
was variable. This is not surprising given that these cells have
a mixture of channels possessing mutant and wild-type SK2
subunits. Figure 7D shows a particularly clear example in
which 500-nm light resulted in a doubling of EPSP amplitude,
consistent with the expected transition from the MAQ-un-
blocked to MAQ-blocked state. EPSP amplitude could be
repeatedly increased and decreased with 500- and 380-nm
light, respectively (Fig. 7E). The control of EPSP size with
light demonstrates that the SK2 Q339C-containing channels
are incorporated in the postsynaptic membrane and function in
a similar manner as wild-type SK2 channels.
Engineering “designer” light-activated K?channels. A
large number of toxins and small molecules have been found
that selectively block different K?channels. However, there
are still channel types with no selective blockers, and it
remains difficult to find blockers that unambiguously discrim-
inate between different members of a given K?channel family.
By genetically engineering a cysteine attachment site into a
Fig. 7. PhotoregulationofSK2channelsinhippocampalslices.A:wholecellrecordingsfromhippocampalCA1neuronsexpressingSK2Q339CinaMAQ-treatedslice.
A depolarizing pulse (top) elicited tail currents (bottom) that were larger in 380-nm (green) than in 500-nm light (violet). B: light has no effect on currents measured
from a nonexpressing cell in a MAQ-treated slice. C: summary data for photocontrol of the SK-mediated tail current. On average, 500-nm light reduced the tail current
measured 100 ms after the pulse by 30 ? 19% (n ? 12; average ? SD) for cells expressing SK2 Q339C and by 3 ? 9% (n ? 14; average ? SD) for nonexpressing
cells. *Significant difference (P ? 0.01; paired t-test). D: average excitatory postsynaptic potentials (EPSP) waveform from a SK2 Q339C-expressing neuron in 500-nm
494OPTICAL CONTROL OF MAMMALIAN K?CHANNELS
J Neurophysiol • VOL 106 • JULY 2011 • www.jn.org
single K?channel subunit, we are generating photoswitch-
ready channels with built-in pharmacological specificity. Sub-
sequent treatment of engineered channels with MAQ allows
specific, precise, and acute photopharmacological regulation of
Photoisomerization of MAQ from trans to cis shortens the
molecule from ?20 to ?13 Å. This shortening will change the
position of the quaternary ammonium moiety with respect to its
binding site in the pore. To maximize photocontrol of current
through the targeted K?channel, MAQ must be properly
positioned on the channel protein such that it can reach the pore
in the trans but not the cis configuration. When the SPARK
channel was engineered, the intramolecular distances between
the Shaker channel pore and several extracellular residues had
already been estimated (Blaustein et al. 2000), making the
selection of an optimal cysteine attachment site (E422C) a
straightforward task (Banghart et al. 2004). Although the K?
channels used here share considerable sequence homology
with Shaker, identifying the optimal attachment position re-
quired some cysteine scanning. Not surprisingly, photosensiti-
zation is acutely dependent on the position of the engineered
cysteine. For example, a single amino acid shift in Kv7.2
completely eliminates photosensitization. The strict positional
requirement has several possible explanations. Some cysteine
positions may tether MAQ too far to enable the quaternary
ammonium to reach the pore. Others may tether MAQ so close
to the pore that photoisomerization to the cis configuration fails
to reduce the effective concentration of quaternary ammonium
sufficiently to relieve block. Finally, at some positions, the
genetically engineered cysteine may simply be inaccessible to
covalent modification. More structural information about the
channels is needed to distinguish between these possibilities.
The degree of MAQ photoswitching is also dependent on the
affinity for blockade by external quaternary ammonium, which
varies among different K?channels. Once MAQ becomes
tethered, the QA moiety is constrained in space to the volume
of a hemisphere with a radius of ?20 Å, which results in an
effective concentration in the tens of millimolar range (Bang-
hart et al. 2004; Kramer and Karpen 1998). However, the
affinity of some K?channels for external QA is so low (Kd?
100 mM) that the channels remain unblocked, even when the
tethered MAQ is in the trans state. The location of the external
QA binding site is conserved among many K?channels (po-
sition 449 in Shaker), and particular amino acids that determine
high or low QA affinity have been characterized (Heginbotham
and MacKinnon 1992; Kavanaugh et al. 1991). By selecting
the appropriate amino acid for this site, it may be possible to
“tune” the extent of block by trans MAQ as well as the extent
of unblock after photoisomerization to the cis configuration. In
this manner, we have succeeded in altering the extent of
photoswitching in the Shaker channel and its mammalian
homolog Kv1.3. However, our attempts to increase the QA
affinity of Kv4.2 by mutating this site (V489Y) prevented
surface expression of the channel. Thus, although it is likely
that more light-regulated K?channel subunits eventually will
be added to the list of those we have already engineered, it is
difficult to predict which channels will be the most successful.
Applications of designer light-activated K?channels. The
various K?channels used in this study differ in their cell-type
distribution, gating properties, kinetics, and modulatory con-
trol. The different physiological functions of K?channels
suggest that overexpression of light-sensitive versions could be
used not only to suppress action potentials with light, but also
to fine-tune different aspects of cellular electrophysiology. For
example, light-regulated Kv7.2 channels might be useful for
controlling resting potential, light-regulated Kv3.1 for control-
ling accommodation, and light-regulated SK2 for controlling
the size of EPSPs.
K?channel optopharmacology enables selective spatial ma-
nipulation of channel function within parts of a single neuron.
For example, local illumination can improve our understanding
of the role of particular K?channels in dendritic integration in
pyramidal neurons. Photoregulation can also be useful for
probing ion channel function in groups of neurons, such as
tonotopically distributed neurons in the auditory brainstem,
where K?channel gradients play an important role in fre-
quency tuning (Parameshwaran et al. 2001).
Genetic knockouts have provided important information
about ion channel function, but elimination of one type of ion
channel can sometimes result in lethality or developmental
abnormalities. For example, genetic knockout of Kv1.3 in mice
results in alterations in the size and shape of olfactory bulb
glomeruli, leading to enhancement of the sensitivity to odor-
ants in vivo (Fadool et al. 2004). Genetic knockout of certain
K?channels can induce compensatory changes in the expres-
sion levels of other ion channels. For example, knockout of
both Kv3.1 and Kv3.3 disrupts normal thalamocortical oscil-
lations (Espinosa et al. 2008), but eliminating either subunit
individually has little effect, possibly resulting from compen-
satory upregulation of the other subunit or changes in the
expression level of entirely different channels. Our approach
provides the means for unambiguously revealing the role of a
given subunit. In principle, replacement of the native Kv3.1
with the cysteine-containing mutant in knockin mice should
provide a “clean” method for acute and reversible optophar-
macological control of the channel, without inducing compen-
satory changes in the expression levels of other proteins.
The optopharmacological strategy described here requires an
exogenous synthetic photoswitch molecule. The photoswitch is
modular in nature, consisting of a reactive moiety, a photoi-
somerizable linker and a ligand that binds to channel or
receptor. This modularity allows flexibility in the design of
each functional group, yielding a combinatorial toolkit for the
optopharmacological regulation of protein function. The in-
creasing availability of structural and pharmacological data
suggests that this optopharmacology strategy may be extended
to other types of channels and receptors, opening a new
window into understanding the function of these signaling
proteins in neurons and other cells.
We thank S. Szobota, J. Patti, and H. Janovjak for useful discussion and A.
Desai for purification of AAV. E. Isacoff (UC Berkeley), B. Rudy (New York
University), A. Tzingounis (University of Connecticut), and T. Jentsch (Leibniz-
Institute, Berlin) generously provided plasmids.
This work was supported by the National Eye Institute (Grant EY-018957
to R. H. Kramer), the National Institute of Mental Health (Grant MH-088484
to R. H. Kramer), and the UC Berkeley Nanomedicine Development Center for
Optical Control of Biological Function (National Eye Institute Grant PN2-EY-
495 OPTICAL CONTROL OF MAMMALIAN K?CHANNELS
J Neurophysiol • VOL 106 • JULY 2011 • www.jn.org
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