Channelrhodopsins: Molecular Properties and Applications

© 2008 Bamberg
Channelrhodopsins: Molecular
Properties and Applications
Ernst Bamberg, PhD 1,2 , Christian Bamann, PhD 1 ,
Katrin Feldbauer, MA 1 , sonja Kleinlogel, PhD 1 ,
Julia spitz, MA 1 , Dirk Zimmermann, PhD 1 ,
Phil Wood, PhD 1 , and Georg Nagel, PhD 3
1 Max-Planck-Institute of Biophysics
Frankfurt, Germany
2 Johann Wolfgang Goethe-University
Institut für Biophysikalische Chemie
Frankfurt, Germany
3 University of Würzburg, Botanik I
Würzburg, Germany

Introduction
Channelrhodopsin-1 and -2 (ChR1,2) occur in the
eyespot of the unicellular alga Chlamydomonas reinhardtii.
Both are retinal binding proteins involved in
the photoreception of the alga, leading to its phototactic
behavior.
The overall sequence homology of ChR1,2 to other
microbial rhodopsins in the seven transmembrane
section is 15% to 18%, compared with the most
prominent representative of this class, the lightdriven
proton-pump bacteriorhodopsin (bR) from
Halobacterium salinarum. Focusing on the sequence of
the putative ion pathway, an 85% homology is found,
suggesting that the ChRs should act as a proton pump
in the same way as bR (Fig. 1). When expressed in
oocytes from Xenopus laevis or in human embryonic
kidney cell line 293 (HEK293) cells, ChR1,2 have
recently been shown to operate as light-gated cation
channels (Nagel et al., 2002; Nagel et al., 2003).
With their seven transmembrane motif and lightgated
opening, ChR1 and ChR2 are unique and represent
a new class of ion channels.
a
b
Figure 1. a, Sequence homology of helix 3, the putative ion
pathway of ChR2, and helix 7, with the lysine as the retinalbinding
residue via the Schiff base. Bacteriorhodopsin (bR)
and other microbial rhodopsins (sensory rhodopsins, SRs) are
compared with ChR2. b, Schematic representation of ChR2
and bR in the plasma membrane.
© 2008 Bamberg
Channelrhodopsins: Molecular Properties and Applications
In addition to its biological function, ChR2 has
emerged as an excellent tool for neurobiological
applications: ChR2 can be expressed functionally
in neural cells in culture as well as in living animals
(Boyden et al., 2005; Nagel et al., 2005). Under
physiological conditions, illumination causes the
depolarization of neural cells, opening up new ways
to activate the cells in a noninvasive manner with
hitherto unknown precision in terms of temporal
and spatial resolution. Although this approach to the
optogenic control of neural cells has already been
documented in numerous recent publications (Bi et
al., 2006; Adamantidis et al., 2007; Arenkiel et al.,
2007; Petreanu et al., 2007; Zhang et al., 2007; Lagali
et al., 2008), little is known about the molecular
mechanisms of ChR2.
Important parameters for understanding these
molecular mechanisms include the single channel
conductance and the spectroscopic properties of
ChR2. The single-channel properties, such as conductance
and lifetime, were obtained by undertaking
noise analysis. As will be described below, the
single-channel properties are in full agreement with
the kinetics of the macroscopic photocurrents
and the kinetics of the photocycle, obtained by flashphotolysis
of the purified protein. Furthermore, under
certain circumstances, ChR2 acts as a light-driven
proton pump, as could be expected from the sequence
homology with the light-driven proton pump bR
and the similarities in their photocycle (Bamann et
al., 2008).
In the second part of this chapter, we will discuss
new ChR molecules that might be useful for
further applications.
Results and Discussion
Electrophysiological description
When expressed in oocytes from Xenopus laevis or
HEK293, ChR2 shows an inward rectifying behavior
under voltage-clamp conditions (Fig. 2). The
action spectrum has a maximal current at 470 nm
(blue light). The current time course is characterized
by an initial transient current that decays to a
stable stationary value. The reversal potential of the
current-voltage curve corresponds precisely to the
gradient of the permeating cation. If illumination
is repeated with different time delays, the transient
currents recover to their initial value with a time
constant of about 7 s. This might reflect the dark
adaptation of the protein, i.e., after 7 s the original
ratio between the cis and the trans form of the retinal
chromophore before illumination is reached. After
the light stimulus is removed, the current decays to
zero within approximately 10 ms. This time course
15
NOtEs

NOtEs
16
a
b
Figure 2. a, Photocurrents obtained from transfected HEK293
cells (I cell ) under whole-cell patch–clamp conditions. b, Current
voltage (I-V) curve, showing the inwardly rectifying behavior
of ChR2. Physiological electrolyte conditions: 110 mM Na + ,
5 mM K + , pH 7.4.
represents the mean lifetime of the channel. (See
Spectroscopy, below, for a more detailed description.)
Under current clamp conditions (depending on other
variables), researchers found that the illumination
causes a depolarization of the HEK293 cells in the
range of several tens of millivolts (Nagel et al., 2003).
These experiments were the starting point for further
studies on neural cells (Boyden et al., 2005; Nagel et
al., 2005).
Single-channel conductance
The most important properties of an ion channel are
its single-channel conductance, ion selectivity, and
kinetics. Using these parameters, one can obtain
the specific activity and an estimate of the expression
level of the channels in the target cell. Classical
patch–clamp studies that sought to obtain the single
channel conductance failed. This led us to the conclusion
that the single channel conductance must
be unusually low in the range of femtosiemens (fS).
Stationary noise analysis of the photocurrent is an
alternative method for studying single channel properties.
Figure 3 shows the power spectra of ChR2,
expressed in HEK293 cells, obtained under wholecell
patch–clamp conditions. Subtracting the spectra
obtained with illumination from that obtained in the
Figure 3. a, Power spectra of the photocurrents in the presence
(red) and absence (black) of light. The inserts show the
original current trace both with (red) and without (black) light.
b, Difference spectrum showing Lorentzian behavior. The
currents were measured under whole-cell patch–clamp
conditions (HEK293 cells). Conditions: high guanidine (Gua+)
gradient (1 mM inside, 200 mM outside), because of the
higher permeability of guanidine + .
© 2008 Bamberg

dark shows Lorentzian behavior. Assuming a twostate
(open-closed) model, single-channel conductance,
as well as open time, can be calculated from
the amplitude of the power spectrum at zero Hertz
S(0), the corner frequency f c , the variance σ2 , and
the calculated open probability P o
(1)
(2)
where I is the macroscopic photocurrent and i is the
current through a single channel. At –60 mV, we
obtained a single channel conductance of ca. 100 fS
and a lifetime of the open state of ca. 10 ms, which
agrees quite well with the kinetics of the photocurrents,
that is, the time course of the current after
switching off the light.
Spectroscopy
As all other rhodopsins, ChR2 is expected to undergo
a photocycle after illumination. This photocycle
starts at the ground state, and different intermediate
products appear in the light. Using flash-photolysis,
these intermediates and their kinetics can be obtained
and analyzed. In order to carry out these
experiments, the protein has to be expressed in an
appropriate system that allows the purification of
sufficient amounts of ChR2. Here Pichia pastoris
was chosen. The absorption spectrum of the protein
purified from this yeast agrees well with the action
spectrum of the photocurrents with a maximum
at 470 nm. Time-resolved spectra obtained using
laser flash photolysis demonstrated the existence of a
very short-living product, absorbing at 410 nm. This
M-like state indicates a deprotonation of the Schiff
base where the retinal is bound to the protein on
helix 7 (as observed for other microbial rhodopsins).
The most dominant intermediate appears with a rise
time of approximately 0.2 ms, and then decays in 10
ms to an inactivated state, which reaches the ground
state within 7 s. The 10 ms decay corresponds to the
open time of the channel, whereas the slow, 7 s process
can be assigned to the reisomerization process
of the retinal. These kinetics correspond well to the
time behavior of the current obtained from electrophysiological
studies. Under continuous light, the
slow process is not visible. This indicates that the
inactivated state is photoactive: An absorption of a
second (sequential) photon leads to the protein’s fast
© 2008 Bamberg
Channelrhodopsins: Molecular Properties and Applications
return to the ground state. The flash photolysis results
are summarized in the photocycle presented in
Figure 4 (Bamann et al., 2008).
a
b
Figure 4. a, Absorbance changes (∆A) obtained by flash-photolysis.
ChR2 was purified from Pichia pastoris and solubilized
in 0.2% decylmaltoside, 20 mM Tris-HEPES, pH 7.4, and
100 mM NaCl. The upper trace shows the time-dependence
of the kinetic intermediates from the global fit analysis (Bamann
et al., 2008). b, Photocycle of ChR2 as derived from flashphotolysis
and electrophysiological experiments.
Channelrhodopsin: a light-driven
proton pump?
The demonstration of the photocycle and the high-
sequence homology with bacteriorhodopsin for the
ion pathway in helix 3 raise the question, Does ChR2
also act as a light-driven proton pump? That is to
say, does a vectorial proton transport that is directly
coupled to the intermediates of the photocycle
exist? In order to investigate this putative pump
17
NOtEs

NOtEs
18
mode, ChR2 was reconstituted in lipid vesicles.
The proteoliposomes were adsorbed to planar lipid
membranes, as described earlier (Bamberg et al.,
1979). This experimental approach was chosen
because the background conductance of the compound
membrane is small compared with the situation in
which ChR2 was measured in the transfected HEK293
cells. Therefore, a superior signal-to-noise ratio can
be expected. Illuminating the compound membranes
yields transient photocurrents owing to the capacitive
coupling, which can be converted into DC-coupling by
adding the appropriate ionophores. Doing this results in
a comparably small stationary current, obtained in the
absence of any ion gradient and electrical potential difference
under short-circuit conditions. To ensure that
protons were the sole permeating cations, Tris-HEPES
(Sigma, Taufkirchen, Germany) was used as electrolyte.
Because light is the sole energy source, the resulting
current can only have been caused by the proton
pumping activity. Symmetrical addition of monovalent
cations had no effect on the pump currents.
Combining the results described above, ChR2
appears to have a dual function as an ion pump
and an ion channel. In the pump mode, the proton
translocation is directly coupled to the photocycle.
However, the pump efficiency is weak because the
transport of monovalent cations (protons included)
is largely uncoupled from the time course of the
520 nm intermediate that is typical for the open
channel. This inefficiency leads to a much faster
passive flow during the lifetime of the 520 nm intermediate
in the photocycle (Fig. 4); that is, 10 4 cations
per photocycle are transported in the channel mode,
as determined by noise analysis, and only one proton
per photocycle in the pump mode. Another characteristic
of the channel mode, its ion translocation,
depends exclusively on the electrochemical gradient
and therefore represents the channel properties, as
previously described (Nagel et al., 2003).
Applications and new constructs
Because of its unique properties as a light-gated,
inwardly directed cation channel, ChR2 has become
a highly sought-after tool for neural applications. It
has opened the way to controlling neural activity
simply by virtue of the light-induced depolarization
of the cell (Boyden et al., 2005). When ChR2 was
expressed in cultivated hippocampal cells, it became
active without the addition of any chromophore retinal,
as had already been demonstrated for HEK293
cells (Nagel et al., 2003). This indicates that hippocampal
cells are able to provide retinal synthesis.
The next step was to accomplish an expression in
living animals. For the first demonstration of ChR2’s
action in living animals, we chose Caenorhabditis
elegans, in which ChR2 was expressed in the muscle
cells (Nagel et al., 2005). After illumination, the
worm showed light-induced contractions, which can
be interpreted either as depolarization of the cell or
as a light-induced [Ca ++ ] increase. In this particular
case, adding retinal was necessary because the worm
has no internal mechanism for retinal synthesis.
Recently, several research groups worldwide have
succeeded in expressing ChR2 in the brain or in the
photoreceptor-deficient retina of rodents. In the
latter case, the first promising steps were made to restore
vision in the blind animals (Bi et al., 2006; Lagali et
al., 2008).
In order to improve the application of ChR2, new
ChR2 molecules with higher light sensitivity will be
necessary. The wild type needs a relatively large photon
flux to reach the depolarization of the target cells.
Greater efficiency can be reached by mutations where
the lifetime of the open state is increased. This has
been verified for the ChR2 mutant H134 to R, where
the lifetime of the channel is increased by a factor
of two, while the single channel conductance shows
no variation, as was demonstrated by noise analysis
(discussed in Single channel conductance, above).
Mutants such as this will be potentially useful for the
recovery of vision and for drug screening assays.
References
Adamantidis AR, Zhang F, Aravanis AM, Deisseroth K,
De Lecea L (2007) Neural substrates of awakening
probed with optogenetic control of hypocretin
neurons. Nature 450:420-424.
Arenkiel BR, Peca J, Davison IG, Feliciano C,
Deisseroth K, Augustine George J, Ehlers MD,
Feng G (2007) In vivo light-induced activation
of neural circuitry in transgenic mice expressing
channelrhodopsin-2. Neuron 54:205-218.
Bamann C, Kirsch T, Nagel G, Bamberg E (2008)
Spectral characteristics of the photocycle of channelrhodopsin-2
and its implication for channel
function. J Mol Biol 375:686-694.
Bamberg E, Apell HJ, Dencher NA, Sperling W,
Stieve H, Lauger P (1979) Photocurrents generated
by bacteriorhodopsin on planar lipid bilayers.
Biophys Struct Mech 5:277-292.
Bi A, Cui J, Ma Y-P, Olshevskaya E, Pu M, Dizhoor
AM, Pan Z-H (2006) Ectopic expression of a
microbial-type rhodopsin restores visual responses
in mice with photoreceptor degeneration. Neuron
50:23-33.
© 2008 Bamberg

Boyden ES, Zhang F, Bamberg E, Nagel G,
Deisseroth K (2005) Millisecond-timescale, genetically
targeted optical control of neural activity. Nat
Neurosci 8:1263-1268.
Lagali PS, Balya D, Awatramani GB, Munch TA,
Kim DS, Busskamp V, Cepko CL, Roska B (2008)
Light-activated channels targeted to ON bipolar
cells restore visual function in retinal degeneration.
Nat Neurosci 11:667-675.
Nagel G, Ollig D, Fuhrmann M, Kateriya S,
Musti AM, Bamberg E, Hegemann P (2002)
Channelrhodopsin-1: a light-gated proton
channel in green algae. Science 296:2395-2398.
Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili
N, Berthold P, Ollig D, Hegemann P, Bamberg E
(2003) Channelrhodopsin-2, a directly light-gated
cation-selective membrane channel. Proc Natl
Acad Sci U S A 100:13940-13945.
Nagel G, Brauner M, Liewald JF, Adeishvili N,
Bamberg E, Gottschalk A (2005) Light activation
of channelrhodopsin-2 in excitable cells of
Caenorhabditis elegans triggers rapid behavioral
responses. Curr Biol 15:2279-2284.
Petreanu L, Huber D, Sobczyk A, Svoboda K (2007)
Channelrhodopsin-2-assisted circuit mapping
of long-range callosal projections. Nat Neurosci
10:663-668.
Zhang F, Wang L-P, Brauner M, Liewald JF, Kay
K, Watzke N, Wood PG, Bamberg E, Nagel G,
Gottschalk A, Deisseroth K (2007) Multimodal
fast optical interrogation of neural circuitry.
Nature 446:633-639.
© 2008 Bamberg
Channelrhodopsins: Molecular Properties and Applications
19
NOtEs