“Vision” in Single-Celled Algae
Suneel Kateriya,1 Georg Nagel,2 Ernst Bamberg,2 and Peter Hegemann1
1
Institut für Biochemie, Universität Regensburg, 93040 Regensburg; and 2Max-Planck-Institut für Biophysik, 60439 Frankfurt am Main, Germany
Photosynthetic unicellular algae have a unique visual system. In Chlamydomonas reinhardtii, the
pigmented eye comprises the optical system and at least five different rhodopsin photoreceptors.
Two of them, the channelrhodopsins, are rhodopsin-ion channel hybrids switched between
closed and open states by photoisomerization of the attached retinal chromophore. They
promise to become a useful tool for noninvasive control of membrane potential and
intracellular ion concentrations.
ision in animals evolved differently in vertebrates and
invertebrates. Both use rhodopsin, a seven-transmembrane
(7-TM) helix protein with covalently linked retinal, as the primary photoreceptor. In vertebrates, after light excitation,
rhodopsin activates a G protein (transducin) and a subsequent
phosphodiesterase, resulting in the hydrolysis of cGMP and
closure of the cGMP-regulated cation channels. The Na+/Ca2+
influx into the photoreceptor cell (rods or cones) is inhibited,
which leads to hyperpolarization of the plasma membrane.
Invertebrate rhodopsins also activate G proteins. These, however, activate phospholipase C. Phospholipase C releases diacylglycerol, which in turn activates the transient receptor
potential/transient receptor potential-like channels. Na+/Ca2+
influx is enhanced, and the plasma membrane is depolarized.
In 1968, the zoologist Richard M. Eakin (2) had already presented convincing arguments that vertebrate and invertebrate
vision developed from the light-sensing system of ancestral
unicellular flagellates. These are motile eukaryotic microorganisms such as unicellular algae, gametes of macroalgae, fungal
zoospores (18), or protozoa. However, so far it has been impossible to obtain rhodopsins from any lower eukaryote. In contrast, rhodopsins from prokaryotes, especially those from
halobacteria (archaea), have been studied intensively during
the past three decades. Microbial and animal rhodopsins are
structurally related in the sense that they comprise a 7-TM helix
but show no sequence homology. Two microbial rhodopsin
prototypes, bacteriorhodopsin and halorhodopsin, are lightdriven ion pumps specific for H+ or Cl, respectively, whereas
the sensory rhodopsins SRI and SRII mediate photophobic
reactions by coupling to specific transducer proteins (halobaterial transducers HTRI and HTRII). As in Escherichia coli
chemotaxis, these transducers activate a response regulator
(RR) that in turn switches the flagellar motor. This type of nonelectrical signal transmission between sensor and motor organ
is known as “two-component signaling.”
“Vision” in lower eukaryotes
When botanists in St. Petersburg, Russia studied motility
and phototaxis of unicellular green algae from the shores of
the Neva River, they had already noticed that most species
contain a characteristic yellow spot in the equatorial position
of the cell body (4). This spot seemed to be the light-sensitive
0886-1714/04 5.00 © 2004 Int. Union Physiol. Sci./Am. Physiol. Soc.
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“organ,” which was therefore named the “eye spot” (Fig. 1A).
Eye spots contain a large amount of carotenoids, which led to
the assumption that carotenoids serve as sensory photoreceptors. Only 100 years later, Foster and Smyth (6) made convincing arguments that the eye spot functions as an optical
device, forming the functional “eye” only in conjunction with
the photoreceptor proteins and biochemical downstream elements (Fig. 1B). Vesicle layers of low and high refractive index
reflect the light, causing a variable contrast that depends on
the direction of light incidence. Consequently, for photoreceptor molecules located at the surface of the eye spot, light
absorption changes during rotation of the alga. Moreover,
intensity and color modulation during helical swimming
depends on the swimming direction relative to the light source
and is minimized when algae directly approach or retreat
from the light source (12).
In a key experiment, Foster et al. (5) restored phototaxis in
“blind” Chlamydomonas cells by addition of retinal, thus
showing for the first time that the photoreceptor is rhodopsin.
Later several groups applied different retinal analogs to these
blind cells, demonstrating that algal rhodopsins possess
microbial type all-trans retinal chromophores as found at that
time only in archaeal rhodopsins. This statement is still valid
and has been expanded to include rhodopsins from eubacteria (proteorhodopsins) and fungi [Alomyces reticulatus (18)
and Neurospora crassa].
Light absorption of algal rhodopsins triggers photoreceptor
currents that have been studied intensively with the suction
pipette technique. In the colonial alga Volvox carterii, flashinduced photoreceptor currents are strongly pH dependent
and mainly carried by H+ (1). However, in single-celled
species like Chlamydomonas the H+ current is hidden by a
secondary Ca2+ current that rises almost with the same kinetics before it rapidly decays after a few milliseconds. Since the
H+-carried photoreceptor current is small at physiological pH
and the cells depolarize at acidic pH, this current is most
clearly seen when recorded directly from the eye with a pH of
4 only in the pipette (3). Under physiological conditions, only
the fast Ca2+-carried photoreceptor current is able to trigger
voltage-sensitive channels in the flagellar membrane, which in
turn cause massive Ca2+ influx into the flagella. This sudden
Ca2+ influx induces a switch of flagellar motion from breaststroke swimming to symmetrical flagellar undulation that is
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seen under the microscope as a phobic response.
Analysis of the stimulus-response curve of the Chlamydomonas photoreceptor currents led to the suggestion that
they are based on two photosystems, one of which is more
active at low flash intensities, whereas the other dominates at
high flash energies (3). Moreover, at high flash energies the
delay between flash and beginning of the photocurrent is
extremely small (<50 Ps; Ref. 13), which was explained by a
direct connection between rhodopsin and the ion channel in
a receptor-ion channel complex (9). At low flash intensities
(<1% rhodopsin bleaching) the photoreceptor current is
delayed by several milliseconds (1), suggesting that the lowintensity photoreceptor system involves a signal amplification
system that activates an eye spot channel indirectly (1, 3).
Algal rhodopsins
Initially, two retinal proteins were purified from Chlamydomonas eye spot membranes and sequenced. Surprisingly,
they show some homology to invertebrate opsins with a quite
conserved retinal binding site and G protein-activating
domain (10). Both Chlamydomonas opsin-related proteins
(Cop1 and Cop2; Fig. 2A) are encoded by one gene transcript,
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FIGURE 1. A: a Chlamydomonas cell with two flagella, a large chloroplast
(green), and the yellow/orange eyespot. B: eye function under consideration
of channelrhodopsin 1 (ChR1), channelrhodopsin 2 (ChR2), and a voltage- or
H+-gated Ca2+ channel (VGCC). The voltage change, '\, is transmitted along
the membrane and sensed by VGCCs in the flagellar membrane.
which undergoes alternative splicing in a light-dependent
manner (7). However, it was shown by an antisense approach
that neither Cop1 nor Cop2 is the primary photoreceptor for
phototaxis (8). The function of both is not yet known.
Later, searching a Chlamydomonas genome database, three
research groups almost simultaneously discovered two cDNA
sequences encoding large apoproteins [originally named
Cop3 and Cop4 (11) but also called CSRA and CSRB (20) or
Acop1 and Acop2 (21)] with some homology to microbial
opsins (Fig. 2B). A sequence comparison between Cop3,
Cop4, and the proton pump bacteriorhodopsin and molecular
modeling of the new proteins (11, 16, 20, 21) suggested that
the new rhodopsins might function as ion transporters. The
amino acids that form the H+-conducting network in bacteriorhodopsin are conserved in Cop3 and Cop4, whereas the rest
of the sequences are different. To test for such a function, the
cop3 mRNA was expressed in oocytes of Xenopus laevis
where functional expression for other microbial type
rhodopsins was previously demonstrated (14, 15, 19). Cop3expressing oocytes showed a light-gated conductance, which
was studied in detail by a two-electrode voltage-clamp technique (16). The observed transport activity was purely passive
and directly dependent on the membrane voltage and the H+
concentration gradient (Fig. 3, A and C). Outward photocurrents could be observed at high extracellular pH or low intracellular pH. The conductance was highly selective for protons,
and no other monovalent or divalent ion was found permeating. The current was stable in the light and decayed with a
time constant of W = 35 ms (19°C) after light was switched off.
The different temperature dependence of photocurrent amplitude and relaxation supported the notion that the conductance is purely passive. The amplitude of the current was
graded with the light intensity, and the currents only saturated
when all rhodopsin was activated (>1020 photon˜m2˜s2). The
action spectrum is rhodopsin shaped, with a maximum in the
green at 500 nm. These experiments left no doubt that the
oocytes had expressed an ion channel with intrinsic light sensor or, in other words, a rhodopsin with intrinsic ion conductance. According to its newly discovered activity, this prototype photoreceptor (Cop3) was renamed channelrhodopsin 1
(ChR1).
For the sake of identifying the activity of Cop4, cop4 mRNA
was also injected into Xenopus oocytes. Again, photocurrents
were recorded on these oocytes (Fig. 3, B and D; Ref. 17).
However, the cells became not only conductive for H+ but
also, most surprisingly, for monovalent and divalent cations
like Na+, K+, and Ca2+. As demonstrated by use of the giant
patch-clamp method, i.e., under cell-free conditions, the
channel activity is independent of any soluble factor or
endogenous protein of the oocyte, and photocurrents
recorded from two different mammalian cell lines, transiently
expressing Cop4, supported this notion. From these data it was
concluded that Cop4 acts as a cation-selective channel and
therefore was renamed channelrhodopsin 2 (ChR2; Ref. 17).
The current kinetics under patch-clamp conditions were
almost indistinguishable from whole cell recordings. The
superior time resolution of the patch-clamp experiments also
allowed the analysis of the opening of ChR2 channels, which
FIGURE 2. Opsin-related proteins in Chlamydomonas:
opsin-related protein (Cop)1 (GenBank accession no.
S60158) and Cop2 (AAG02503) (A), the channelrhodopsins
Cop3/Chop1 (channelopsin 1, AF385748) and Cop4/Chop2
(channelopsin 2, AF461397) (B), and the hypothetical signal
transducing rhodopsins Cop5 (AY272055), Cop6, and Cop7
(C). TR, transducer; RR, response regulator; AC/GC, adenylate or guanylate cyclase.
ChRO molecules are reacting to an “inactive state,” ChRI, that
is not competent for immediate light activation. The recovery
of ChRD from ChRI is slow but is accelerated by extracellular
H+ or negative potential. These findings allowed the conclusion that both channelrhodopsins are most active at low pH
and highly negative membrane potentials.
Function of channelrhodopsins in living algae
Sineshchekov and colleagues (20) generated transformants
in which the ratio of ChR1 and its homolog ChR2 was
changed by an antisense approach. In ChR1-deprived cells
photocurrents at high flash intensities were reduced, whereas
in ChR2-deprived cells photocurrents at low flash energies
were reduced. The authors concluded that ChR1 mediates the
behavioral high-intensity response (photophobic response),
whereas ChR2 is responsible for the low-light response (phototaxis). However, this claim appears to be controversial for
FIGURE 3. A and B: transmembrane arrangement of ChR1 (A)
and ChR2 (B). C and D: photocurrents generated by bluegreen light (500 nm, ChR1, formerly named Cop3) or blue
light (450 nm, ChR2, formerly named Cop4) and recorded by
two-electrode voltage clamp at different membrane voltages
from ChR1- (C) and ChR2- (D) expressing oocytes. Experiments were carried out at external pH = 7.5 and internal pH
= 7.3, 100 mM NaCl2, 5 mM KCl, 2 mM CaCl2, and 1 mM
MgCl2. Light is indicated by grey horizontal bars. C was modified from Ref. 16; D was modified from Ref. 17.
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was found to proceed without visible delay. The time constant
was determined to be 200 Ps or faster. Surprisingly, and in
contrast to ChR1, the conductance inactivates in continuous
light to a smaller steady-state level (Fig. 3D). The steady-state
activity is specifically controlled by pH and membrane potential. Closing of the ion channel is decelerated by intracellular
H+, whereas recovery from desensitization is accelerated by
extracellular H+ or negative membrane potential.
From the described activities of the channelrhodopsins, we
have concluded that both undergo a photocycle similar to that
of other microbial-type rhodopsins. According to the current
model (Fig. 4, A and B), photochemical isomerization causes
conversion of the dark form (ChRD) into a primary photoproduct ChRK on a picosecond timescale. This is necessary to
achieve an acceptable quantum efficiency. Then within
microseconds the conducting “open state” ChRO is generated.
In the case of ChR1, ChRO quantitatively converts back to the
resting state ChRD. In the case of ChR2, at least part of the
FIGURE 4. A and B: photocycle schemes for ChR1 (A) and
ChR2 (B). ChRD, dark form; ChRK, primary photoproduct;
ChRO, open or active ion-conducting state. ChRI, inactive
state. C and D: depolarization of ChR2-expressing cells by
blue light (450 nm), as indicated by black bars. C: an oocyte
expressing the NH2-terminal half of ChR2 (ChR2-315) in a
solution containing (in mM): 110 NaCl, 5 KCl, 2 CaCl2, and
1 MgCl2, pH 7.6. D: whole cell patch-clamp recording (in
current-clamp mode) of a human embryonic kidney 293 cell,
transiently expressing ChR2-315. Pipette solution was (in
mM) 140 KCl, 5 EGTA, 2 MgCl2, and 10 HEPES, pH 7.4. Bath
solution was (in mM) 140 NaCl, 2 MgCl2, 1 CaCl2, and 10
mM HEPES, pH 7.4 (19). hX, Light.
More rhodopsin sequences
Even if the conductance of ChR1/2 measured in Xenopus
oocytes does not depend on the large COOH-terminal extension (16, 17), this extension might serve as a hinge to a secondary protein. In fact, three sequences with homology to
prokaryotic transducer proteins are found in the Chlamydomonas genome. Very surprisingly, all three sequences are
connected to rhodopsin-like sequences that have so far
escaped identification. All three are more related to SRI and
SRII from halobacteria than to ChR1 and ChR2. We have provisionally named these sequences Cop5, Cop6, and Cop7 (Fig.
2C). The homology between Cop5 and Cop7 is 30% from helix
3 to 7, and the homology between Cop5 and the well-studied
SRII from N. pharaonis is 25%. Moreover, most amino acids
that interact with retinal are conserved (Table 1). Overall conservation of the transducer is higher, and the catalytically most
prominent boxes H, X, D, G, and N can be easily identified
(Table 1). This was quite surprising since such microbial-type
transducers have not been identified in any higher plant yet.
Prokaryotic transducers couple to so-called response regulators, RRs, constituting the heart of the two-component signaling system. In Cop5 and Cop7, such RR sequences are found
in frame downstream of the transducer. The sequence homology to the RR CheY from E. coli is 35%, and the phosphate
acceptor motif is conserved (Table 1). Most surprisingly, in
Cop5 the RR is followed by an adenylate or guanylate cyclase
domain (AC/GC in Fig. 2C). The AC/GC sequence is only 20%
homologous to the AC from T. brucei, but the catalytic domain
is highly conserved (Table 1). In Cop5 we see the unique case
TABLE 1.
Prototype Protein Sequence
Chlamyopsin-5 (AY272055) (1425 aa)
Conserved sequence signature in Chlamyopsin-5
Sensory Rhodopsin II N. pharaonis (1H68A)
25 % Homologous with Cop-5 (56-303 aa)
Helix 3 and 7
Histidine Kinase E. coli PhoQ (1ID0A)
40% Homologous with Cop-5 (390-550 aa)
H, X, D, G, and N boxes
Response Regulator E. coli CheY (1EAYA)
35% Homologous with Cop-5 (705-820 aa)
Phosphate acceptor motif
Adenylate Cyclase T. brucei (1FX2A)
20 % Homologous with Cop-5 (860-1055 aa)
Catalytic unit (highly conserved)
Guanylate Cyclase B. taurus
P55203 (885-1015 aa)
15 % Homologous with Cop-5 (870-1010 aa)
Catalytic unit
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the following reasons. As already mentioned above, the delay
between flash and beginning of the ChR2-mediated photocurrent is, like for ChR1, in the range of microseconds,
whereas in low light the delay in Chlamydomonas is milliseconds. In bright light both ChR1 and ChR2 are rapidly
degraded, similarly as the photophobic response disappears,
whereas phototactic sensitivity fades away much more slowly.
Therefore, from our perspective it is likely that both channelrhodopsins control photophobic responses and only indirectly
influence phototaxis. The primary phototaxis photoreceptor
still remains to be discovered. But knockout mutants and more
behavioral studies are needed to fully understand ChR1 and
ChR2 function.
Under the assumption that ChR1 and ChR2 are both
responsible for phobic responses, several mysteries still have
to be solved. First, in Chlamydomonas the fast photoreceptor
current is a Ca2+ current that is quite insensitive to the extracellular pH (3). Thus it must be carried by a secondary conductance and not by ChR1 or ChR2 (Fig. 1B). This conductance also awaits molecular identification. Second, if ChR1
and ChR2 are responsible for phobic responses, which
rhodopsin is triggering phototaxis? Sineshchekov et al. (20)
argued that ChR2 as the phototaxis receptor might couple to
a transducer protein like the archaeal transducers HTRs,
which are activated by their sensory rhodopsins, SRI or SRII.
Applications for heterologous expression of channelrhodopsins in animal cells
Immediately after ChR1 was identified as a light-gated ion
channel, it was suggested that channelrhodopsins might be
used for the modulation of membrane potential and cytoplasmic pH of cells other than Chlamydomonas (16). Similar
applications become even more obvious for ChR2 with its
large light-gated permeability to mono- and divalent cations
(17). Because the truncated versions (ChR1-346 and ChR2315) show equal ion-conducting function (16, 17), the 7-TM
core protein is sufficient for the conductance. In fact, ChR2315 has been used to depolarize Xenopus oocytes by illumination with blue light under physiological conditions (Fig.
4C). Activation of ChR2 or the 7-TM part alone increased
cytoplasmic Ca2+ concentration to such an extent that the
oocyte-endogenous Ca2+-sensitive chloride channels were
activated (17). Later, ChR2-315 was also expressed in two different mammalian cell lines, BHK and human embryonic kidney (HEK) 293 (17). A large, light-gated ionic conductance
was obtained in both cases. In addition, illumination led to
strong depolarization (Fig. 4D). In this respect HEK 293 are of
special interest, because this cell line does not express other
Ca2+-sensitive ion channels. Therefore, HEK 293 cells may be
perfused with a cytoplasmic CaCl2 solution for measuring
Ca2+ conductances without interference from endogenous
Ca2+-sensitive ion channels. Heterologous expression of the
small, 315-amino acid core protein (ChR2-315) might
become a useful tool for light-activated manipulation of mammalian cells.
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where all four elements, i.e., rhodopsin, transducer, RR, and
effector, are encoded by one open reading frame that possibly
is translated into one large protein with a total of 11
transmembrane segments (as was determined with the
software program available at http://www.ch.embnet.org
/cgi-bin/TMPRED_from_parser). We have no indication yet
under which conditions these rhodopsins with potential linked
enzymatic activity (enzymerhodopsins) are expressed. The only
hint is one partial Cop5 cDNA clone that appears in the
Chlamydomonas expressed sequence tag database http://
www.kazusa.org.jp/en/-plant/chlamy/EST/). If all three genes,
cop5, -6, and -7, are expressed either simultaneously or under
certain physiological conditions, the number of opsin-related
proteins in Chlamydomonas expands to seven. The assumption
that one of these new rhodopsins contributes to phototaxis
seems to be justified. But other functions like control of retinal
biosynthesis or developmental processes should also be taken
into account.
The finding that rhodopsin is used for phototaxis in archaea,
eubacteria, green algae, and fungal zoospores might support
the speculation that rhodopsin evolved from archaea via
eukaryotic flagellates up to animal rhodopsins. The fact that
microbial-type rhodopsins (type I), no matter whether they
occur in archaea or green algae, have very little homology to
animal-type rhodopsins (type II) might point to an independent evolution. Therefore, it is conceivable that animal
rhodopsins have developed from other rhodopsin-related proteins that originally were not sensing light (chemoreceptors
and others). Cop1 and Cop2 might fall into this category.