Article

ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 10, pp. 1555-1570 © The Author(s) 2023. This article is an open access publication.

Published in Russian in Biokhimiya, 2023, Vol. 88, No. 10, pp. 1880-1897.

1555

REVIEW

Channelrhodopsins: From Phototaxis to Optogenetics

Elena G. Govorunova and Oleg A. Sineshchekov

e-mail: oleg_sinesh@yahoo.com

Received June 24, 2023

Revised July 9, 2023

Accepted July 9, 2023

Abstract— Channelrhodopsins stand out among other retinal proteins because of their capacity to generate passive ionic

currents following photoactivation. Owing to that, channelrhodopsins are widely used in neuroscience and cardiology

as instruments for optogenetic manipulation of the activity of excitable cells. Photocurrents generated by channelrhodop-

sins were first discovered in the cells of green algae in the 1970s. In this review we describe this discovery and discuss

the current state of research in the field.

DOI: 10.1134/S0006297923100115

Keywords: rhodopsins, ion channels, photoreception, membrane potential, flagellate algae, neurons

Abbreviations: ChR, channel rhodopsin; EPC,early photoreceptor current; LPC,late photoreceptor current.

* To whom correspondence should be addressed.

INTRODUCTION

Bacteriorhodopsin, the focus of L. A. Drachev’s

early research [1-3], was the first retinal protein iden-

tified in a microbe rather than in mammalian tissues.

Thediscovery of bacteriorhodopsin by D. Oesterhelt and

W. Stoeckenius in the early 1970s [4] was a sensation,

and the ease of isolation of this protein in large quantities

from the source organism, haloarchaeon Halobacterium

salinarum, and its remarkable stability have made it a

favorable object of many biophysical, biochemical, and

structural studies [5-8]. It would not be an exaggeration

to say that today we know about bacteriorhodopsin more

than not only about any other retinal protein, but also

more than about any integral membrane protein, a model

of which it represents. In contrast to visual rhodopsins

that fulfil their photosensory function by activation of

an enzymatic transduction cascade, bacteriorhodopsin

is an electrogenic proton pump that transports protons

from the cytoplasm to the external medium and thus pro-

vides basic means of solar energy utilization in haloar-

chaeal cells.

When photoelectric activity of bacteriorhodopsin

was discovered, it stimulated search for similar pro-

cesses in other microorganisms, including eukaryotes.

Most motile pro- and eukaryotes respond to light with

a change in their swimming pattern [9, 10]. The first

sensory rhodopsins, SRI [11-13] and SRII [14], have

been discovered in H. salinarum. Their photoexcitation

does not lead to generation of transmembrane electrical

currents but activates an enzymatic cascade, eventual-

ly causing motor responses. In contrast to haloarchaea

that change their reversal frequency in response to light,

unicellular green flagellate algae demonstrate genuine

phototaxis, i.e., oriented movement along the direction

of the light beam [15]. Discussion of this phenomenon

with E. N. Kondratieva, a well-known expert on pho-

totroph microorganisms [16], and successful registration

of photoinduced changes in the membrane potential of

chroloplasts isolated from a higher plant [17, 18], in-

spired research on photosensing in green algae in the

Department of Physico-Chemical Biology of the Mos-

cow State University under the guidance of F. F. Litvin.

However, attempts to detect electrical stages of photo-

sensory transduction by insertion of microelectrodes

into algal cells, as used for recording electrical processes

associated with photosynthesis, have been unsuccess-

ful[19]. This goal could only be achieved by the devel-

opment of a new method for extracellular recording us-

ing a suction pipette by the corresponding author of this

review [20, 21].

Using this method, it has been shown that light in

the spectral range of phototaxis evokes transmembrane

photoreceptor potential in the region overlaying the eye-

spot – a cellular organoid made of carotenoid globules

that acts as a modulator of photoreceptor illumination

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during helical swimming of the cell. Isotonic propagation

of the depolarizing photoreceptor potential to the flagel-

lar membrane triggers a regenerative electrical response

similar to an action potential in neurons. Theselection

of the object, the green alga Haematococcus pluvialis of

a relatively large cell size and with the elastic cell wall,

was a major factor in this work’s success. Subsequently

the corresponding author of this review has shown that

both stages of this photoelectric sensory cascade could

be recorded directly in a cell suspension[22], which en-

abled its detection in many other green and cryptophyte

algae [23-27].

In contrast to bacteriorhodopsin, the main compo-

nent of purple membranes that constitute ~50% of the

cell surface, photoreceptor proteins that guide algal pho-

totaxis are present in their cells in very low concentra-

tions [28], so that their purification by biochemical meth-

ods is not feasible. Analysis of phototaxis action spectra

by K. Foster led to a hypothesis that these photorecep-

tors are retinal proteins [29]. A large body of indirect ev-

idences obtained during the 1980s and 1990s, including

restoration of phototaxis in “blind” mutants of the green

alga Chlamydomonas reinhardii deficient in carotenoid

biosynthesis upon the addition of exogenous retinal pro-

vided the first experimental validation of this hypothesis

[30-32]. Identification of Chlamydomonas photoreceptor

proteins or, rather, genes encoding them, at the molec-

ular level has been achieved only upon the development

of high-throughput methods of polynucleotide sequenc-

ing. It turned out that the genome of C. reinhardtii en-

codes not just one, but two such proteins [23, 33-35].

Remarkably, protein sequences, isomer composition of

the chromophore, and primary photochemical reac-

tions of C. reinhardtii rhodopsins turned out to be closer

to bacteriorhodopsin than to animal visual rhodopsins.

Analysis of photoreceptor currents in C. reinhardtii ge-

netic transformants with reduced amounts of rhodop-

sins showed that these proteins serve as photoreceptors

in phototaxis and the photophobic (photoshock) motile

response of this alga, although photoreceptor currents

generated by them differ in their properties [23, 28].

But only expression of C. reinhardtii rhodopsin

genes in animal cells, such as Xenopus oocytes, carried

out by G. Nagel, P. Hegemann, and E. Bamberg, has

revealed their unique properties [33, 34] (earlier this ap-

proach was successfully used also to study bacteriorho-

dopsin [36]). It tuned out that unlike the latter, C. rein-

hardtii rhodopsins passively transport cations (not only

protons, but also sodium, potassium, and, to a lesser

extent, calcium ions) across the cell membrane, i.e., act

as light-gated cation channels, the only such channels

known so far. This property was highlighted by introduc-

ing a new name, “channelrhodopsin(ChR)”, to desig-

nate these proteins, which rapidly replaced the earlier

suggested names, “Chlamydomonas sensory rhodopsins”

[23] and “archaeal-type rhodopsins” [35].

ChRs became very popular after they had been

shown to stimulate neuronal activity upon illumination

[37-39]. This technique, knows as “optogenetics”, has

revolutionized neuroscience and related fields of re-

search [40-43]. Moreover, it is anticipated that ChRs

might be used for gene therapy to cure many neurolog-

ical, psychiatric, and cardiovascular disorders [44, 45].

The prospect of vision restoration in patients with de-

generative retinal conditions looks most promising [46].

Photoactivated proton pumps similar to bacteriorho-

dopsin are also used in optogenetics as the tools for in-

hibition of neuronal activity [47], although they trans-

port less than one charge per captured photon, generate

smaller currents than ChRs and therefore require illu-

mination of longer duration and/or higher intensity to

change the membrane potential.

High-throughput sequencing of genomes and tran-

scriptomes led to identification of ChRs in several other

major eukaryotic lineages besides green algae. More-

over, it turned out that phototrophic [48] and even het-

erotrophic [49] protists possess ChRs that selectively

conduct anions. It also looks like cation selectivity in-

dependently emerged at least twice in ChR evolution,

and in one case the proteins preserved many features

of bacteriorhodopsin [50]. Finally, last year ChRs were

found that are more permeable for K

than for Na

[51],

and ChR diversity might not yet be exhausted.

In retinal ion pumps like bacteriorhodopsin, all steps

of the ion transport are tightly linked to specific photo-

chemical conversions of the photocycle monitored by

optical methods in purified proteins or membranes[52].

In contrast to this, in ChRs of different families photo-

currents correlate with different photochemical conver-

sions, and this correlation provides information on func-

tional mechanisms of the channels [50, 53-55].

In this review we will discuss methods of record-

ing ChR activity in native cells, the photoelectric sen-

sory cascade in flagellate algae, the photoreceptor role

of ChRs in phototaxis, and, briefly, their diversity and

optogenetic applications. For more detail of ChR molec-

ular mechanisms we recommend other reviews [56-59].

Also, many reviews cover the history and principles of

optogenetics [42, 60-65].

METHODS FOR REGISTRATION

OF ChR ACTIVITY IN PROTIST CELLS

Photoinduced changes in the membrane potential

in retinal rodes and cones brought about by closing cy-

clic-nucleotide-gated channels can be recorded with in-

tracellular electrodes [66]. However, the small cellular

volume of flagellate algae makes this approach not feasi-

ble for the study of ChR activity. Some green flagellates

such as H. pluvialis possess an elastic cell wall, which

makes it possible to suck their cells into a micropipette.

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Fig. 1. Principles of recording and examples of photoelectric signals recorded in the cells of green flagellate algae: a-c)principles of the suction

pipette technique(a), measurements in non-oriented cell suspensions (b), and measurements in pre-oriented cell suspensions(c); d-f)examples

ofphotocurrent traces recoded from an individual cell(d), a non-oriented cell suspension(e), and a pre-oriented cell suspension(f). The arrows

inpanels(a-c) show the time of the excitation flash. Designations: PC,photoreceptor current; RR,regenerative response; rec.,recorded.

This does not lead to formation of a gigaOhm seal that

is necessary for voltage clamp, but asymmetric distri-

bution of ChR molecules in the cell membrane allows

registration of their photoinduced responses [20, 21].

Thirteen years later this approach (known as the suction

pipette technique, Fig.1a) was applied to a C.reinhardtii

mutant lacking outer layers of the cell wall [67], and in

eight more years, to a Volvox carteri mutant [68]. C.rein-

hardtii is a model object of many biological studies, in

the genome of which the first channelrhodospin genes

were subsequently identified [23, 33-35].

The development of the suction pipette technique

enabled the discovery of photoinduced depolarization

of the membrane in green flagellate algae mediated by

their phototaxis receptors [20,21], which laid the foun-

dation for the development of optogenetics twenty-five

years later. However, application of the suction pipette

technique is not only labor-consuming, but also limited

to cells with the elastic cell wall, rare among flagellates.

In addition, sucking a cell into a pipette may activate its

mechanoreceptors [69] and in any case cannot be con-

sidered as a physiological condition. Asymmetric local-

ization of molecular generators of photocurrents in dif-

ferent regions of the cell membrane, revealed by using

the suction pipette technique, led to the hypothesis that

a total photocurrent of thousands of cells in a suspen-

sion can be recorded even without artificial increase of

the electrical resistance between different regions of the

membrane by the glass of the pipette.

In the first modification of this assay, a photoex-

citation flash is directed along the line connecting the

electrodes (Fig. 1b). Photoreceptor current generated

by the cells oriented with their photoreceptors toward

the light source exceeds that by the cells in the oppo-

site orientation, and the difference signal is detected by

the electrodes immersed in the cell suspension. Another

modification of this assay uses preorientation of the cells

with the light that causes phototaxis, or with gravity, di-

rected at the 90° angle to the excitation flash (Fig. 1c).

In this case the electrodes register a projection of the

photocurrent on the direction of the preorienting factor,

which allows instantaneous measurements of the orien-

tation degree [22, 70, 71].

Both modifications of this suspension assay allow

recording under fully physiological conditions and are

not limited by the cell size and the cell wall structure.

Such versatility and technical simplicity allowed us-

ing this method to study many C. reinhardtii mutants

with defects in the eyespot size [72] and photomotil-

ity [73, 74]. Moreover, the suspension assay was used

to confirm that phototaxis restoration in “blind” carot-

enoid-deficient mutants upon the addition of exogenous

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BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

retinal indeed reflects reconstitution of functional pho-

toreceptor proteins rather than, for instance, retinal in-

corporation in the eyespot [31]. Furthermore, the sus-

pension assay enabled the discovery and characterization

of channelrhodopsin-mediated photoinduced electrical

cascades in several other green algae, including members

of the genera Spermatozopsis, Hafniomonas, Polytomella,

Mesostigma, and Platymonas, and in the phylogenetically

distant cryptophyte alga Cryptomonas [24-27]. Finally,

the suspension method was used as an indirect approach

to study gravitaxis [22] and chemotaxis [75] in C. rein-

hardtii, and provided a foundation for the development

of an express bioassay for detection of water pollution

with heavy metals and formaldehyde [76, 77]. A more

detailed technical description of both methods, the suc-

tion pipette technique, and the suspension assay, can be

found in the earlier published reviews [25, 70, 71].

Electrophysiological investigation of photorecep-

tor currents allowed characterization of photoreceptor

proteins in flagellate algae even before their genes were

cloned. Approximation of the light dependence of pho-

toreceptor current produced the value of 0.8 Å for the

product of the quantum yield and the optical cross sec-

tion, which is close to that of other retinal proteins [78].

The recovery of photoreceptor current after a saturating

flash provided an estimate of the duration of the photo-

receptor pigment’s photocycle (100 ms). Analysis of the

dependence of photoreceptor current amplitude on the

orientation of the polarization plane of the light stimu-

lus showed that the retinal chromophore is orientated in

parallel with the membrane plane [79, 80]. The action

spectra of photoreceptor current and photomotility of

H. pluvialis reveal a complex multiband structure, with

the most red-shifted band at 550 nm [20, 21]. This result

led to the conclusion that in flagellates the photorecep-

tor system might consist of several pigments acting as

an antenna for a bacteriorhodopsin-type protein. Sub-

sequently, a contribution of two photoreceptor proteins

in phototaxis has directly been shown in the model alga

C. reinhardtii [23], and even more candidates for this role

have been found in some other algae. However, all these

proteins are light-gated ion channels rather than photo-

activated ion pumps like bacteriorhodopsin [33,34].

PHOTOELECTRIC CASCADE IN PHOTOTAXIS

OF GREEN FLAGELLATE ALGAE

The photoelectric signal recorded in H. pluvialis cells

sucked into a micropipette comprises a gradual primary

photoreceptor current and a secondary regenerative re-

sponse that is driven by the primary current and develops

in the “all or nothing” manner. Comparison of the signs

of these signal components upon suction of different

parts of the cell into the pipette revealed that the prima-

ry inward photocurrent flows only across a small patch

of the plasma membrane overlaying the eyespot [20, 21,

67]. This intracellular structure originally was thought

to contain photoreceptor pigment [81], but later it was

found to play only an accessory role as a shading/reflect-

ing device [29, 82, 83].

After cloning of ChR genes, immunofluorescent

methods could be used to determine intracellular local-

ization of the encoded proteins. It has been shown that

both C. reinhardtii ChRs are mostly confined to the eye-

spot region [35, 84, 85], which is fully consistent with

the results of electrophysiological studies. Proteomic

analysis of the isolated eyespot preparations also showed

the presence of ChRs [86]. According to the current

view, ChR molecules are embedded in the plasma mem-

brane that shows specific ultrastructure in the eyespot

region [87]. In C. reinhardtii cells grown under a light-

dark cycle, immunofluorescence microscopy also shows

ChRs in the flagella and basal bodies, and their amount

in these organelles depends of the phase of the cycle

[88,89]. However, the functional state of ChRs found in

these locations is not yet known.

Flagellates rotate around their longitudinal axis

during swimming, so that the photoreceptor patch of

the plasma membrane experiences a periodic change

in illumination [9, 29, 90-94]. These conditions can be

modeled in a cell held on a micropipette by periodic il-

lumination at the 1 Hz frequency. At the light intensities

eliciting phototaxis in H. pluvialis, the beat frequency

of the cis-flagellum (the one closest to the eyespot) in-

creases, whereas that of the trans-flagellum decreases af-

ter switching the light on, and the opposite responses are

observed after switching the light off [78, 90, 95]. Such

asymmetric changes in the flagella beat frequency in a

freely swimming cell are expected to steer the cells in the

direction of light, i.e., lead to phototaxis. Photoinduced

motor responses of C. reinhardtii flagella have been stud-

ied in more detail and involve changes of not only beat

frequency, but also amplitude and velocity [96-99].

The regenerative electrical response occurs in the

flagellar membrane and therefore is described in the

literature as “the flagellar current” [32, 67, 70]. Paral-

lel registration of flagella beating and photoelectric re-

sponses in a cell held on a micropipette has shown that

the regenerative response triggers a change in the flagella

beating mode from a ciliary stroke to undulation, ob-

served during the photophobic response in a freely swim-

ming cell [20, 21, 78, 95, 100]. The photophobic response

consists of a sudden stop and/or a random change in the

swimming direction and is observed when the stimulus

intensity and/or duration exceeds a certain threshold [9,

10, 91, 101, 102].

The amplitude of the regenerative response is prac-

tically independent on the intensity of the light stim-

ulus, but the duration of the lag period from the be-

ginning of illumination till the onset of this response

shows an inverse dependence on it. The integral under

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Fig. 2. Properties of the photoreceptor current components in the green flagellate alga Chlamydomonas reihardtii: a)the kinetics of the early and

late photoreceptor currents, recorded from a cell suspension; b)the light dependence of the peak amplitude of photoreceptor current (right Y axis)

and an integral under the photocurrent curve (left Y axis).

the photocurrent curve till the onset of the regenerative

response is constant at any light intensity and increases

under red background illumination that hyperpolarizes

the membrane due to photosynthesis [19, 78, 79, 103].

These results show that the regenerative response is trig-

gered by translocation of a certain number of charges

across the membrane that depolarizes it by a few mV.

The strict dependence of the regenerative response

on the Ca

concentration in the medium and its sensi-

tivity to blockers of Ca

channels suggest that it is medi-

ated by opening of potential-gated Ca

channels in the

flagellar membrane [20, 21, 67]. Analysis of the regen-

erative response amplitude in C. reinhardtii cells during

flagella regrowth after their amputation has shown that

these channels are distributed over the entire flagella

length [104]. Selection of a mutant deficient in the regen-

erative response [74] enabled cloning of the gene cav2,

encoding these channels [105]. Immunofluorescent mi-

croscopy shows that the CAV2 protein is predominantly

located in the proximal part of the flagella.

PHOTORECEPTOR CURRENT COMPONENTS

Experiments with nanosecond laser flash excitation

and high acquisition rate have revealed that photore-

ceptor current comprises two components with different

properties [25, 32, 78, 95, 106, 107]. These components

are resolved in the current traces recorded from all tested

organisms, which indicates common principles of their

photoreceptor systems and signal transduction chains.

The rise of the early photoreceptor current (EPC) is

limited only by the response time of the setup (<30 μs

for the suction pipette technique [106] and <3 μs for the

suspension assay [31]). In contrast, the late photorecep-

tor current(LPC) occurs after a delay up to several mil-

liseconds, the duration of which depends on the stimulus

intensity. At least two kinetic components can also be

resolved in the photocurrent decay. The light saturation

level of the EPC is determined only by photochemical

processes of photon absorption, whereas that of LPC is

observed at ~1000-fold lower intensities. Only LPC but

not EPC depends on temperature and red background

illumination that hyperpolarizes the membrane by ac-

tivating photosynthesis [19, 106]. Finally, LPC shows a

much stronger dependence on the Ca

concentration in

the medium, as compared to EPC [25]. Because of that,

the actual kinetics of EPC can be determined by calcu-

lating the difference between the traces recorded before

and after these treatments (Fig.2a).

The maximal amplitudes of EPC and LPC mea-

sured at saturation yield the ratio <1 : 10. However,

membrane depolarization is proportional not to the max-

imal photocurrent, but to the number of charges trans-

ported across the membrane, i.e., the integral under the

photocurrent trace. Owing to slow closing of the LPC

channels, the membrane depolarization brought about

by LPC at saturation is practically equal to that pro-

duced by EPC atsaturation, although the levels of their

saturation differ 1000 times (Fig.2b).

channels that mediate LPC are located in the

plasma membrane near the eyespot region, but not nec-

essarily within it. These secondary Ca

channels are the

primary reason of the very high, nearly “single-quan-

tum” photosensitivity of phototaxis [108]. In contrast to

the Ca

channels in the flagellar membrane, these chan-

nels are not voltage-gated, because they generate gradu-

al current (i.e., the current amplitude of which depends

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Fig. 3. A schematic depiction of photosensory transduction in phototaxis of flagellate algae. ChR1 and2, channelrhodopsins1 and2, respectively;

EPC and LPC, early and late photoreceptor currents, respectively.

on the stimulus intensity). At least 43 genes encoding

channels have been found in the C. reinhardtii ge-

nome [109], but functions of only few of them are known.

Several Ca

-binding proteins but no Ca

channels have

been detected in the eyespot preparations [86, 110].

One of possible reasons for this failure is the insufficient

sensitivity of proteomics for detection of integral mem-

brane proteins. It is also possible that these presumable

channels are not homologous to any other known Ca

channels and therefore their sequences cannot be recog-

nized as such by bioinformatic analysis.

Mechanisms by which EPC activates the second-

ary Ca

channels in the eyespot region are not yet clear.

Heterotrimeric GTPases, and Ca

-dependent protein

kinases and phosphatases have been detected in prepa-

ration of isolated eyespots [111-113]. However, these

proteins can participate in other sensory cascades, such

as that activated by photoexcitation of phototropin, also

found in the eyespot preparations [86, 110]. Phototro-

pin has been shown to mediate photoregulation of the

eyespot size and the cellular content of ChRs [114].

The lack of a lag period and the high level of light

saturation led to the conclusion that EPC reflects an ion

flow through rhodopsin molecules themselves [32,103].

Attempts to determine the ionic selectivity of channel-

rhodopsins in C. reinhardtii [115, 116] and V. carteri[68]

using the suction pipette technique have not been suc-

cessful, because photocurrent recording in the algae

cannot be carried out under voltage clamp, and because

of the difficulty to separate EPC and LPC in these ex-

periments. Ionic selectivity of these and tens of other

channelrhodopsins has been determined only by patch

clamp measurements upon channelrhodopsin expression

in cultured animal cells.

Identification of ChRs genes in C. reinhardtii and

the development of methods for genetic transforma-

tion of this model alga allowed verification of the role

of ChRs as photoreceptor proteins in phototaxis and the

photophobic response. Using the method of RNA inter-

ference, K.-H. Jung in the laboratory of J. L. Spudich

created transformants with a decreased content of each

of the two ChRs, initially named Chlamydomonas sen-

sory rhodopsins A and B [23,28], better known today

as ChRs 1 and 2 (ChR1 and ChR2), respectively. Re-

markably, knocking down of ChR1 led to stimulation of

ChR2 expression, which led to a further increase in the

ChR2/ChR1 ratio in the ChR1 transformant. Analysis of

photoelectric signals recorded from transformant cells in

response to a laser flash showed that kinetics of photore-

ceptor current generated by ChR1 is limited only by the

time response of the measuring system, whereas the cur-

rent generated by ChR2 appears after a light-dependent

delay. In other words, ChR1 generated current with the

kinetics of EPC, and ChR2, current with the kinetics

of LPC. Moreover, ChR1 current exhibited high light

saturation like EPC, whereas ChR2 current, low satu-

ration like LPC. The action spectra of ChR1 and ChR2

photocurrents were also different, which reflected their

different absorption spectra: the maximum of ChR1 cur-

rent was observed at 510 nm, and that of ChR2 current,

at470 nm. These results were corroborated by absorption

spectroscopy of purified proteins obtained by hetero-

logous expression of their genes [84, 117, 118]. Measure-

ments of photoorientation by photoelectric recording in

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cell suspensions and of photophobic response by motion

analysis showed that both ChRs mediate both photomo-

tility responses [23, 28]. A light scattering assay for pho-

toorientation in an independently created transformant

with a reduced ChR1 content confirmed photoreceptor

role of this protein [84]. The low light saturation of LPC

mostly generated by ChR2 suggest that the latter is the

primary photoreceptor in phototaxis, whereas ChR1,

mostly responsible for EPC, primarily contributes to the

photophobic response that is observed at higher light

intensities (Fig. 3). It has to be however noted that the

ratio of ChR1 and ChR2 is different in different strains

[23, 84, 119], which may influence their relative contri-

butions in photomotility responses.

The method of RNA interference allows to reduce

the amount of the encoded protein in the cell, but not to

prevent its biosynthesis completely. The latter has been

achieved first with insertional mutagenesis [85, 120],

and later with targeted disruption of ChR genes [119,

121, 122]. In a strain with the ChR1 gene disrupted by

zinc finger nuclease, the sensitivity of photoorientation

was decreased over 3 orders of magnitude, whereas dis-

ruption of ChR2 gene produced a smaller effect, and

knocking out both ChR1 and ChR2 genes practically

abolished the response [122]. When the CRISPR/Cas9

system was used for genome editing, knockout of either

ChR gene had little effect on the sensitivity of photoori-

entation, but disruption of both genes eliminated the

response [119]. The difference between these results is

most probably explained by the difference between the

amounts of ChR1 and ChR2 in the parental strains and

the degree of compensatory upregulation of one rhodop-

sin upon suppression of the other one.

The CRISPR/Cas9 genome editing method enabled

not only knocking out ChR genes, but also creating

C. reinhardtii strains carrying point mutations of each

of them[119]. All tested mutants exhibited reduction of

photoorientation sensitivity, although the cellular levels

of mutant proteins were also reduced in most cases, as

compared to control. A ChR2 mutant with reduced rel-

ative permeability for protons compared to Na

, as de-

termined by patch clamp analysis in model mammalian

cells, generated photoreceptor currents only in the pres-

ence of Na

in the medium, which confirmed the impor-

tance of proton currents for phototaxis in the wild type.

THE ROLE OF THE CYTOPLASMIC FRAGMENT

Molecules of all known ChRs consist of the rho-

dopsin domain, formed by seven transmembrane α-heli-

ces, and a cytoplasmic fragment made of an almost equal

number of amino acid residues. Heterologous expression

studies show that the rhodopsin domain is necessary and

sufficient for channel conductance, although at least in

one ChR the cytoplasmic fragment has been found to

influence the kinetics of channel current [123]. Almost

exclusively, only rhodopsin domains are used for optoge-

netic applications and functional studies in heterologous

systems. Functional analysis of the cytoplasmic fragment

has only become possible after the development of ge-

nome editing methods in C. reinhardtii cells [121]. Trun-

cation of the last 78 C-terminal amino acid residues of

ChR1 leads to displacement of the eyespot to a more an-

terior position, and the same effect is observed after dis-

ruption of the entire ChR1 gene [122]. Moreover, the at-

tachment of a fluorescent tag to the C-terminus impedes

trafficking of ChR1 to the plasma membrane. These ob-

servations confirm the earlier proposed hypothesis that

the cytoplasmic fragments are responsible for the correct

cellular localization of ChRs.

The important role of the cytoplasmic fragment in

the function of microbial rhodopsins has been demon-

strated earlier in sensory rhodopsins from the blue-green

alga Anabaena [124]. Modification of the cytoplasmic

fragment in a channelrhodopsin has been shown to in-

fluence photocurrent kinetics [123]. Phosphorylation of

amino acid residues in the cytoplasmic fragment of ChR1

and ChR2 has been detected by mass-spectrometric

analysis C. reinhardtii eyespot preparations [110]. Rapid

changes in the degree of ChR1 phosphorylation correlate

with the sign of phototaxis, i.e., swimming towards or

away from the light source [125]. Obviously, localization

of the photoreceptors, as well as changes in photocurrents

they generate determine the phototaxis sign.

Bioinformatic analysis of the cytoplasmic fragments

of ChRs from some protists (but not C. reinhardtii) has

identified various protein domains known to participate

in enzymatic cascades in other organisms [126, 127].

However, no evidence of the functional role of these do-

mains as parts of ChR fusion proteins has been obtained

so far. In addition, the cytoplasmic fragments of some

green algae channelrhodopsins exhibit SUMO (small

ubiquitin-like modifier) binding sites, and biochemi-

cal studies confirm reversible binding of this protein to

ChR1 linked to regulation of its stability in C. reinhardtii

cells [128]. Finally, immunoprecipitation shows that the

cytoplasmic fragments of ChR1 and ChR2 bind the small

GTPase CrARL11. CrARL11 belongs to the Arf family,

other members of which are known to mediate trafficking

of proteins to the cilia and flagella in animal cells [128].

ChR DIVERSITY

AND OPTOGENETIC APPLICATIONS

Recording of photoreceptor currents in green flag-

ellate algae has revealed a wide distribution of ChRs in

members of this taxonomic group [25-27]. Green algae

(chlorophytes) are the closest relatives of land plants,

which justified genome sequencing in many of their spe-

cies [129, 130] including those earlier studied by electro-

GOVORUNOVA, SINESHCHEKOV1562

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

physiological methods. Bioinformatic analysis of the data

obtained has identified >200 individual ChR sequences

in these microorganisms (their lists can be found in [127]

and [131]), but only few of them have been studied by

patch clamp recording upon heterologous expression in

model cells. The great majority of the latter are primarily

proton channels like C. reinhardtii ChRs, and some of

them also conduct Na

, K

and, to a lesser extent, Ca

[26, 27, 33, 132, 133]. Collectively, these proteins are re-

ferred to as “cation channelrhodopsins” (CCRs) [58,

131, 134]. These ChRs are widely used for photostimula-

tion of neurons and other excitable cells [40-42].

All tested cryptophytes are capable of photoorien-

tation [135-137], and photoreceptor currents similar to

those in green flagellates have been recorded from one

fresh-water species [24]. The fully sequenced genome

of the marine cryptophyte Guillardia theta encodes tens

of rhodopsins, among which there are ChRs [48, 138],

proton pumps [139] and proteins that do not transport

ions[24]. At least five G. theta ChRs function as cation

channels when their genes are expressed in mammalian

cells [138, 140, 141], and many their homologs are found

in other cryptophytes [142]. According to their sequenc-

es and details of the transport mechanism, these proteins

are closer to haloarchaeal proton pumps than chlorophyte

ChRs, and therefore have been named “bacteriorhodop-

sin-like cation channelrhodopsins” (BCCRs) [50]. Their

similarity to haloarchaeal proton-pumping rhodopsins

is further corroborated by their trimeric structure [143,

144], whereas chlorophyte ChRs form dimers [145-147].

Most BCCRs generate small currents upon expression in

mammalian cells, but at least one their representative,

known as ChRmine (also known as Rhodomonas lens

cation channelrhodopsin1, RlCCR1), is a popular op-

togenetic tool for neuronal activation using green light

[148, 149].

Another group of G. theta ChRs exhibit exclusively

anion selectivity [48]. Their homologs have been found

in many other cryptophytes and are known as “anion

channelrhodopsins” (ACRs) [150, 151]. As the Cl

–

con-

centration in the soma of mature neurons is low, opening

of these channels leads to Cl

–

influx across the mem-

brane and inhibition of spiking. ACRs have successfully

been used as optogenetic inhibitors of neuronal activity

and behavior in worms, insects, fish, and mammals [48,

152-158]. In addition, they have been applied for opto-

genetic control of guard cell movements and growth di-

rection in higher plants [159, 160].

Besides cryptophytes, haptophyte [49] and even

some chlorophyte [127] algae have been shown to pos-

sess ACRs. Moreover, homologous proteins (or rather

genes that encode them) have been found in heterotroph

organisms from the infrakingdom of stramenopiles [49,

131, 161]. Most if not all of these organisms develop fla-

gella at least at one stage of their life cycle. It is plausible

that ACRs, as well as CCRs, acts as photoreceptors in

their phototaxis, but this hypothesis requires experimen-

tal verification.

Using ACRs, ~200 sequences of which are current-

ly known [127, 131], as optogenetic inhibitors is limited

by the direction of their photocurrents in recipient cells.

The Cl

–

concentration in the axon terminals is typically

higher than in the soma, so ACR photoexcitation depo-

larizes the membrane and may even lead to triggering

back-propagating action potentials [162-165]. These side

effects can be reduced by the addition of soma-targeting

motifs that decrease trafficking of ACRs to the terminals

[163, 165, 166], but complete elimination of this unde-

sired trafficking has not yet been possible.

That is why the discovery of ChRs more permeable

for K

than for Na

last year attracted much attention

of the optogenetic community. The first two proteins of

this functional class, known as “kailum channelrhodop-

sins” (KCRs), have been identified in the heterotrophic

stramenopile Hyphochytrium catenoides [51], and later

their homologs were found in some other organisms

[167, 168]. The results of the first experiments on inhibi-

tion of neuronal and cardiomyocyte activity using KCRs

look very promising [51, 167, 169], so these proteins are

anticipated to become the primary molecular instrument

for his purpose.

It is important to note that only one function of

channelrhodopsins is currently used in optogenetics –

their direct channel activity. Their second function that

is dominant in native algal cells, generation of the late

(delayed) photoreceptor Ca

current with 1,000-fold

magnification, has not yet found its practical applica-

tion, because the channels involved have not yet been

identified at the molecular level.

CONCLUSION

In the 1970s, when the studies on photoelectric ac-

tivity of bacteriorhodopsin and yet unidentified ChRs

were launched at the Moscow State University, it was im-

possible to predict the emergence of the entire new field

of biomedicine known today as optogenetics and based

on application of these proteins to manipulate the mem-

brane potential with light. At present, only one function

of phototaxis receptors (their channel activity) is used in

optogenetics. A complex and highly sensitive photosen-

sory cascade that mediates phototaxis is still awaiting its

applications. Nevertheless, this example serves as a very

convincing demonstration of the importance of funda-

mental studies for scientific and technical progress.

Contributions. E.G.G. writing the text; O.A.S. con-

ceptualization and supervision of the work, writing and

editing the text.

Funding. The work was supported by the private

funds of the authors.

CHANNELRHODOPSINS 1563

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Acknowledgments. We thank all colleagues who con-

tributed publications cited in this review.

Ethics declarations. The authors declare no conflicts

of interest. This article does not contain description of

studies with the involvement of humans or animal subjects.

Open access. This article is licensed under a Cre-

ative Commons Attribution 4.0 International License,

which permits use, sharing, adaptation, distribution, and

reproduction in any medium or format, as long as you

give appropriate credit to the original author(s) and the

source, provide a link to the Creative Commons license,

and indicate if changes were made. The images or other

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erwise in a credit line to the material. If material is not

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your intended use is not permitted by statutory regula-

tion or exceeds the permitted use, you will need to obtain

permission directly from the copyright holder. To view

a copy of this license, visit http://creativecommons.org/

licenses/by/4.0/.

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