Article

ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 10, pp. 1571-1579 © Pleiades Publishing, Ltd., 2023.

Russian Text © The Author(s), 2023, published in Biokhimiya, 2023, Vol. 88, No. 10, pp. 1898-1907.

1571

Retinal-Based Anion Pump

from the Cyanobacterium Tolypothrix campylonemoides

Tatyana I. Rokitskaya

1,a

*, Aleksey A. Alekseev

, Fedor M. Tsybrov

, Sergej M. Bukhalovich

Yuri N. Antonenko

1,b

*, and Valentin I. Gordeliy

3,c

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Moscow Region, Russia

Institut de Biologie Structurale (IBS), Université Grenoble Alpes, CEA, CNRS, Grenoble, France

e-mail: rokitskaya@belozersky.msu.ru

e-mail: antonen@belozersky.msu.ru

e-mail: valentin.gordeliy@gmail.com

Received June 30, 2023

Revised September 15, 2023

Accepted September 15, 2023

Abstract— In this work, TcaR rhodopsin from the cyanobacterium Tolypothrix campylonemoides was characterized. Analysis

of the amino acid sequence of TcaR revealed that this protein possesses a TSD motif that differs by only one amino acid

from the TSA motif of the known halorhodopsin chloride pump. The TcaR protein was expressed in E.coli, purified, and

incorporated into proteoliposomes and nanodiscs. Functional activity was measured by electric current generation through

the planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one side of the membrane surface, as well as

by fluorescence using the voltage-dependent dye oxonolVI. We have shown that TcaR rhodopsin functions as a powerful

anion pump. Our results show that the novel microbial anion transporter, TcaR, deserves deeper investigation and may be

of interest both for fundamental studies of membrane proteins and as a tool for optogenetics.

DOI: 10.1134/S0006297923100127

Keywords: microbial rhodopsin, photosensitive ion pump, optogenetics, proteoliposomes, bilayer lipid membrane

Abbreviations: BLM, planar bilayer lipid membrane; CCCP,carbonyl cyanide m-chlorophenyl hydrazone; PLs,proteoliposomes.

* To whom correspondence should be addressed.

INTRODUCTION

Microbial rhodopsins comprise a large family of

light-sensitive α-helical proteins with covalently bound

retinal as a cofactor. These proteins have been found in

microorganisms from all domains of life including vi-

ruses [1]. One of the main functions of these proteins is

their ability to perform light-driven transport of protons,

cations, and anions. The retinal-containing proteins

participate in energy storage by the marine microor-

ganisms, moreover, according to rough estimates, con-

tribution of the rhodopsin-based photosynthesis to the

total bioenergetics of biosphere is comparable or even

exceeds contribution of the chlorophyll-based photosyn-

thesis [2]. However, other functions of the proteins from

this family are known: sensory functions, ability to form

light-gated ion channels in the membrane[3], and even

regulation of enzymatic activity [4]. Structural-func-

tional studies of new microbial rhodopsins are very im-

portant for the development of new optogenetic tools[5].

Metagenomic analysis is often used for searching new

rhodopsins; and during the search for new objects a lot

of attention is paid to unusual pattern of the conserved

amino acid residues. The most studied light-driven pro-

ton pump, bacteriorhodopsin of Halobacterium sali-

narum, hasDTD motif (D85, T89, andD96– key ami-

no acid residues for proton transport)[6].

The gene encoding TcaR opsin have been found in

the course of bioinformatics search of the open metage-

nomic databases [7]. It has been located in the genome

of Tolypothrix campylonemoides VB511288 cyanobacte-

ria isolated from the green biofilm found on the façade

ROKITSKAYA et al.1572

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

of the building in the East-Indian city Santiniketan.

Analysis of the TcaR amino acid sequence reveals the

presence of the TSD motif. Rhodopsins from cyanobac-

teria with such motif have been characterized previously

[8-10], they have the ability to pump anions in bacterial

cells in response to illumination. The TSD motif differs

by only one amino acid from the motif of the known

chloride pump of the halorhodopsin, TSA[11].

In this study we present the data of functional stud-

ies of the TcaR rhodopsin using electrophysiological

[12, 13] and optical methods, including experiments on

determination of its ion selectivity in lipid vesicles and

nanodiscs. Electrophysiological approach used in our

study was developed by L. A. Drachev and A. D. Kaulen,

and later was widely applied for the investigation of rho-

dopsins in the modification suggested by Bambergetal.

[14-16]. The method is based on measuring of electric

photoresponses from the macroscopic planar bilayer

lipid membrane (BLM) with bound proteoliposomes

containing retinal proteins. This measuring approach

has been widely accepted due to the fact that it has high

sensitivity and, same as optical techniques, allows moni-

toring fast transient processes.

MATERIALS AND METHODS

Materials. Reagents from Sigma-Aldrich (USA)

were used in this work (unless indicated otherwise).

Proteoliposomes with TcaR rhodopsin. Nucleo-

tide sequence encoding TcaR (GenBank accession

no. JXC B00 000 000) was optimized for expression in

Escherichia coli cells using GeneArt software (Thermo

Fischer Scientific, USA). Nucleotide sequence at the

5′-end was optimized with the help of the RNA Web-

Server (Institute of Theoretical Chemistry, Vienna

University) with the goal to reduce the possibility of

hairpin formation at the RNA regions including ribo-

some binding sites. The gene was commercially synthe-

sized (Eurofins, Luxemburg) and cloned into an ex-

pression vector pET15b (Novagen, USA) at XbaI and

XhoI restriction sites. Hence, the gene was inserted in

front of the LEHHHHHH sequence (polyhistidine se-

quence used later for nickel-chelate affinity chromatog-

raphy). Expression in the cells of the E. coli C41(DE3)

strain and purification of the TcaR protein was car-

ried out according to the protocol described previ-

ously [17].

For preparation of proteoliposomes, a 1% (w/v)

solution of a soybean azolectin in chloroform (Sigma,

USA) was added to a glass flask. Chloroform was re-

moved with the help of a rotary evaporator and vacu-

um pump. The obtained thin lipid film formed on the

glass flask walls was resuspended in a solution contain-

ing 0.1 M NaCl (Applichem, Germany) and 2% (w/v)

sodium cholate to produce final concentration of azolec-

tin 1% (w/v). Lipid suspension was subjected to ultra-

sound treatment for 5 min at 4°C, followed by fast ad-

dition of a solubilized rhodopsin to final concentration

0.7 mg/ml and detergent-absorbing Bio-Beads SM-2

(Bio-Rad, USA). The obtained mixture was mixed in an

orbital shaker for 2h with minimal illumination, followed

by replacement of the beads (this procedure was repeat-

ed for at least four times).

Nanodiscs with TcaR. Assembly of nanodiscs and

incorporation of TcaR was carried out using standard

techniques described previously in the literature [18].

Dimyristoyl phosphatidylcholine (DMPC) (Avanti Po-

lar Lipids, USA) was used as a lipid. An elongated vari-

ant of the apolipoprotein-1 protein named MSP1E3D1

was used at a molar ratio DMPC/MSP1E3D1/

TcaR = 100/2/3. Dry lipid film obtained by dissolving

of DMPC powder in chloroform followed by evapora-

tion on a rotary evaporator (Heidolph, Germany) was

rehydrated by adding a detergent solution of 100 mM

CHAPS to obtain a lipid/detergent ratio of 1 : 2. Next,

a nanodisc-forming protein MSP1E3D1 was added to

the solution of the target protein TcaR solubilized in

micelles. The obtained mixture was incubated for 1 h at

room temperature, followed by addition of a BioBeads™

sorbent (Bio-Rad) in order to remove detergent at a ra-

tio of 1 g of the sorbent per 40 mg of detergent. Deter-

gent removal was carried out for 3h.

Planar bilayer lipid membrane was formed from

a decane solution, which contained 2% diphytanoyl

phosphatidylcholine and 0.04% (w/v) dimyristoyl ethyl-

phosphatidylcholine (Avanti Polar Lipids), on an aper-

ture in a partition separating a Teflon cell with a buffer

solution into two parts [19]. Diameter of the aperture

was 0.8 mm. Composition of a buffer solution varied and

is presented in figure captions. All experiments were

performed at room temperature (23-25°C).

Electric current was recorded under voltag-clamp

conditions. Potential difference was applied to silver

chloride electrodes connected through agar bridges into

the Teflon cell to both sides of the membrane. Current

was measured with the help of a patch-clamp amplifier

OES-2 (OPUS, Russia), digitized with a NI-DAQmx

(National Instruments, USA), and analyzed using a

commercial WinWCP Strathclyde Electrophysiology

Software, developed by J. Dempster (Strathclyde Uni-

versity, United Kingdom).

A halogen lamp Novaflex (World Precision Instru-

ments, USA) with power density 0.77W/cm

was used

for BLM illumination. The lamp illuminated the cell

from the front (cis) side, proteoliposomes were added to

BLM from the cis-side.

Proteoliposomes (PLs) with incorporated retinal

protein TcaR were added to the cis-side of the cell, which

was closer to illuminator. A high-ohmic electrode was in

the opposite part of the cell on the trans-side. Following

addition of 25-50 μl of TcaR-proteoliposomes to BLM

RETINAL-BASED ANION PUMP 1573

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

and 60-min incubation in a buffer solution containing

10 mM Mes and 10 mM Tris (pH ≈ 7), lipid membrane

was illuminated with a light in visible range of the spec-

trum. The BLM formed from the mixture of diphyta-

noyl phosphatidylcholine and cationic lipid dimyristoyl

ethyl-phosphatidylcholine has been previously shown to

facilitate adsorption of the negatively charged liposomes.

Significance of differences in data sets was evaluated

with the Student’s t-test.

Measurement of potential generation in suspension

of proteoliposome containing TcaR was carried out by

measuring fluorescence of the potential-sensitive dye ox-

onol VI (final concentration 2 μM) in a buffer containing

100 mM NaH

and 1 M NaCl (pH 7.2) as described

previously [19]. Fluorescence intensity was re corded with

a Panorama Fluorat-02 spectrofluorimeter (Lumex, Rus-

sia), excitation wavelength 590nm and emission wave-

length 620 nm (slits– 5 nm) at 15°C. After reaching the

prescribed temperature 10-20 μl of proteoliposomes was

added to the cuvette, which was further illuminated with

a 1-mW green laser. To monitor generation of pH gra-

dient in the liposomes, fluorescence of pH-sensitive

dye, 9-aminoacridine (excitation– 425 nm, emission–

455 nm) was used.

Absorption spectra of TcaR were recorded with a

Specord50 spectrophotometer (Analytik, Germany).

RESULTS AND DISCUSSION

According to the literature data, pump activity of

the retinal proteins is usually monitored using a simple

technique based on measuring changes of pH in a lipo-

some suspension with the help of a pH electrode, how-

ever, this method has low sensitivity and requires large

amounts of the protein. A more complicated method

exhibits a significantly higher sensitivity, in this method

proteoliposomes are attached to the surface of a planar

bilayer lipid membrane, but are not fused with it and re-

main intact on the BLM surface. It was shown that in

such system illumination induces generation of transient

electric potentials [12, 13] and transient currents across

the BLM[14-16], which could be recorded with regu-

lar macroscopic electrodes. Addition of protonophores

to such system usually results in the appearance of sta-

tionary photocurrents through the BLM, because the

electrical accessibility of the inner aqueous phase of the

proteoliposomes increases[16,20].

After the start of illumination, a small and fast

change of the BLM current towards negative values was

observed (black curve, Fig. 1,aandb), which was fol-

lowed by a return to the initial current values within a

second time scale. Further addition of a choline chloride

(Fig. 1a) or potassium fluoride (KF) (Fig. 1b) resulted

in the immediate increase of the transient current as a

response to switching illumination (light-gray curves),

as well as in continuous increase of the transient current

with time (dark-gray curves, recorded after 30 min).

The sign of the observed transient current for PL with

TcaR corresponds to the one for the PL with bacteri-

orhodopsin and indicates several possible ways of the

protein functioning: pumping of protons or monovalent

cations inside liposomes, pumping of monovalent an-

ions outside of liposomes, or some other possibilities.

Following the addition of tributyltin, which is a trans-

porter of monovalent anions [21, 22], to both sides of

the membrane resulted in the significant increase of

not only initial, but also of the stationary current as a

response to illumination (dotted curves). Addition of

another known anion transporter, prodigiosin [23], in-

stead of tributyltin, also resulted in the increase of initial

transient and stationary BLM photocurrents (Fig. 1c).

The results of experiments with the adsorbed PL on the

effects of addition of 50 mM chloride ions followed by

addition of 1 μg/ml of tributyltin on the amplitude of

the initial photoresponse of BLM (4 experiments) are

presented in Fig. 2. Initial photocurrent in the presence

of 50 mM of chloride anion was taken as one unit. It can

be seen that the addition of chloride ions causes increase

of the initial current (p < 0.0003), and the following ad-

dition of anion transporters leads to the current increase

(p<0.006).

When an experiment was conducted in the medi-

um with sulfate anion as a main anion in the electro-

lyte [medium composition: 10 mM Mes, 10 mM Tris, and

100 mM Na

(pH 7.0)], prolonged incubation of li-

posomes with the BLM also resulted in their adsorp-

tion on the surface of the planar lipid membrane, which

was manifested by the appearance of a transient current

on illumination (Fig. 3, black curve). However, in this

case the amplitude of the transient current was low-

er in comparison to the one observed in the presence

of chloride anion. Addition of protonophore (carbonyl

cyanide m-chlorophenyl hydrazone, CCCP) increased

the stationary current upon illumination (light-gray

curve), and following addition of 60mM sodium chlo-

ride (dark-gray curve) resulted in significant increase

in the initial current induced in response to the illumi-

nation. Prodigiosin added at the end of the experiment

significantly increased the stationary BLM photocurrent

(Fig.3, dotted curve).

In all experiments with adsorption of proteolipo-

somes on BLM, addition of potassium chloride, sodi-

um chloride, choline chloride, or potassium fluoride, as

well as addition of the transporters of monovalent anions

(tributyltin and prodigiosin) resulted in the significant

increase of the transient photocurrent. However, small

transient photocurrents were observed after attachment

of liposomes on the planar lipid membranes even in the

absence of monovalent anions. It is likely related to the

presence of chloride anions inside liposomes and their

leakage from the electrodes’ agar bridges to the solution.

ROKITSKAYA et al.1574

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 1. Measurements of electrical current through BLM with adsorbed proteoliposomes containing TcaR at BLM potential 0mV. Period of mem-

brane illumination is shown with solid black line. a)BLM current during incubation with liposomes (50μl) for 60min (black curve); light-gray

curve– BLM current immediately after addition of 50mM choline chloride; dark-gray curve– after 30-min incubation with choline chloride;

dotted curve– after addition of 1μg/ml of tributyltin. b)BLM current during incubation with liposomes (25μl) for 60min (black curve); light-

gray curve– BLM current immediately after addition of 50mM KF; dark-gray curve– after 30-min incubation with KF; dotted curve– after ad-

dition of 1μg/ml of tributyltin. c)BLM current during incubation with liposomes (25μl) for 60min (black curve); BLM current immediately after

addition of 50mM choline chloride; dark-gray curve– after 30-min incubation with choline chloride; dotted curve– after addition of 150nM

of prodigiosin. Buffer solution contained 10mM Mes and 10mM Tris(pH7.0).

RETINAL-BASED ANION PUMP 1575

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 2. Summarized results of four experiments with TcaR-proteoli-

posomes investigating effect of addition of 50 mM chloride ions and

following addition of 1 μg/ml of tributyltin on amplitude of the initial

BLM photoresponse. Initial photocurrent in the presence of 50 mM

of chloride anion was taken as one unit.

Fig. 3. Electric current in BLM with adsorbed proteoliposome contain-

ing TcaR in the medium containing 100mM Na

. Period of mem-

brane illumination with white light is shown by solid black line. BLM

current during incubation with liposomes (25μl) for 120min (black

curve); light-gray curve– BLM current after addition of 2μMCCCP;

dark-gray curve – after addition of 60 mM NaCl; dotted curve –

after addition of 150 nM prodigiosin. Buffer contained 10 mM Mes,

10mM Tris, and 100 mMNa

(pH7.0).

We prepared membrane nanodiscs with incorporated

TcaR protein and measured photoresponses of BLM in

their presence in different media. Nanodiscs does not

have inner space filled with water, hence, the equivalent

electric scheme in this case could differ from the exper-

iments with liposomes. The results of measuring BLM

photocurrent in the buffer with 100 mM NaCl (Fig. 4a),

100 mM Na

(Fig. 4b), and 100 mM potassium glu-

conate (Fig. 4c) after addition of 10 μl of nanodiscs

at the cis-side of BLM are presented in Fig. 4. Nano-

discs rapidly adsorbed on the BLM, and already after

20 min BLM currents appeared in response to illumi-

nation. It is clearly seen that the significant transient

currents were observed only in the case of the medium

with sodium chloride (Fig. 4a; light-gray curve). In the

presence of sodium sulfate or potassium gluconate the

observed photocurrents were significantly lower (light-

gray curves in Figs. 4,bandc). Moreover, in the case

of 100 mM Na

(Fig. 4b) the first rapid response

has even opposite sign. Addition of sodium chloride at

the end of each experiment resulted in appearance of a

large immediate current change in response to illumina-

tion (Fig. 4, b and c; dark-gray curves), or further in-

crease of the initial current (Fig. 4a). This result allows

us suggesting that sodium, potassium, sulfate, and glu-

conate ions are not transported by the pump. The sta-

tionary BLM photocurrent increased significantly in the

presence of CCCP protonophore or anion transporter

tributyltin (medium-gray and dotted curves in Fig. 5).

In conclusion these experiments indicate the ability of

the retinal-containing protein TcaR to transport chloride

anions across the membrane in response to illumination.

The data presented in Fig.1b allow us suggesting the

ability of the TcaR protein to transport fluoride anions.

Transport of hydroxyl anion by this protein also cannot

be excluded. It was shown previously that the mem-

brane fragments with halorhodopsin that has similar se-

quence of important amino acids (the TSA motif) could

generate photocurrent in the presence Cl

–

, Br

–

, and

–

, but not in the presence of SO

2–

, F

–

, and NO

–

[24].

However, depending on the illumination conditions ha-

lorhodopsin could function as a light-driven chloride

pump or proton pump [25]. According to our results,

transport of anions by the TcaR exposed to light is re-

alized from inside of the liposomes to outside (which

is equivalent to the transport into the cell cytoplasm).

Hence, a direction of the active transport of anions by

the TcaR and halorhodopsin is the same.

It was shown previously that the fluorescent dyes

such as potential-sensitive oxonolVI and ΔpH-sensitive

9-acridine amine(9-AA) could be used for measuring ac-

tivity of the rhodopsins in suspension [19,26]. Typical re-

sponse of oxonolVI fluorescence on addition to the sus-

pension of proteoliposomes containing TcaR is presented

in Fig.5a. Significant decrease of the fluorescence signal

is observed during illumination with green light, which

suggests generation of membrane potential on the lipo-

somes. OxonolVI is an anionic dye and decrease of its

fluorescence indicates generation of potential with plus

sign inside the liposomes, which corresponds to the same

polarity as in the similar experiments with bacteriorho-

dopsin[26]. It should be mentioned that the sensitivity

of this method is significantly lower that of the meth-

od based on the measurement of photocurrent on BLM

[19], hence, generation of large fluorescence responses

ROKITSKAYA et al.1576

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 4. Time-dependencies of electric current in BLM with adsorbed nanodiscs containing TcaR at BLM potential 0mV. Period of the membrane

illumination with white light is shown with solid black line. a)BLM current during incubation with nanodiscs (10μl) for 5min (black curve);

light-gray curve– after 20-min incubation with nanodiscs; medium-gray curve– after addition of 2μM of CCCP; dotted line– after addition of

1μg/ml tributyltin; dark-gray curve– after addition of NaCl (to concentration 200mM). Buffer solution contained 10mM Mes, 10mM Tris, and

100mMNaCl(pH7.0). b)Similar current time-dependencies in buffer solution containing10mM Mes, 10mM Tris, 100mM Na

(pH7.0).

c)Similar current time-dependencies in buffer solution 10mM Mes, 10mM Tris, 100mM potassium gluconate (pH7.0).

RETINAL-BASED ANION PUMP 1577

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 5. Ion-transporting properties of TcaR in proteoliposome suspen-

sion measured by changes of fluorescence of 2 μM oxonolVI(a) and

4 μM 9-amino acridine(b). 10μl of proteoliposomes were suspend-

ed in 1 ml of medium containing 1 M NaCl and 100 mM NaH

(pH7.2). Period of membrane illumination with the light of green LED

is shown with solid black line.

Fig. 6. Absorption spectra of TcaR in nanodiscs in the presence of

varying concentration of potassium chloride. Measurement medium

contained 10mM Mes and 10mM Tris (pH6.5). Potassium chloride

concentrations: 0mM (black line), 10mM (light-gray line), 50mM

(dark-gray line), and 150mM (dashed line).

in the suspension of proteoliposomes withTcaR (Fig.5a)

indicates high efficiency of the TcaR pump. Similar ex-

periments using the 9-AA dye demonstrated that no pH

gradient is formed in this system (Fig.5b).

A series of absorption spectra of TcaR in the ab-

sence of chloride anions (black curve) and in the pres-

ence of increasing concentrations of chloride anions

(gray and dotted curves) is presented in Fig.6. Position

of absorption maximum of the TcaR protein in nano-

discs is at 531 nm, and a 5-nm shift of the absorption

maximum towards short wavelength occurs after addi-

tion of 50-150mM of chloride ions. Furthermore, opti-

cal density at the absorption maximum increased. These

data are in good agreement with the effect of chloride

anions on the absorption spectra of halorhodopsin in-

vestigated previously [8, 9, 27] and provide support to

the notion that TcaR is an effective chloride pump.

Analysis of amino acid sequence of TcaR and re-

sults obtained in this study allow us suggesting that

TcaR exhibits functional similarity with halorhodopsin.

Halorhodopsin participates in maintenance of salt bal-

ance in bacteria, and, it could be assumed, that physi-

ological role of TcaR in cyanobacteria is similar. Con-

sidering that halorhodopsin is used in optogenetics,

TcaR could also find its application niche among the

used optogenetic tools. It seems that the TcaR protein

deserves more detailed and comprehensive investigation

including investigation of peculiarities of its photocycle,

as well as its structure.

Contributions. V.I.G. concept of the study; A.A.A.,

T.I.R., Y.N.A., S.M.B., F.M.Ts. conducting experi-

ments; Y.N.A., V.I.G., T.I.R. writing text of the paper.

All authors participated in discussion of the obtained re-

sults and editing of the final text of the paper.

Funding. This work was financially supported by

the Russian Science Foundation (grants 23-24-00038,

21-64-00018) and by the Ministry of Science and

Higher Education of the Russian Federation (project

AAAA-A19-119031390114-5).

Ethics declarations. The authors declare no conflict

of interests in financial or any other sphere. This article

does not contain any studies with human participants

or animals performed by any of the authors.

REFERENCES

1. Gushchin, I., and Gordeliy, V. (2018) Microbial rhodop-

sins, Subcell Biochem., 87, 19-56, doi: 10.1007/978-981-

10-7757-9_2.

ROKITSKAYA et al.1578

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

2. Gomez-Consarnau, L., Raven, J. A., Levine, N. M.,

Cutter, L.S., Wang, D., Seegers, B., Aristegui, J., Fuhr-

man, J. A., Gasol, J. M., and Sanudo-Wilhelmy, S. A.

(2019) Microbial rhodopsins are major contributors to the

solar energy captured in the sea, Sci. Adv., 8, eaaw8855,

doi:10.1126/sciadv.aaw8855.

3. Spudich, J. L., Sineshchekov, O. A., and Govorunova,

E.G. (2014) Mechanism divergence in microbial rhodop-

sins, Biochim. Biophys. Acta, 1837, 546-552, doi:10.1016/

j.bbabio.2013.06.006.

4. Mukherjee, S., Hegemann, P., and Broser, M. (2019)

Enzymerhodopsins: novel photoregulated catalysts for

optogenetics, Curr. Opin. Struct. Biol., 57, 118-126,

doi:10.1016/j.sbi.2019.02.003.

5. Kirpichnikov, M. P. and Ostrovskiy (2019) Optoge-

netics and vision, Herald Russ. Acad. Sci., 89, 34-38,

doi:10.1134/S1019331619010039.

6. Luecke, H., Schobert, B., Richter, H. T., Cartailler, J.P.,

and Lanyi, J.K. (1999) Structure of bacteriorhodopsin at

1.55A resolution, J.Mol. Biol., 291, 899-911, doi:10.1006/

jmbi.1999.3027.

7. Das, S., Singh, D., Madduluri, M., Chandrababunaidu,

M.M., Gupta, A., Adhikary, S.P., and Tripathy, S. (2015)

Draft genome sequence of bioactive-compound-produc-

ing cyanobacterium Tolypothrix campylonemoides strain

VB511288, Genome Announc., 3, e00226-15, doi:10.1128/

genomeA.00226-15.

8. Hasemi, T., Kikukawa, T., Watanabe, Y., Aizawa, T.,

Miyauchi, S., Kamo, N., and Demura, M. (2019) Pho-

tochemical study of a cyanobacterial chloride-ion pump-

ing rhodopsin, Biochim. Biophys. Acta, 1860, 136-146,

doi:10.1016/j.bbabio.2018.12.001.

9. Yun, J. H., Park, J. H., Jin, Z., Ohki, M., Wang, Y., Lupa-

la, C.S., Liu, H., Park, S.Y., and Lee, W. (2020) Struc-

ture-based functional modification study of a cyanobac-

terial chloride pump for transporting multiple anions,

J.Mol. Biol., 432, 5273-5286, doi:10.3390/ma13143061.

10. Astashkin, R., Kovalev, K., Bukhdruker, S., Vaganova,S.,

Kuzmin, A., Alekseev, A., Balandin, T., Zabelskii, D.,

Gushchin, I., Royant, A., Volkov, D., Bourenkov, G.,

Koonin, E., Engelhard, M., Bamberg, E., and Gordeliy, V.

(2022) Structural insights into light-driven anion pumping

in cyanobacteria, Nat. Commun., 13, 6460, doi:10.1038/

s41467-022-34019-9.

11. Oesterhelt, D., Tittor, J., and Bamberg, E. (1992) Aunify-

ing concept for ion translocation by retinal proteins, J.Bio-

energ. Biomembr., 24, 181-191, doi:10.1007/BF00762676.

12. Drachev, L. A., Jasaitis, A. A., Kaulen, A. D., Kondrash-

in, A. A., Liberman, E.A., Nemecek, I.B., Ostroumov,

S.A., Semenov, A.Yu., and Skulachev, V.P. (1974) Direct

measurement of electric current generation by cytochrome

oxidase, H

-ATPase and bacteriorhodopsin, Nature,

249, 321-324, doi:10.1038/249321a0.

13. Drachev, L. A., Frolov, V. N., Kaulen, A.D., Liberman,

E. A., Ostroumov, S. A., Plakunova, V. G., Semenov,

A.Y., and Skulachev, V.P. (1976) Reconstitution of bio-

logical molecular generators of electric current. Bacteri-

orhodopsin, J.Biol. Chem., 251, 7059-7065, doi:10.1016/

S0021-9258(17)32940-X.

14. Bamberg, E., Apell, H.J., Dencher, N.A., Sperling, W.,

Stieve, H., and Lauger, P. (1979) Photocurrents generated

by bacteriorhodopsin on planar bilayer membranes, Bio-

phys. Struct. Mech., 5, 277-292, doi:10.1007/BF02426663.

15. Bamberg, E., Butt, H. J., Eisenrauch, A., and Fend-

ler,K. (1993) Charge transport of ion pumps on lipid bi-

layer membranes, Q.Rev. Biophys., 26, 1-25, doi:10.1017/

s0033583500003942.

16. Friedrich, T., Geibel, S., Kalmbach, R., Chizhov, I.,

Ataka, K., Heberle, J., Engelhard, M., and Bamberg, E.

(2002) Proteorhodopsin is a light-driven proton pump

with variable vectoriality, J. Mol. Biol., 321, 821-838,

doi:10.1016/s0022-2836(02)00696-4.

17. Shevchenko, V., Mager, T., Kovalev, K., Polovinkin, V.,

Alekseev, A., Juettner, J., Chizhov, I., Bamann, C.,

Vavourakis, C., Ghai, R., Gushchin, I., Borshchevskiy,V.,

Rogachev, A., Melnikov, I., Popov, A., Balandin, T.,

Rodriguez-Valera, F., Manstein, D.J., Bueldt, G., Bam-

berg, E., and Gordeliy, V. (2017) Inward H

pump xe-

norhodopsin: mechanism and alternative optogenetic ap-

proach, Sci. Adv., 3, e1603187, doi:10.1126/sciadv.1603187.

18. Schuler, M. A., Denisov, I. G., and Sligar, S.G. (2013)

Nanodiscs as a new tool to examine lipid-protein inter-

actions, Methods Mol. Biol., 974, 415-433, doi: 10.1007/

978-1-62703-275-9_18.

19. Rokitskaya, T. I., Maliar, N. L., Siletsky, S. A., Gor-

deliy, V., and Antonenko, Y. N. (2022) Electrophysio-

logical characterization of microbial rhodopsin transport

properties: electrometric and ΔpH measurements us-

ing planar lipid bilayer, collodion film, and fluorescent

probe approaches, Methods Mol. Biol., 2501, 259-275,

doi:10.1007/978-1-0716-2329-9_12.

20. Rokitskaya, T. I., Maliar, N., Kovalev, K.V., Volkov, O.,

Gordeliy, V.I., and Antonenko, Y.N. (2021) Rhodopsin

channel activity can be evaluated by measuring the pho-

tocurrent voltage dependence in planar bilayer lipid mem-

branes, Biochemistry (Moscow), 86, 409-419, doi:10.1134/

S0006297921040039.

21. Selwyn, M. J., Dawson, A. P., Stockdale, M., and

Gains, N. (1970) Chloride-hydroxide exchange across

mitochondrial, erythrocyte and artificial lipid mem-

branes mediated by trialkyl-and triphenyltin compounds,

Eur. J. Biochem., 14, 120-126, doi: 10.1111/j.1432-1033.

1970.tb00268.x.

22. Antonenko, Y. N. (1990) Electrically silent anion transport

through bilayer lipid membrane induced by tributyltin and

triethyllead, J.Membr. Biol., 113, 109-113, doi:10.1007/

BF01872884.

23. Sato, T., Konno, H., Tanaka, Y., Kataoka, T., Nagai,K.,

Wasserman, H. H., and Ohkuma, S. (1998) Prodigios-

ins as a new group of H

/Cl

–

symporters that uncouple

proton translocators, J. Biol. Chem., 273, 21455-21462,

doi:10.1074/jbc.273.34.21455.

RETINAL-BASED ANION PUMP 1579

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

24. Bamberg, E., Hegemann, P., and Oesterhelt, D. (1984)

Reconstitution of the light-driven electrogenic ion-

pump halorhodopsin in black lipid membranes, Bio-

chim. Biophys. Acta, 773, 53-60, doi: 10.1016/0005-2736

(84)90549-2.

25. Bamberg, E., Tittor, J., and Oesterhelt, D. (1993)

Light-driven proton or chloride pumping by halorhodop-

sin, Proc. Natl. Acad. Sci. USA, 90, 639-643, doi:10.1073/

pnas.90.2.639.

26. Antonenko, Y. N., Denisov, S. S., Silachev, D. N.,

Khailova, L. S., Jankauskas, S. S., Rokitskaya, T. I.,

Danilina, T. I., Kotova, E. A., Korshunova, G. A.,

Plotnikov, E. Y., and Zorov, D. B. (2016) A long-linker

conjugate of fluorescein and triphenylphosphonium as

mitochondria-targeted uncoupler and fluorescent neu-

ro- and nephroprotector, Biochim. Biophys. Acta, 1860,

2463-2473, doi:10.1016/j.bbagen.2016.07.014.

27. Steiner, M., Oesterhelt, D., Ariki, M., and Lanyi,

J. K. (1984) Halide binding by the purified halorho-

dopsin chromoprotein. I. Effects on the chromophore,

J.Biol. Chem., 259, 2179-2184, doi:10.1016/S0021-9258

(17)43334-5.