3.E.1 The Ion-translocating Microbial Rhodopsin (MR) Family

Members of the MR family catalyze light-driven ion translocation across microbial cytoplasmic membranes or serve as light receptors. Among the high resolution structures for members of the MR family are the archaeal proteins, bacteriorhodopsin (Luecke et al., 1999), sensory rhodopsin II (Royant et al., 2001) and halorhodopsin (Kolbe et al., 2000) as well as an Anabaena cyanobacterial sensory rhodopsin (3.E.1.8.a) (Vogeley et al., 2004). Homologues include putative fungal chaparone proteins, a retinal-containing rhodopsin from Neurospora crassa (Maturana et al., 2001), a H+-pumping rhodopsin from Leptosphaeria maculans (Waschuk et al., 2005), retinal-containing proton pumps isolated from marine bacteria (Béjà et al., 2000), a green light-activated photoreceptor in cyanobacteria that does not pump ions and interacts with a small (14 kDa) soluble transducer protein (Jung et al., 2003; Vogeley et al., 2004) and light-gated H+ channels from the green alga, Chlamydomonas reinhardtii (Nagel et al., 2002). The N. crassa NOP-1 protein exhibits a photocycle and conserved H+ translocation residues that suggest that this putative photoreceptor is a slow H+ pump (Brown et al., 2001; see also Brown, 2004 and Waschuk et al., 2005). Allosteric structural changes in the photocycle are mediated by a sliding movement of a transmembrane helix (Takeda et al. 2004). MR proteins such as SRII exhibit fast internal motion and residual conformational entropy (O'Brien et al. 2020). Procedures for the formation of thin (mono-) and thick (multi-) layers from materials containing BR and BR/nanoparticle hybrids have been reviewed (Oleinikov et al. 2020) and their usefulness in optogenetic studies have been reviewed (Kandori 2021).  The molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in anion- and cation-selective channelrhodopsins have been defined (Govorunova et al. 2021). Concerted motions and molecular functions of Llight-driven ion-pumping rhodopsins have been reviewed (Mizutani 2021). An outward proton pumping rhodopsin with a record in thermostability has been made by amino acid mutations (Yasuda et al. 2022). Dynamic aspects of bacteriorhodopsin as a typical membrane protein have been studied by  site-directed solid-state 13C NMR (Saitô et al. 2004). Ion-pumping microbial rhodopsin proteins have been classified using a machine learning approach (Selvaraj et al. 2023). The surface proton current observed in bacteriorhodopsin purple membranes has been explained (Silverstein 2023). There has been widespread use of proton-pumping rhodopsin in Antarctic phytoplankton (Andrew et al. 2023).

The Anabaena sensory rhodopsin exhibits light-induced interconversion between 13-cis and all trans states (Vogeley et al., 2004). The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis. Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor's soluble 14-kilodalton transducer.

Most proteins of the MR family are all of about the same size (250-350 amino acyl residues) and possess seven TMSs with their N-termini on the outside and their C-termini on the inside. There are 8 subfamilies in the MR family: (1) bacteriorhodopsins pump protons out of the cell; (2) halorhodopsins pump chloride (and other anions such as bromide, iodide and nitrate) into the cell; (3) sensory rhodopsins, which normally function as receptors for phototactic behavior, are capable of pumping protons out of the cell if dissociated from their transducer proteins; (4) the fungal chaparones are stress-induced proteins of ill-defined biochemical function, but this subfamily also includes a H+-pumping rhodopsin (Waschuk et al., 2005); (5) the bacterial rhodopsin, called proteorhodopsin, is a light-driven proton pump that functions as does bacteriorhodopsins; (6) the N. crassa retinal-containing receptor serves as a photoreceptor (Zhai et al., 2001); (7) the green algal light-gated proton channel, channelrhodpsin-1, (8) sensory rhodopsins from cyanobacteria and (9) light-activated rhodopsin guanylyl cyclases. A phylogenetic analysis of microbial rhodopsins and a detailed analysis of potential examples of horizontal gene transfer have been published (Sharma et al., 2006). Microbial rhodopsins have a Trp residue in the middle of TMS3, which is homologous to W86 of bacteriorhodopsin (BR), is well conserved among microbial rhodopsins with various light-driven functions, and it serves as a gate-keeper in many microbial rhodopsins (Nagasaka et al. 2020). Roles of functional lipids in the bacteriorhodopsin photocycle in various delipidated purple membranes have been examined (Zhong et al. 2022).

Bacterio- and halorhodopsins pump 1 H+ and 1 Cl- per photon absorbed, respectively. Specific transport mechanisms and pathways have been proposed (see Kolbe et al., 2000; Lanyi and Schobert, 2003; Schobert et al., 2003). The mechanism involves (1) photo-isomerization of the retinal and its initial configurational changes, (2) deprotonation of the retinal Schiff base and the coupled release of a proton to the extracellular membrane surface, and (3) the switch event that allows reprotonation of the Schiff base from the cytoplasmic side. Six structural models describe the transformations of the retinal and its interaction with water 402, Asp85, and Asp212 in atomic detail, as well as the displacements of functional residues farther from the Schiff base. The changes provide rationales for how relaxation of the distorted retinal causes movements of water and protein atoms that result in vectorial proton transfers to and from the Schiff base (Lanyi and Schobert, 2003). Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin (Royant et al., 2000). Bacteriorhodopsin activity as a function of its local environment has been quantified with a raman-based assay (Leighton and Frontiera 2023).

A marine bacterial rhodopsin has been reported to function as a proton pump. However, it most closely resembles sensory rhodopsin II of archaea as well as an Orf from the fungus Leptosphaeria maculans (AF290180). These proteins exhibit 20-30% identity with each other.  Sensory rhodopsins are widespread in the microbial world, but they exhibit different modes of signaling in different organisms, including interaction with other membrane proteins, interaction with cytoplasmic transducers and light-controlled Ca2+ channel activity. Work on cyanobacteria, algae, fungi and marine proteobacteria has shown that the common design of these proteins allows rich diversity in their signaling mechanisms (Spudich 2006). Kwon et al. 2020 describe a class of bacterial rhodopsins containing the 3 omega motif. This motif forms a stack of three nonconsecutive aromatic amino acids that correlates with the B-C loop orientation, and is shared among the phylogenetically close ion pumps such as the NDQ motif-containing sodium-pumping rhodopsin, the NTQ motif-containing chloride-pumping rhodopsin, and some proton-pumping rhodopsins including xanthorhodopsin.  Kwon et al. 2020 reviewed research on these omega rhodopsins.

The association of sensory rhodopsins with their transducer proteins appears to determine whether they function as transporters or receptors. Association of a sensory rhodopsin receptor with its transducer occurs via the transmembrane helical domains of the two interacting proteins. There are two sensory rhodopsins in any one halophilic archaeon, one (SRI) that responds positively to orange light but negatively to blue light, the other (SRII) that responds only negatively to blue light. Each transducer is specific for its cognate receptor. An x-ray structure of SRII complexed with its transducer (HtrII) at 1.94 Å resolution is available (Gordelly et al., 2002).  Molecular and evolutionary aspects of the light-signal transduction by microbial sensory receptors have been reviewed (Inoue et al. 2014). 

Sol-gel immobilization of proteins in transparent inorganic matrices provide a liposomal system in which the liposome provides membrane structure. Two transmembrane proteins, bacteriorhodopsin (bR) and F0F1-ATP synthase have been incorporated into such a matrix called proteogels; if containing only bRho, a stable proton gradient forms when irradiated with visible light, whereas proteogels containing proteoliposomes with both bRho and an F0F1-ATP synthase couple the photo-induced proton gradient to the production of ATP (Luo et al. 2005). Thus, the liposome/sol-gel architecture can harness the properties of transmembrane proteins and enable a variety of applications, from power generation and energy storage to the powering of molecular motors.

Channelrhodopsin-1 (ChR1) or channelopsin-1 (Chop1; Cop3; CSOA) of C. reinhardtii is most closely related to the archaeal sensory rhodopsins. It has 712 aas with a signal peptide, followed by a short amphipathic region, and then a hydrophobic N-terminal domain with seven probable TMSs (residues 76-309) followed by a long hydrophilic C-terminal domain of about 400 residues. Part of the C-terminal hydrophilic domain is homologous to intersectin (EH and SH3 domain protein 1A) of animals (AAD30271).

Chop1 serves as a light-gated proton channel and mediates phototaxis and photophobic responses in green algae (Nagel et al., 2002). Based on this phenotype, Chop1 could be assigned to TC category #1.A, but because it belongs to a family in which well-characterized homologues catalyze active ion transport, it is assigned to the MR family. Expression of the chop1 gene, or a truncated form of this gene encoding only the hydrophobic core (residues 1-346 or 1-517) in frog oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel, passively but selectively permeable to protons. This channel activity may generate bioelectric currents (Nagel et al., 2002).

A homologue of ChR1 in C. reinhardtii is channelrhodopsin-2 (ChR2; Chop2; Cop4; CSOB). This protein is 57% identical, 10% similar to ChR1. It forms a cation-selective ion channel activated by light absorption. It transports both monovalent and divalent cations. It desensitizes to a small conductance in continuous light. Recovery from desensitization is accelerated by extracellular H+ and a negative membrane potential. It may be a photoreceptor for dark adapted cells (Nagel et al., 2003). A transient increase in hydration of transmembrane α-helices with a t(1/2) = 60 μs tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t(1/2) = 10 μs), with the latter being reprotonated by aspartic acid 156 (t(1/2) = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. E90 deprotonates exclusively in the nonconductive state. The observed proton transfer reactions and the protein conformational changes relate to the gating of the cation channel (Lórenz-Fonfría et al. 2013).

Most of the MR family homologues in yeast and fungi are of about the same size and topology as the archaeal proteins (283-344 amino acyl residues; 7 putative transmembrane α-helical segments), but they are heat shock- and toxic solvent-induced proteins of unknown biochemical function. They have been suggested to function as pmf-driven chaperones that fold extracellular proteins (Zhai et al., 2001), but only indirect evidence supports this postulate. The MR family is distantly related to the 7 TMS LCT family (TC #2.A.43) (Zhai et al., 2001). It is a part of the TOG superfamily which includes G-protein coupled receptors (GPCRs) (Yee et al. 2013), and the conclusioin of homology between MRs and GPCRs has been extensively confirmed (Shalaeva et al. 2015).

Archaerhodopsin-2 (aR2), a retinal protein-carotenoid complex found in the claret membrane of Halorubrum sp. aus-2, functions as a light-driven proton pump. Trigonal and hexagonal crystals revealed that trimers are arranged on a honeycomb lattice (Yoshimura and Kouyama, 2008). In these crystals, the carotenoid bacterioruberin binds to crevices between the subunits of the trimer. Its polyene chain is inclined from the membrane normal by an angle of about 20 degrees and, on the cytoplasmic side, it is surrounded by helices AB and DE of neighbouring subunits. This peculiar binding mode suggests that bacterioruberin plays a structural role for the trimerization of aR2. When compared with the aR2 structure in another crystal form containing no bacterioruberin, the proton release channel takes a more closed conformation in the P321 or P6(3) crystal; i.e., the native conformation of protein is stabilized in the trimeric protein-bacterioruberin complex.

A crystallographic structure of xanthorhodopsin at 1.9 Å resolution revealed a dual chromophore, the geometry of the carotenoid and the retinal (Luecke et al., 2008). The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as approximately 45%, and the 46 degrees angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer. At 1.9 Å resolution, the structure revealed a light-driven proton pump with a dual chromophore.  Ion-transporting rhodopsins of marine bacteria have been reviewed (Inoue et al. 2014).

Most residues participating in the trimerization are not conserved in bacteriorhodopsin, a homologous protein capable of forming a trimeric structure in the absence of bacterioruberin. Despite a large alteration in the amino acid sequence, the shape of the intratrimer hydrophobic space filled by lipids is highly conserved between aR2 and bacteriorhodopsin. Since a transmembrane helix facing this space undergoes a large conformational change during the proton pumping cycle, it is feasible that trimerization is an important strategy to capture special lipid components that are relevant to the protein activity (Yoshimura and Kouyama, 2008).

Ion-pumping bacterial rhodopsins functioning as outward H+ or Na+ and inward Cl- pumps convert light energy into transmembrane electrochemical potential differences. The H+, Na+, and Cl- pumps possess conserved respective DTE, NDQ, and NTQ motifs in helices C, which likely serve as their functional determinants, and this has been verified (Inoue et al. 2016). Phylogenetic analyses suggested that a H+ pump was the common ancestor from which Cl- pumps emerged followed by Na+ pumps. Inoue et al. 2016 proposed that successful functional conversion was achieved when these amino acid sequences changed, possibly accompanied by other changes. 

Nango et al. 2016 used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bRho from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bRho transports protons uphill against a transmembrane concentration gradient.

Brho (BR) has light-independent lipid scramblase activity (Verchère et al. 2017). This activity occurs  at a rate >10,000 per trimer per second, comparable to that of other scramblases including bovine rhodopsin and fungal TMEM16 proteins. BR scrambles fluorescent analogues of common phospholipids but does not transport a glycosylated diphosphate isoprenoid lipid. In silico analyses suggested that membrane-exposed polar residues in transmembrane helices 1 and 2 of BR may provide the molecular basis for lipid translocation by coordinating the polar head-groups of transiting phospholipids. Consistent with this possibility, molecular dynamics simulations of a BR trimer in a phospholipid membrane revealed water penetration along transmembrane helix 1 with the cooperation of a polar residue (Y147 in transmembrane helix 5) in the adjacent protomer. These findings suggest that the lipid translocation pathway may lie at or near the interface of the protomers of the BR trimer (Verchère et al. 2017). 

Electronic current passes through bR-containing artificial lipid bilayers in solid 'electrode-bilayer-electrode' structures. The current through the protein is more than four orders of magnitude higher than would be estimated for direct tunneling through 5-nm water-free peptides. Jin et al. 2006 found that electron transport (ET) occurs only if retinal or a close analogue is present in the protein. As long as the retinal can isomerize after light absorption, there is a photo-ET effect. The contribution of light-driven proton pumping to the steady-state photocurrents is negligible. Possibly this is relevant to the early evolutionary origin of halobacteria (Jin et al. 2006).

Parvularcula oceani xenorhodopsin (PoXeR) was the first light-driven inward proton pump with a brho topology and structure, binding retinal to TMS 7. Ultrafast pump-probe spectroscopy revealed that the isomerization time of retinal is 1.2 ps, considerably slower than those of other microbial rhodopsins (180-770 fs). Following the production of J, the K intermediate was formed at 4 ps. Proton transfer occurred on a slower time-scale. While a proton was released from Asp216 into the cytoplasm, no proton-donating residue was identified on the extracellular side. A branched retinal isomerization (from 13-cis-15-anti to 13-cis-15-syn and all-trans-15-anti) occurred simultaneously with proton uptake. Thus, retinal isomerization is the rate-limiting process in proton uptake, and the regulation of pKa of the retinal Schiff base by thermal isomerization enables uptake from the extracellular medium (Inoue et al. 2018). Tamogami 2023 introduce a useful experimental method for measuring rapid transient pH changes with photoinduced proton uptake/release using transparent tin oxide (SnO2) or indium-tin oxide (ITO) electrodes. The unique pH-dependent behavior of the photoinduced proton transfer sequence as well as the vectoriality of proton transport in proteorhodopsin (PR) from marine eubacteria was also described. Through intensive ITO experiments over a wide pH range, in combination with photoelectric measurements using Xenopus oocytes or a thin polymer film 'Lumirror,' they made several interesting observations on photoinduced proton transfer in PR: 1) proton uptake/release sequence reversal and potential proton translocation direction reversal under alkali conditions, and 2) fast proton release from D227, a secondary counterion of the protonated retinal Schiff base at acidic pH values (Tamogami 2023).

Rhodopsins with enzymatic activity are present in microbes; three different types are known: light-activated guanylyl cyclase opsins (Cyclop) in fungi (TC# 3.E.1.5.1), light-inhibited two-component guanylyl cyclase opsins (2c-Cyclop) in green algae, and rhodopsin phosphodiesterases (RhoPDE) in choanoflagellates (TC# 3.E.1.5.2) (Tian et al. 2022). They are integral membrane proteins with eight TMSs, different from the other microbial (type I) rhodopsins with 7 TMSs. A classification as type Ib rhodopsins for opsins with 8 TMSs and type Ia for the ones with 7 TMSs has been proposed (Tian et al. 2022). Kojima and Sudo 2023 propposed that animal and microbial rhodopsins convergently evolved from their distinctive origins as multi-colored retinal-binding membrane proteins whose activities are regulated by light and heat but independently evolved for different molecular and physiological functions in the cognate organism. However, bioinformatic research in the Saier lab suggested that these proteins all evolved from a common ancestor (Yee et al. 2013; Shlykov et al. 2012).

New sensory rhodopsins, resembling proteorhodopsins (see TC# 3.E.1.6), display many unusual amino acid residues, including those around the retinal chromophore; most strikingly, a tyrosine in place of a carboxyl counterion of the retinal Schiff base on helix C (Saliminasab et al. 2023). Experimental data, bioinformatic sequence analyses, and structural modeling suggest that the tyrosine/aspartate complex counterion contributes to a complex water-mediated hydrogen-bonding network that couples the protonated retinal Schiff base to an extracellular carboxylic dyad. These SRs interact with Htr-like transducers (Saliminasab et al. 2023) but show greater sequence similarity with proteorhodopsins that archaeal sensory rhodopsins that also interact with Htr-like transducers (Saliminasab et al. 2023)

The generalized transport reaction for bacterio- (and some sensory) rhodopsins is:

H+ (in) + hν → H+ (out)

That for halorhodopsin is:

Cl- (out) + hν → Cl- (in)

That for xenorhodopsin is:

H+ (out) + hν  → H+ (in)

 



This family belongs to the Transporter-Opsin-G protein-coupled receptor (TOG) Superfamily.

 

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Shionoya, T., M. Singh, M. Mizuno, H. Kandori, and Y. Mizutani. (2021). Strongly Hydrogen-Bonded Schiff Base and Adjoining Polyene Twisting in the Retinal Chromophore of Schizorhodopsins. Biochemistry 60: 3050-3057.

Shlykov, M.A., W.H. Zheng, J.S. Chen, and M.H. Saier, Jr. (2012). Bioinformatic characterization of the 4-Toluene Sulfonate Uptake Permease (TSUP) family of transmembrane proteins. Biochim. Biophys. Acta. 1818: 703-717.

Siletsky, S.A., M.D. Mamedov, E.P. Lukashev, S.P. Balashov, D.A. Dolgikh, A.B. Rubin, M.P. Kirpichnikov, and L.E. Petrovskaya. (2016). Electrogenic steps of light-driven proton transport in ESR, a retinal protein from Exiguobacterium sibiricum. Biochim. Biophys. Acta. 1857: 1741-1750.

Silverstein, T.P. (2023). Lee''s transient protonic capacitor cannot explain the surface proton current observed in bacteriorhodopsin purple membranes. Biophys Chem 301: 107096.

Sineshchekov, O.A., E.G. Govorunova, H. Li, Y. Wang, and J.L. Spudich. (2023). Channel Gating in Kalium Channelrhodopsin Slow Mutants. J. Mol. Biol. 168298. [Epub: Ahead of Print]

Sineshchekov, O.A., Trivedi, V.D., Sasaki, J., and J.L. Spudich. (2005). Photochromicity of Anabaena sensory rhodopsin, an atypical microbial receptor with a cis-retinal light-adapted form. J. Biol. Chem. 280: 14663-14668.

Soto-Rodríguez, J. and F. Baneyx. (2018). Role of the Signal Sequence in Proteorhodopsin Biogenesis in E. coli. Biotechnol Bioeng. [Epub: Ahead of Print]

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Suomivuori, C.M., A.P. Gamiz-Hernandez, D. Sundholm, and V.R.I. Kaila. (2017). Energetics and dynamics of a light-driven sodium-pumping rhodopsin. Proc. Natl. Acad. Sci. USA. [Epub: Ahead of Print]

Takabatake, A., N. Kawazoe, and S. Izawa. (2015). Plasma membrane proteins Yro2 and Mrh1 are required for acetic acid tolerance in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 99: 2805-2814.

Takeda, K., Y. Matsui, N. Kamiya, S. Adachi, H. Okumura, and T. Kouyama. (2004). Crystal structure of the M intermediate of bacteriorhodopsin: allosteric structural changes mediated by sliding movement of a transmembrane helix. J. Mol. Biol. 341: 1023-1037.

Tamogami, J. (2023). [A Study on Mechanisms Underlying Proton Transport in Proton Pump-type Microbial Rhodopsins]. Yakugaku Zasshi 143: 111-118.

Taniguchi, Y., T. Ikehara, N. Kamo, H. Yamasaki, and Y. Toyoshima. (2007). Dynamics of light-induced conformational changes of the phoborhodopsin/transducer complex formed in the n-dodecyl β-D-maltoside micelle. Biochemistry 46: 5349-5357.

Tanimoto S., Sugiyama Y., Takahashi T., Ishizuka T. and Yawo H. (2013). Involvement of glutamate 97 in ion influx through photo-activated channelrhodopsin-2. Neurosci Res. 75(1):13-22.

Tashiro, R., K. Sushmita, S. Hososhima, S. Sharma, S. Kateriya, H. Kandori, and S.P. Tsunoda. (2021). Specific residues in the cytoplasmic domain modulate photocurrent kinetics of channelrhodopsin from Klebsormidium nitens. Commun Biol 4: 235.

Tian, Y., S. Gao, and G. Nagel. (2022). In Vivo and In Vitro Characterization of Cyclase and Phosphodiesterase Rhodopsins. Methods Mol Biol 2501: 325-338.

Tomita, H., E. Sugano, N. Murayama, T. Ozaki, F. Nishiyama, K. Tabata, M. Takahashi, T. Saito, and M. Tamai. (2014). Restoration of the majority of the visual spectrum by using modified Volvox channelrhodopsin-1. Mol Ther 22: 1434-1440.

Tsukamoto, T., K. Mizutani, T. Hasegawa, M. Takahashi, N. Honda, N. Hashimoto, K. Shimono, K. Yamashita, M. Yamamoto, S. Miyauchi, S. Takagi, S. Hayashi, T. Murata, and Y. Sudo. (2016). X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION. J. Biol. Chem. 291: 12223-12232.

Tsunoda, S.P., D. Ewers, S. Gazzarrini, A. Moroni, D. Gradmann, and P. Hegemann. (2006). H+ -pumping rhodopsin from the marine alga Acetabularia. Biophys. J. 91: 1471-1479.

Umegawa, Y., S. Kawatake, M. Murata, and S. Matsuoka. (2023). Combined effect of the head groups and alkyl chains of archaea lipids when interacting with bacteriorhodopsin. Biophys Chem 294: 106959.

Urui, T., K. Hayashi, M. Mizuno, K. Inoue, H. Kandori, and Y. Mizutani. (2024). - Reisomerization Preceding Reprotonation of the Retinal Chromophore Is Common to the Schizorhodopsin Family: A Simple and Rational Mechanism for Inward Proton Pumping. J Phys Chem B 128: 744-754.

Verchère, A., W.L. Ou, B. Ploier, T. Morizumi, M.A. Goren, P. Bütikofer, O.P. Ernst, G. Khelashvili, and A.K. Menon. (2017). Light-independent phospholipid scramblase activity of bacteriorhodopsin from Halobacterium salinarum. Sci Rep 7: 9522.

Vogeley, L., O.A. Sineshchekov, V.D. Trivedi, J. Sasaki, J.L. Spudich, and H. Luecke. (2004). Anabaena sensory rhodopsin: a photochromic color sensor at 2.0 Å. Science 306: 1390-1393.

Volkov, O., K. Kovalev, V. Polovinkin, V. Borshchevskiy, C. Bamann, R. Astashkin, E. Marin, A. Popov, T. Balandin, D. Willbold, G. Büldt, E. Bamberg, and V. Gordeliy. (2017). Structural insights into ion conduction by channelrhodopsin 2. Science 358:.

Volz, P., N. Krause, J. Balke, C. Schneider, M. Walter, F. Schneider, R. Schlesinger, and U. Alexiev. (2016). Light and pH-induced changes in structure and accessibility of transmembrane helix B and its immediate environment in Channelrhodopsin-2. J. Biol. Chem. [Epub: Ahead of Print]

Ward, M.E., L. Shi, E. Lake, S. Krishnamurthy, H. Hutchins, L.S. Brown, and V. Ladizhansky. (2011). Proton-detected solid-state NMR reveals intramembrane polar networks in a seven-helical transmembrane protein proteorhodopsin. J. Am. Chem. Soc. 133: 17434-17443.

Waschuk, S.A., A.G. Bezerra, Jr., L. Shi, and L.S. Brown. (2005). Leptosphaeria rhodopsin: bacteriorhodopsin-like proton pump from a eukaryote. Proc. Natl. Acad. Sci. USA 102: 6879-6883.

Watanabe, S., T. Ishizuka, S. Hososhima, A. Zamani, M.R. Hoque, and H. Yawo. (2016). The regulatory mechanism of ion permeation through a channelrhodopsin derived from Mesostigma viride (MvChR1). Photochem Photobiol Sci 15: 365-374.

Watanabe, Y., E. Sugano, K. Tabata, A. Hatakeyama, T. Sakajiri, T. Fukuda, T. Ozaki, T. Suzuki, T. Sayama, and H. Tomita. (2021). Development of an optogenetic gene sensitive to daylight and its implications in vision restoration. NPJ Regen Med 6: 64.

Weinert, T., P. Skopintsev, D. James, F. Dworkowski, E. Panepucci, D. Kekilli, A. Furrer, S. Brünle, S. Mous, D. Ozerov, P. Nogly, M. Wang, and J. Standfuss. (2019). Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography. Science 365: 61-65.

Wietek, J., J.S. Wiegert, N. Adeishvili, F. Schneider, H. Watanabe, S.P. Tsunoda, A. Vogt, M. Elstner, T.G. Oertner, and P. Hegemann. (2014). Conversion of channelrhodopsin into a light-gated chloride channel. Science 344: 409-412.

Wu, K., J.H. Dawe, and J.P. Aris. (2000). Expression and subcellular localization of a membrane protein related to Hsp30p in Saccharomyces cerevisiae. Biochim. Biophys. Acta. 1463: 477-482.

Yang, J., L. Zu, G. Li, C. Zhang, Z. Ge, W. Wang, X. Wang, B. Liu, N. Xi, and L. Liu. (2023). Upconversion optogenetics-driven biohybrid sensor for infrared sensing and imaging. Acta Biomater. [Epub: Ahead of Print]

Yang, Q. and D. Chen. (2023). Na Binding and Transport: Insights from Light-Driven Na-Pumping Rhodopsin. Molecules 28:.

Yang, T., W. Zhang, J. Cheng, Y. Nie, Q. Xin, S. Yuan, and Y. Dou. (2019). Formation Mechanism of Ion Channel in Channelrhodopsin-2: Molecular Dynamics Simulation and Steering Molecular Dynamics Simulations. Int J Mol Sci 20:.

Yasuda, S., T. Akiyama, K. Kojima, T. Ueta, T. Hayashi, S. Ogasawara, S. Nagatoishi, K. Tsumoto, N. Kunishima, Y. Sudo, M. Kinoshita, and T. Murata. (2022). Development of an Outward Proton Pumping Rhodopsin with a New Record in Thermostability by Means of Amino Acid Mutations. J Phys Chem B 126: 1004-1015.

Yee, D.C., M.A. Shlykov, A. Västermark, V.S. Reddy, S. Arora, E.I. Sun, and M.H. Saier, Jr. (2013). The transporter-opsin-G protein-coupled receptor (TOG) superfamily. FEBS J. 280: 5780-5800.

Yeh, V., T.Y. Lee, C.W. Chen, P.C. Kuo, J. Shiue, L.K. Chu, and T.Y. Yu. (2018). Highly Efficient Transfer of 7TM Membrane Protein from Native Membrane to Covalently Circularized Nanodisc. Sci Rep 8: 13501.

Yoshimura, K. and T. Kouyama. (2008). Structural role of bacterioruberin in the trimeric structure of archaerhodopsin-2. J. Mol. Biol. 375: 1267-1281.

Zabelskii, D., A. Alekseev, K. Kovalev, V. Rankovic, T. Balandin, D. Soloviov, D. Bratanov, E. Savelyeva, E. Podolyak, D. Volkov, S. Vaganova, R. Astashkin, I. Chizhov, N. Yutin, M. Rulev, A. Popov, A.S. Eria-Oliveira, T. Rokitskaya, T. Mager, Y. Antonenko, R. Rosselli, G. Armeev, K. Shaitan, M. Vivaudou, G. Büldt, A. Rogachev, F. Rodriguez-Valera, M. Kirpichnikov, T. Moser, A. Offenhäusser, D. Willbold, E. Koonin, E. Bamberg, and V. Gordeliy. (2020). Viral rhodopsins 1 are an unique family of light-gated cation channels. Nat Commun 11: 5707.

Zabelskii, D., N. Dmitrieva, O. Volkov, V. Shevchenko, K. Kovalev, T. Balandin, D. Soloviov, R. Astashkin, E. Zinovev, A. Alekseev, E. Round, V. Polovinkin, I. Chizhov, A. Rogachev, I. Okhrimenko, V. Borshchevskiy, V. Chupin, G. Büldt, N. Yutin, E. Bamberg, E. Koonin, and V. Gordeliy. (2021). Structure-based insights into evolution of rhodopsins. Commun Biol 4: 821.

Zamani, A., S. Sakuragi, T. Ishizuka, and H. Yawo. (2017). Kinetic characteristics of chimeric channelrhodopsins implicate the molecular identity involved in desensitization. Biophys Physicobiol 14: 13-22.

Zhai, Y., W.H.M. Heijne, D.W. Smith, and M.H. Saier, Jr. (2001). Homologues of archaeal rhodopsins in plants, animals and fungi: structural and functional predications for a putative fungal chaperone protein. Biochim. Biophys. Acta 1511: 206-223.

Zhang, W., A. Brooun, M.M. Mueller, and M. Alam. (1996). The primary structures of the Archaeon Halobacterium salinarium blue light receptor sensory rhodopsin II and its transducer, a methyl-accepting protein. Proc. Natl. Acad. Sci. USA 93: 8230-8235.

Zhang, W., T. Yang, S. Zhou, J. Cheng, S. Yuan, G.V. Lo, and Y. Dou. (2019). Molecular Dynamics Simulation of Transmembrane Transport of Chloride Ions in Mutants of Channelrhodopsin. Biomolecules 9:.

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Zhong, Y.R., T.Y. Yu, and L.K. Chu. (2022). Roles of functional lipids in bacteriorhodopsin photocycle in various delipidated purple membranes. Biophys. J. 121: 1789-1798.

Examples:

TC#NameOrganismal TypeExample
3.E.1.1.1

Bacteriorhodopsin. Proton efflux occurs via a transient linear water-molecule chain in a hydrophobic section of the Brho channel between Asp96 and Asp85 (Freier et al., 2011). Local-global conformational coupling in brho has been proposed as a transport mechanism from crystal structures of the nine states in the bacteriorhodopsin photocycle (Lanyi and Schobert 2004). The protein can be converted into a chloride uptake pump by a single amino acid substitution at position 85.  However, halorhodopsin (3.E.1.2.1), which pumps chloride ions (Cl-) into the cell, apparently does not use hydrogen-bonded water molecules for Cl- transport (Muroda et al. 2012).  Nango et al. 2016 used time-resolved serial femtosecond crystallography and an x-ray free electron laser to visualize conformational changes in bRho from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bRho transports protons uphill against a transmembrane concentration gradient. Nango et al. 2016 have created a 3-d movie of structural changes in the protein showing that an initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. Brho has light-independent lipid scramblase activity (Verchère et al. 2017). This activity occurs  at a rate >10,000 per trimer per second, comparable to that of other scramblases including bovine rhodopsin and fungal TMEM16 proteins. BR scrambles fluorescent analogues of common phospholipids but does not transport a glycosylated diphosphate isoprenoid lipid. In silico analyses suggested that membrane-exposed polar residues in transmembrane helices 1 and 2 of BR may provide the molecular basis for lipid translocation by coordinating the polar head-groups of transiting phospholipids. Consistent with this possibility, extensive coarse-grained molecular dynamics simulations of a BR trimer in a phospholipid membrane revealed water penetration along transmembrane helix 1 with the cooperation of a polar residue (Y147 in transmembrane helix 5) in the adjacent protomer. These findings suggest that the lipid translocation pathway may lie at or near the interface of the protomers of the BR trimer (Verchère et al. 2017). Retinal isomerization has been observed in the using a femtosecond x-ray laser (Nogly et al. 2018). S-TGA-1, a halobacterium-derived glycolipid, has the highest specificity to bRho, with a nanomolar dissociation constant (Inada et al. 2019). Weinert et al. 2019 recorded the structural changes in bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release. From 10 to 15 milliseconds onwards, large additional structural rearrangements, up to 9 Å on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid-96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle (Weinert et al. 2019).  The effect of membrane composition on the orientation and activity of bR has been reported (Palanco et al. 2017).  Efficient transfer of bRho from native membranes to covalently circularized nanodiscs has been accomplished (Yeh et al. 2018). The oligomeric status of BRho plays a role in the photocycle associated with short-range processes, such as retinal isomerization and deprotonation of the protonated Schiff base at the retinal pocket (Kao et al. 2019). Functional bacteriorhodopsin is efficiently solubilized and delivered to membranes by the chaperonin, GroEL (Deaton et al. 2004). Thirty one unfolded bR states in the presence of the retinal chromophore have been identified during denaturation (Jacobson et al. 2020). A transient protonic capacitor explains the bacteriorhodopsin membrane experiment of Heberle et al. 1994. Thus, after proton release by an integral membrane protein, long-range proton transfer along the membrane surface is faster than proton exchange with the bulk water phase  as predicted by the TELP theory (Lee 2023). The head groups and alkyl chains of phospholipids are essential for boundary lipids and greatly influence the biological function of bRho (Umegawa et al. 2023).

Archaea

Bacteriorhodopsin of Halobacterium salinarum

 
3.E.1.1.10

Schizorhodopsin of 202 aas and 7 TMSs. The crystal structure reveals the mechanism of inward proton pumping (Higuchi et al. 2021). Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H+ pumps (Higuchi et al. 2021). Strongly hydrogen-bonded Schiff base and adjoining polyene twisting in the retinal chromophore of schizorhodopsins has been observed (Shionoya et al. 2021).

Szr of Candidatus Lokiarchaeota archaeon

 
3.E.1.1.11

Mastigocladopsis repens rhodopsin chloride pump (inward pumping).  The 3-d structure is known to 3.3 Å resolution (Besaw et al. 2020). It may transport both Cl- and Br- (Kikukawa 2021).

Rhodopsin chloried pump of Mastigocladopsis repens

 
3.E.1.1.13

Xenorhodopsinof 229 aas and 7 TMSs.  The structures have been determined for all intermediates of the photocycle;  7ZNB, C, D, E, G, H and I, all A - C. Thus, a comprehensive function-structure study of the light-driven bacterial inward proton pump, xenorhodopsin from Bacillus coahuilensis, in all major proton-conducting states was carried out (Kovalev et al. 2023). The structures revealed that proton translocation is based on proton wires regulated by internal gates. The wires serve as both selectivity filters and the translocation pathways for protons. The cumulative results suggest a general concept of proton translocation. Kovalev et al. 2023 demonstrated the use of serial time-resolved crystallography at a synchrotron source with sub-millisecond resolution for rhodopsin studies, opening the door for principally new applications. 

Xenorhodopsin of Bacillus coahuilensis

 

 
3.E.1.1.14

Synechocystis halorhodopsin of 234 aas and 7 TMSs.  The 3-D structure is known (Astashkin et al. 2022). MRs can pump various monovalent ions like Na+, K+, Cl-, I-, and NO3-. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR).  Astashkin et al. 2022 presented the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. The data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics (Astashkin et al. 2022).

SyHR of Synechocystis sp. PCC 7509

 
3.E.1.1.15

Schizorhodopsin 4 of 110 aas and 7 TMSs.  The protein pumps H+ into the cytoplasm of the cell.  Cis-trans reisomerization preceding reprotonation of the retinal chromophore Is common to the schizorhodopsin family. A simple and rational mechanism for inward proton pumping has been proosed (Urui et al. 2024), and the 3-D structure is known (Higuchi et al. 2021).

Schizorhodopsin 4 of an Asgard group archaeon

 
3.E.1.1.16

Microbial (bacterial) rhodopsin, Bop2, of 246 aas and 7 TMSs.  It has no proton-pumping activity but is potentially capable of functioning as a sensory SRII-like protein. The chromophore contains 36.5% all-trans-, 7.6% 11-cis- and 56.4% 13-cis-retinal in the dark and 30.1% 11-cis- and 47.7% 13-cis-retinal upon illumination with >460 nm light (Sudo et al. 2011).  Haloquadratum walsbyi (Hw) survives at high MgCl2 concentrations, with a total of three MRhos identified, including (1) a high-acid-tolerance light-driven proton outward pump, HwBR, (2) a chloride-insensitive chloride pump, HwHR, and (3) HwMR, the sole magnesium-sensitive MRho among all tested MRho proteins from Haloarchaea. D84 is one of the key residues mediating such magnesium ion association in HwMR. Sequence analysis and molecular modeling suggested HwMR to have an extra H8 helix in the cytosolic region like those in signal-transduction-type MRho of deltarhodopsin-3 (dR-3) and Anabaena sensory rhodopsin (ASR) (Ko et al. 2022).

Bop2 of Haloquadratum walsbyi

 
3.E.1.1.17

Cyanorhodopsin, CyR, of 254 aas and 7 TMSs.  These proteins are found in non-marine cyanobacterial strains.  They catalyze light-driven outward H+ pumping (Hasegawa et al. 2020).

CyR of Calothrix sp. NIES-2098

 
3.E.1.1.2Archaerhodopsin-2 (aR2) (a retinal protein-carotenoid complex) (Yoshimura and Kouyama, 2007).EuryarchaeotaaR2 of Halorubrum sp. aus-2 (P29563)
 
3.E.1.1.3

"Middle" rhodopsin or bacteriorhodopsin 1 (Brhol); it has 11-cis-retinal and shows intermediate properties between Brho and sensory rhodopsin II (Sudo et al., 2011). Its structure is known to 2.0 Å resolution following crystalization using polymer-bounded lipid nanodiscs (Broecker et al. 2017). It may pump Cl- and Br- into the cell (Kikukawa 2021).  See also Ko et al. 2022.

Euryarchaeota

Middle rhodopsin of Haloquadratum walsbyi (G0LFX8)

 
3.E.1.1.4

Archaerhodopsin 3, archaerhodopsin-3, AR3, Aop3, BACR3 of 258 aas and 7 TMSs.  It pumps protons in response to light absorption (Saint Clair et al. 2012) and is 86% identical to 3.E.1.1.2. Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization (Bada Juarez et al. 2021). Opsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes (Mei et al. 2021).  Khangholi et al. 2021 studied photoactivation of cell-free expressed archaerhodopsin-3 in a model cell membrane and concluded that it functions as a channel with open and closed states and a pore radius of 0.3 nm.

Archaea

AR3 of Halorubrum sodomense

 
3.E.1.1.5

Bacteriorhodopsin I (HmBRI) of 250 aas and 7 TMSs. The structure is known to 2.5 Å resolution, revealing the usual BRI fold but with several modifications (Shevchenko et al. 2014). Expression in E. coli membranes does not affect the overall structure.

Bacteriorhodopsin I of Haloarcula marismortui

 
3.E.1.1.6

Sensory rhodopsin (green-light-activated photoreceptor; does not transport ions) (Jung et al., 2003). Has all-trans-retinal when dark adapted, but 11-cis-retinal when light adapted due to reversible interconversion (Sineshchekov et al., 2005).  Anabaena sensory rhodopsin, a photochromic sensor that interacts with a soluble 14-kDa cytoplasmic transducer that is encoded on the same operon, interconverts between all-trans-15-anti and 13-cis-15-syn retinal forms depending on the wavelength of illumination, although only the former participates in a photocycle with a signaling M intermediate (Dong et al. 2016). A mutation in the cytoplasmic half-channel of the protein, replacing Asp217 with Glu (D217E), results in the creation of a light-driven, single- photon, inward proton transporter. Dong et al. 2016 presented the 2.3 A structure of dark-adapted D217E ASR, which reveals changes in the water network surrounding Glu217, as well as a shift in the carbon backbone near retinal-binding Lys210, illustrating a possible pathway leading to the protonation of Glu217 in the cytoplasmic half-channel, located 15 A from the Schiff base.

 

Bacteria

Sensory rhodopsin of Anabaena (Nostoc) sp. PCC7120

 
3.E.1.1.7

H+-pumping electrogenic bacteriorhodopsin of 250 aas and 7 TMSs (Kamo et al. 2006).  The delta rhodopsin (dR), a microbial light-driven proton-pumping rhodopsin from Haloterrigena turkmenica, was expressed and localized in the vacuolar membrane of Saccharomyces cerevisiae by conjugation with a vacuolar membrane-localized protein. Vacuoles with dR were isolated from S. cerevisiae, and the light-driven proton pumping activity was evaluated based on the pH change outside the vacuoles. A light-induced increase in the intracellular ATP content was observed in yeast harboring vacuoles with dR (Daicho et al. 2024).

Brho of Haloterrigena turkmenica

 
3.E.1.1.8

Inward H+ pumping xenorhodopsin (bacteriorhodopsin) of 228 aas and 7 TMSs.

Xenorhodopsin of Nanosalina sp. (strain J07AB43)

 
3.E.1.1.9

Rhodopsin, RxR, of 239 aas and 7 TMSs. This rhodopsin is from the thermophilic eubacterium Rubrobacter xylanophilus DSM 9941(T) and was isolated from thermally polluted water. Although R. xylanophilus rhodopsin (RxR) is from an Actinobacterium, it is located between eukaryotic and archaeal rhodopsins in the phylogenetic tree (Kanehara et al. 2017). E. coli cells expressing RxR showed a light-induced decrease in environmental pH and inhibition by a protonophore, indicating that it works as a light-driven outward proton pump. Purified RxR has an absorption maximum at 541 nm and binds all-trans retinal. The pKa values for the protonated retinal Schiff base and its counterion were 10.7 and 1.3, respectively. Of note, RxR showed an extremely high thermal stability in comparison with other proton pumping rhodopsins such as thermophilic rhodopsin TR (by 16-times) and bacteriorhodopsin from Halobacterium salinarum (HsBR, by 4-times) (Kanehara et al. 2017). Hayashi et al. 2020 showed how RxR realizes its exceptionally high stability while retaining its original proton pumping function.  Biochemical synthesis of membrane-spanning lipids is one adaptation that organisms such as thermophilic archaea have evolved to prevent membrane leakiness (Kim et al. 2019).

Rhodoopsin of Rubrobacter xylanophilus

 
Examples:

TC#NameOrganismal TypeExample
3.E.1.2.1

Halorhodopsin Cl- uptake pump; homologous to bacteriorhodopsin (3.E.1.1.1) which can be converted from a proton pump with outwardly directed polarity into a chloride pump with inwardly directed polarity via a single amino acid substitution at position 85.  Cl- transport does not depend on water hydrogen bonded to the chromophore as in the case of bacteriorhodopsin (Muroda et al. 2012).  However, inter-helical hydrogen bonds, mediated by a key arginine residue, largely govern the dynamics of the protein and water groups coordinating the chloride ion (Jardón-Valadez et al. 2014).  Helices E and F probably move considerable during chloride binding and ion transport (Schreiner et al. 2016).

Archaea

Halorhodopsin of Halobacterium salinarum

 
3.E.1.2.2

Chloride-pumping halorhodopsin (a trimer with the carotenoid, bacterioruberin, binding to crevices between adjacent protein subunits in the trimeric assembly; Sasaki et al., 2012). Structure known to 2.0 Å resolution (Kouyama et al., 2010) (PDB# 3A7K)). In addition to Cl-, it pumps Br-, I- and NO3- (Kikukawa 2021).

Archaea

Halorhodopsin of Natronomonas pharaonis (P15647)

 
Examples:

TC#NameOrganismal TypeExample
3.E.1.3.1Sensory rhodopsin I Archaea Sensory rhodopsin I of Halobacterium salinarum
 
3.E.1.3.2

Sensory rhodopsin II or photoreceptor phoborhodopsin (ppR).  The 3-d structure has been solved by NMR (Gautier and Nietlispach 2012). The dynamics of light induced conformational changes have been studied (Taniguchi et al. 2007).

Archaea

Sensory rhodopsin II (phoborhodopsin) of Halobacterium salinarum

 
3.E.1.3.3

Sensory rhodopsin II, SR2 (Sop2; ppR), also called Pharaonis phoborhodopsin. The NMR solution structure of the detergent solubilized protein is in good agreement with the x-ray structure (Gautier et al. 2010). The onformational dynamics of Sensory Rhodopsin II in nanolipoprotein and styrene-maleic acid lipid particles has been studied (Mosslehy et al. 2019). The retinal configuration of ppR intermediates have been studied (Makino et al. 2018).

Archaea

SRII of Natronomonas (Natronobacterium) pharaonis (P42196)

 
3.E.1.3.4

Sensory rhodopsin III, SRIII, of 232 aas and 7 TMSs (Fu et al. 2010).

SRIII of Haloarcula marismortui

 
3.E.1.3.5

Phoborhodopsin (sensory rhodopsin II) of 249 aas and 7 TMSs.  The photochemistry and proton transport have been reviewed (Kamo et al. 2001) and the crystal structure is known (Kandori and Kamo 2002). Rhodopsins have been reviewed (Kandori 2020).

Phoborhodopsin of Halorubrum chaoviator

 
Examples:

TC#NameOrganismal TypeExample
3.E.1.4.1Heat shock protein HSP30 Yeast HSP30 of Saccharomyces cerevisiae
 
3.E.1.4.2

Retinal binding protein, Neurospora Opsin-1, NOP-1 (Bieszke et al. 1999; Bieszke et al. 2007).

Fungi

NOP-1 of Neurospora crassa

 
3.E.1.4.3

H+ pumping rhodopsin (opsin; Ops), of 313 aas and 7 TMSs (Idnurm and Howlett 2001; Waschuk et al., 2005).  Its 3-d strcuture has been solved, and it resembles archaeal rhodopsins more than bacterial rhodopsins (Zabelskii et al. 2021).

Fungi

Rhodopsin of Leptosphaeria maculans (AAG01180)

 
3.E.1.4.4

Acetaularia rhodopsin I, ARI or c102333 of 246 aas.  It exhibits outward H+ pumping activity, and D89 and D100 are essential for pumping activity (Lee et al. 2015). Blue-light causes a shunt of the photocycle under H+ reuptake from the extracellular side (Tsunoda et al. 2006). Similarities and differences of AR with BR have been revealed by detailed electrophysiological studies, revealing among other things, the voltage dependencies of the pump (Tsunoda et al. 2006).

Plants (Chlorophyta)

c102333 of Acetabularia acetabulum (Q1AJZ3)

 
3.E.1.4.5

Opsin 1, Bacteriorhodopsin-like protein

Cryptophyta

Opsin 1 of Guillardia theta (Q2QCJ4)

 
3.E.1.4.6

Possible chaperone membrane protein related to Hsp30, Mrh1 (320 aas; 33% identical to Hsp30p). This protein and its two paralogues, Hsp30 and YR02, are induced by heat shock and are present primarily in the plasma membrane (Wu et al. 2000).  It plays a role in acetic acid tolerance and may be an acetic acid exporter (Takabatake et al. 2015).

Fungi

Mrh1p of Saccharomyces cerevisiae (Q12117)

 
3.E.1.4.7

Cyanorhodopsin of 334 aas and 7 TMSs, Ops1 (Frassanito et al. 2010).

Algae (Glycophyta)

Cyanorhodopsin of Cyanophora paradoxa

 
3.E.1.4.8

Yro2 of 344 aas and 7 TMSs.  Plays a role in acetic acid tolereance and is induced by acetic acid stress and by entry into the stationary phase.  It is 72% identical to Mrh1 (TC# 3.E.1.4.6) which is also believed to be involved in the acetic acid stress response (Takabatake et al. 2015).

Yro2 of Saccharomyces cerevisiae

 
3.E.1.4.9Pentachlorophenone-induced protein, FDD123 Fungi FDD123 of Coriolus versicolor
 
Examples:

TC#NameOrganismal TypeExample
3.E.1.5.1

Bacterio-rhodopsin/guanylyl cyclase 1 fusion protein of 626 aas; light-activated enzyme, RhCG, Gc1 or Cyc1Op. The central bacteriorhodopsin domain with 7 TMSs is linked via an additional TMS to the C-terminal adenylate/guanylate cyclase catalytic domain. CyclOp enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals (Gao et al. 2015).

Gc1 of Blastocladiella emersonii (Aquatic fungus)

 
3.E.1.5.2

Learning/memory process protein of 704 aas and 7 N-terminal TMSs as the rhodopsin (Rh) domain with a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain.  The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an "M" intermediate with a deprotonated Schiff base; its recovery is much faster than that of the enzyme domain (Watari et al. 2019). Mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels have thus been revealed (Watari et al. 2019).

Rh-PDE fusion protein of Salpingoeca rosetta

 
3.E.1.5.3

Rhodopsin-containing uncharacterized protein of 2205 aas with the rhodopsin domain at the N-terminus with 7 or 8 TMSs.  Additional TMSs may exist in the remainder of the protein.  It may be a light inhibited guanlyate cyclase (Tian et al. 2022). The guanylate cyclase domain may be within residues 1120 and 1350.

UP of Volvox reticuliferus

 
Examples:

TC#NameOrganismal TypeExample
3.E.1.6.1

Green-light-absorbing H+ pumping proteorhopdopsin of 249 aas and 8 TMSs. It exhibits variable vectorality: H+ is pumped out at basic pH but not at acidic pH; see Friedrich et al., 2002). It presents a fast proton release and an alkaliphilic photocycle, consistent with its marine origin and the near-surface environment where this bacterium was collected. This proteorhodopsin has been used to measure membrane potentials and electrical spiking in E. coli (Kralj et al., 2011; Ward et al., 2011).  3-d structures of three proteorhodopsins show that they can exist as pentamers or hexamers, depending on the protein (Ran et al. 2013).  Protonation states of several carboxylic acids, the boundaries and distortions of transmembrane α-helices, and secondary structural elements in the loops have been identified (Shi et al. 2009). Proteorhodopsin molecules incorporated into mesostructured silica films exhibit native-like function, as well as enhanced thermal stability compared to surfactant or lipid environments (Jahnke et al. 2018).

Proteobacteria

Green-light-absorbing proteorhodopsin from an uncultured γ-proteobacterium EOAC 31A08

 
3.E.1.6.10

Uncharaacterized bacteriorhodopsin of 289 aas and 7 TMSs.

UP of Parvularcula oceani

 
3.E.1.6.11

Uncharacterized bacteriorhodopsin of 321 aas and 7 TMSs.

UP of Parvularcula oceani

 
3.E.1.6.12

Blue-light absorbing proteorhodopsin (BPR) of 251 aas and 8 TMSs including a cleavable N-terminal TMS. BPR does not rely on the Sec pathway for inner membrane integration (Soto-Rodríguez and Baneyx 2018). The BPR signal sequence is recognized by the signal recognition particle (SRP; a protein that orchestrates the cotranslational biogenesis of inner membrane proteins) and serves as a beneficial "pro" domain rather than a traditional secretory peptide. It is a light-driven proton pump that may have a regulatory rather than energy harvesting function, based on light-induced opening of proton channels to modulate cell physiology depending on light intensity variations. It could therefore be a sensory rhodopsin, potentially associated with a transducer component. It presents a much slower photocycle than that of the green-absorbing proteorhodopsin, probably an adaptation to the intensity of solar illumination at a depth of 75m, where this bacterium was collected. Transport occurs only at pHs above 7 and is unidirectional.


BPR of Gamma-proteobacterium Hot 75m4

 
3.E.1.6.13

Viral rhodopsin II (RhoII) of 211 aas and 7 TMSs, OLPVRII, a VirChR1, a Na+/K+ channel that does not transport Ca2+. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit (Bratanov et al. 2019). The proton donor, E42, is in helix B. The structure is unique among known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels. A photon (hν) causes neural firing.  The 1.4 Å structure has been determined revealing a unique ion transport pathway through the protein (Zabelskii et al. 2020).

OLPVR1 of Organic Lake phycodnavirus

 
3.E.1.6.14

Chloride (Cl-) pumping rhodopsin of 272 aas and 7 TMSs. It may pump Cl-, Br-, I-, and NO3- (Kikukawa 2021).

NTQ rhodopsin of Nonlabens marinus

 
3.E.1.6.15

Bellilinea Na+-pumping rhodopsin, BeNaR, of 269 aas and 7 TMSs. Kurihara et al. 2023 identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli cells expressing this rhodopsin showed light-induced alkalization of the medium only in the presence of Na+, and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na+ pump function across the cellular membrane. Its Na+-pumping activity is greater than that of the known Na+-pumping rhodopsin, KR2. Its photochemical properties included: (i) Visible spectroscopy and HPLC revealed that BeNaR had an absorption maximum at 524 nm with predominantly (>96%) the all-trans retinal conformer. (ii) Time-dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na+-pumping activity. Kurihara et al. 2023 proposed a model for the outward Na+-pumping mechanism of BeNaR. 

BeNaR of Bellilinea caldifistulae

 
3.E.1.6.2

Xanthorhodopsin, a proton pump with a carotenoid antenna, salinixanthin (Lanyi and Balashov 2008). A crystal structure (1.9 Å resolution) is available (Luecke et al., 2008).

Bacteroidetes

Xanthorhodopsin with a salinixanthin chromophore of Salinibacter ruber (Q2S2F8)

 
3.E.1.6.3

Gloeobacter rhodopsin (GT) of 298 aas and 7 TMSs (associates with salinixanthin, the light-harvesting carotenoid antenna of xanthorhodopsin) (Imasheva et al., 2009Hashimoto et al., 2010).  Expression in a chemotrophic E. coli enabled light-driven phototrophic energy generation (Kim et al. 2017). The X-ray crystallographic structure and oligomeric state have been described (Morizumi et al. 2019). The structural characteristics of GR's hydrogen bonding network in the transmembrane domain as well as the displacement of extracellular sides of the TMS resemble those of XR. The pH-dependent pentamer form of GR was documented. Structural motifs (extended helices, 3-omega motif and flipped B-C loop) typical of other characterized rhodopsins, were also identified (Morizumi et al. 2019).  This proton outward pumping rhodopsin interacts with a helix-turn-helix transcriptional regulator and regulats gene expression. ATP-binding cassette (ABC) transporters and the self-regulation of G. violaceus transcriptional regulator (GvTcR) are regulated by light, and gene regulation was observed in G. violaceus using the real-time PCR (Shim et al. 2024).

Bacteria

Rhodopsin of Gloeobacter violaceus (Q7NP59)

 
3.E.1.6.4

Bacteriorhodopsin-like circadian clock related protein (Okamoto and Hastings, 2003)

Dinoflagellates (Alveolata)

BacRhodopsin of Pyrocystis lunula (Q8GZE7)

 
3.E.1.6.5

H+-pumping bacteriorhodopsin, Brho or ESR (Petrovskaya et al. 2010). Photoelectric potential generation correlates with the ESR structure and proposed mechanism of proton transfer (Siletsky et al. 2016). Proteoliposomes with unidirectional orientation using ESR from Exiguobacterium sibiricum have been prepared. Three ESR hybrids with soluble protein domains (mCherry or thioredoxin at the C-terminus and Caf1M chaperone at the N-terminus) were obtained and characterized (Petrovskaya et al. 2023).

Bacteria

Brho of Exiguobacterium sibiricum (B1YFV8)

 
3.E.1.6.6

Proton pumping proteorhodopsin of 253 aas (Kimura et al. 2011).

Bacteroidetes

Proteorhodopsin of Nonlabens dokdonensis (Donghaeana dokdonensis)

 
3.E.1.6.7

Na+ or H+ pumping bacteriorhodopsin, NaR, Kr2 or KR2 (Krokinobacter rhodopsin 2), of 280 aas and 7 TMSs.  It uses light to pump protons or sodium ions from the cell depending on the ionic composition of the medium. In cells suspended in a KCl solution, NaR functions as a light-driven proton pump, whereas in a NaCl solution, it exhibits light-driven sodium ion pumping, a novel activity within the rhodopsin family (da Silva et al. 2015).  A cation switch controls its conformations, and specific interactions of Na+ with the half-channels open an appropriate path for ion translocation (da Silva et al. 2015). Several high resolution x-ray structures have been solved (4XTO, Kato et al. 2015). Putative Na+ binding sites have been identified, and it was shown how protonation and conformational changes gate the ion through these sites toward the extracellular side (Suomivuori et al. 2017). Evidence for homology of this and other microbial rhodopsin with GPCR receptors including mamalian rhodopsins has been presented (Yee et al. 2013; Shalaeva et al. 2015). The 3-d structures have been determined by different groups (3X3B_A,B, 4XT0_A-E; 5JRF_A, and 6RF4_A-E). A covalent bond between Lys-255 and the polypeptide chain is responsible for stable retinal chromophore binding and sodium-pumping activity of KR2,but not for transport activity (Ochiai et al. 2023). Light-driven Na+-pumping rhodopsin (NaR) has been reviewed with an overview of structural and functional studies encompassing ground/intermediate-state structures and photocycle kinetics (Yang and Chen 2023). The review focuses on (1) unraveling the translocation pathway of Na+; (2) examining the role of structural changes within the photocycle, particularly in the O state, in facilitating Na+ transport; and (3) investigating the timing of Na+ uptake/release.

NaR of Dokdonia eikasta (Krokinobacter eikastus)

 
3.E.1.6.8

Proteorhodopsin of 246 aas and 7 TMSs, Pro.  A light-driven Na+ pump (Bertsova et al. 2015).

Proteorhodopsin of Dokdonia sp. PRO95

 
3.E.1.6.9

Bacteriorhodopsin (thermophilic rhodopsin; TR) of 260 aas and 7 TMSs.  53% identical to xanthorhodopsin (TC# 3.E.1.6.2).  It is a photoreceptor protein with extremely high thermal stability and a light-driven electrogenic proton pump. The x-ray crystal structure revealed the presence of a putative binding site for a carotenoid antenna and a larger number of hydrophobic residues and aromatic-aromatic interactions than in most microbial rhodopsins (Tsukamoto et al. 2016). The structural changes upon thermal stimulation involved a thermally induced structure in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. An extracellular LPGG motif between helices F and G may play an important role in thermal stability, acting as a "thermal sensor" (Tsukamoto et al. 2016).

Bacteriorhodopsin of Thermus thermophilus

 
Examples:

TC#NameOrganismal TypeExample
3.E.1.7.1

Channelrhodopsin-1 (chlamyrhodopsin-3) (ChR1; Cop3; CSOA) (light-gated cation (H+, Na+, K+, and Ca2+) channel) (Nagel et al., 2003). TMSs 1 and 2 are the main structures involved in desensitization involving the stabilization of the protein's conformation and the alteration of the charge distribution around the retinal-Schiff base (Zamani et al. 2017). Replacing the glutamate located at the central gate of the ion channel with positively charged amino acyl residues reverses the ion selectivity and allows anion (chloride, Cl-) conduction (Zhang et al. 2019).  zlight-gated channelrhodopsin sparks proton-induced calcium release in guard cells (Huang et al. 2023).  The mechanisms of cation transport and valence selectivity through the channelrhodopsin chimera, C1C2 (ChR1/ChR2), in the high- and low-conducting open states have been examined (Prignano et al. 2024). Electrophysiology measurements identified a single-residue substitution within the central gate, N297D, that increased Ca2+ permeability vs. Na+ by nearly two-fold at peak current, but less so at stationary current. Molecular models of dimeric wild-type C1C2 and N297D mutant channels were examined in both open states and the PMF profiles for Na+ and Ca2+ permeation through each protein using well-tempered/multiple-walker metadynamics were determined. Results of these studies agree well with experimental measurements and demonstrated that the pore entrance on the extracellular side differs from original predictions and is actually located in a gap between helices I and II. Cation transport occurs via a relay mechanism where cations are passed between flexible carboxylate sidechains lining the full length of the pore by side chain swinging, like a monkey swinging on vines. In the mutant channel, residue D297 enhances Ca2+ permeability by mediating the handoff between the central and cytosolic binding sites via direct coordination and side chain swinging. Prignano et al. 2024 also found that altered cation binding affinities at both the extracellular entrance and the central binding sites underlie the distinct transport properties of the low-conducting open state. This facilitates an understanding of ion selectivity and permeation in cation channelrhodopsins.

Algae

Channelrhodopsin-1 of Chlamydomonas reinhardtii

 
3.E.1.7.10

Kalium (potassium) channelrhodoopsin 1, KCR1, of 265 aas and 7 TMSs.  It shows higher selectivity for K+ than for Na+ and therefore is used to silence neurons with light (optogenetics). Replacement of the conserved cysteine residue in the TMS 3 (Cys110) with alanine or threonine results in a >1,000-fold decrease in the channel closing rate (Sineshchekov et al. 2023).

KCR1 of Hyphochytrium catenoides

 
3.E.1.7.11

ChRmine of 318 aas and 7 TMSs. It is a pump-like cation-conducting channelrhodopsin that exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; Kishi et al. 2022 presented the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. The authors applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities (Kishi et al. 2022).

ChRmine of Tiarina fusa

 
3.E.1.7.12

Uncharacterized protein of 931 aas with 7 N-terminal TMSs.

UP of Cafeteria roenbergensis

 
3.E.1.7.13

Cation channelrhodopsin 1, partial [synthetic construct] of 334 aas and 7 TMSs.

Cation-channelrhodopsin 1 (synthetic)

 
3.E.1.7.2

Channelrhodopsin-2 (chlamyrhodopsin-4 of 737 aas and 7 N-terminal TMSs; ChR2; CR2; Cop4; CSOB) (light-gated cation-selective ion channel (both monovalent and divalent cations (H+, Na+, K+, and Ca2+) are transported)) (Nagel et al., 2003). Berndt et al. (2010) showed that ChR2 has two open states with differing ion selectivities. The channel is fairly nonspecific at the beginning of a light pulse, and becomes more specific for protons during longer periods of light exposure. Residues involved in channel closure have been identified (Bamann et al. 2010).  ChR2 is 712 aas long; the MR domain is N-terminal (Lee et al. 2015). The free energy profiles computed for proton transfer to the counterion, either via a direct jump or mediated by a water molecule, demonstrate that, when retinal is all-trans, water and protein electrostatic interactions largely favour the protonated retinal Schiff base state (Adam and Bondar 2018).

   Blue light illumination of ChR2 activates an intrinsic leak channel conductive for cations. Sequence comparison of ChR2 with the related ChR1 protein revealed a cluster of charged amino acids within the predicted transmembrane domain 2 (TM2), which includes glutamates E90, E97 and E101. Charge inversion substitutions altered ChR2 function, replacement of E90 by lysine or alanine resulted in differential effects on H+- and Na+-mediated currents. These results are consistent with this glutamate side chain within TMS2 contributing to ion flux through and the cation selectivity of ChR2 (Ruffert et al., 2011). Glutamate residue-97 lies in the outer pore where it interacts with a cation to facilitate dehydration. This residue is also the primary binding target of Gd3+(Tanimoto et al., 2012).  Channelrhodopsin has been converted into a light-gated chloride channel (Wietek et al. 2014).  TMSs 2, 6 and 7 reorient or rearrange during the photocycle with no major differences near TMSs 3 and 4 at the dimer interface. TMS2 plays a key role in light-induced channel opening and closing in ChR2 (Müller et al. 2015).  Negative charges at the extracellular side of transmembrane domain 7 funnel cations into the pore (Richards and Dempski 2015).  CrChR2, is the most widely used optogenetic tool in neuroscience.  Water efflux and the cessation of the ion conductance are synchronized (Lórenz-Fonfría et al. 2015).  light and pH induce changes in the structure and accessibility of TMSB (Volz et al. 2016). Residues V86, K93 and N258 form a putative barrier to ion translocation. These residues contribute to cation selectivity (V86 and N258), the transition between the two open states (V86), open channel stability, and the hydrogen-bonding network (K93I and K93N) (Richards and Dempski 2017). The x-ray structure is available and reveals much about the mechanism of channel regulation (Gerwert 2017; Volkov et al. 2017). The mechanism of formation of the ion channel of ChR2 has been studed by molecular dynamics simulation and steering (Yang et al. 2019). The effects on ion channel activities of different protonation states of E90 in channelrhodopsin-2 have been described (Cheng et al. 2021). Yang et al. 2023 have designed a TRP-like biohybrid sensor by integrating upconversion nanoparticles (UCNP) and optogenetically engineered cells on a graphene transistor for infrared sensing and imaging. They used UCNP and ChR2 within the sensor in place of TRPs. (Yang et al. 2023). Light activation of ChR2 augments an influx of Na+ with a consequent inhibition of cell growth. In a K+ uptake deficient yeast strain, growth can be rescued in selective medium by the blue light induced K+ conductance of ChR (Höler et al. 2023).

 

Algae

Channelrhodopsin-2, CR2, of Chlamydomonas reinhardtii (Q8RUT8)

 
3.E.1.7.3

Channelrhodopsin-2 (ChR2) of light-gated ion channel. A 6 Å projection map is available (Müller et al., 2011). Glutamate residue-97 lies in the outer pore where it interacts with a cation to facilitate dehydration. This residue is also the primary binding target of Gd3+ (Tanimoto et al., 2012). Cardiomyocytes expressing ChR2 upon optical stimulation depolarize, resulting from alterations of membrane voltage and intracellular calcium (Keshmiri Neghab et al. 2021).

Channelrhodopsin-2 of Volvox carteri (B4Y105)

 
3.E.1.7.4

Channelopsin, MChR1 (Govorunova et al., 2011). In another channelrhodopsin (CrChR2) of this family, an E97A mutation in TMS 2 prevents high affiinity binding of the inhibitor, Gd3+ and interfers with photocurrent, but this ChR1 with an alanine at this position, has low affininty for Gd3+ but normal photocurrent (Watanabe et al. 2016).

Plants

MChR1 of Mesostigma viride (F8UVI5)

 
3.E.1.7.5

Channel rhodopsin of 829 aas and 7 N-terminal TMSs, KnChR.  Channelrhodopsins (ChRs) are light-gated cation (H+, Na+, K+, and Ca2+) channels extensively applied as optogenetics tools for manipulating neuronal activity. All ChRs have a large cytoplasmic domain. The cation channel properties of KnChR from a filamentous terrestrial alga, Klebsormidium nitens, have been described (Tashiro et al. 2021). The C-terminal cytoplasmic domain has a peptidoglycan binding domain (FimV), and modulates the ion channel properties. The channel closure rate is affected by the C-terminus moiety, and truncation of the moiety to various lengths prolongs the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. Tashiro et al. 2021 proposed that electrostatic interaction between the rhodopsin domain and the C-terminal domain accelerates the channel kinetics. Maximal sensitivity was observed at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs.

KnChR of Klebsormidium nitens

 
3.E.1.7.6

Catonic channelrhodopsin 1 of 630 aas and probably 7 N-terminal TMSs. (see Idzhilova et al. 2022 for reference).

Channelrhodopsin 1 of Tetraselmis subcordiformis (Platymonas subcordiformis) (Carteria subcordiformis)

 
3.E.1.7.7

Cationic channelrhodopsin 3 of 511 aas and 7 probable TMSs (see Idzhilova et al. 2022

Cationic channelrhodoopsin 3 of Tetraselmis subcordiformis (Platymonas subcordiformis) (Carteria subcordiformis)

 
3.E.1.7.8

Light-activated cation selective ion channel [synthetic construct] of 344 aas and 7 probable TMSs. It is also called ComV1 or Ex3mV1Co. Channelrhodopsins have been utilized in gene therapy to restore vision in patients with retinitis pigmentosa (Hatakeyama et al. 2023). Channel kinetics (tauon and tauoff) were considerably altered by the replacement of the 172nd amino acid and was dependent on the amino acid characteristics. The size of amino acids at this position correlated with tauon and decay, whereas the solubility correlated with tauon and tauoff. Molecular dynamic simulation indicated that the ion tunnel constructed by H172, E121, and R306 widened due to H172A variant, whereas the interaction between A172 and the surrounding amino acids weakened compared with H172. The bottleneck radius of the ion gate constructed with the 172nd amino acid affected the photocurrent and channel kinetics. The 172nd amino acid in ComV1 is a key residue for determining channel kinetics as its properties alter the radius of the ion gate (Hatakeyama et al. 2023).

ComsV1, synthetic construct

 
3.E.1.7.9

Channel rhodopsin, mVChR1, synthetic construct, of 344 aas with possibly 7 TMSs (Tomita et al. 2014). This system has been used for the development of an optogenetic gene sensitive to daylight and it use in vision restoration for people (Watanabe et al. 2021).

mVChR1, a synthetic construct

 
Examples:

TC#NameOrganismal TypeExample
3.E.1.8.1

Anion-specific light-gated channel rhodopsin of 438 aas, Acr1, lacking measurable cation transport capability (Govorunova et al. 2015). The crystal structure of the light-gated anion channel, GtACR1, revealed a continuous tunnel traversing the protein from extracellular to intracellular sides (Li et al. 2021). The tunnel is the conductance channel closed by three constrictions: C1 in the extracellular half, mid-membrane C2 containing the photoactive site, and C3 on the cytoplasmic side. The crystal structure of bromide-bound GtACR1 revealed structural changes that relax the C1 and C3 constrictions, including a novel salt-bridge switch mechanism involving C1 and the photoactive site. Thus, substrate binding induces a transition from an inactivated state to a pre-activated state in the dark that facilitates channel opening by reducing free energy in the tunnel constrictions. The results provide direct evidence that the tunnel is the closed form of the channel of GtACR1 and shed light on the light-gated channel activation mechanism (Li et al. 2021).  The preferential transport of NO3- by full-length Guillardia theta anion channelrhodopsin 1, ACR1, is enhanced by its extended cytoplasmic domain (Ohki et al. 2023).

Arc1 of Guillardia theta

 
3.E.1.8.2

Anion-specific light-gated channel rhodopsin of 438 aas, Acr2, lacking measurable cation transport capability (Govorunova et al. 2015). Two conserved carboxylates, E159 and D230, play roles in the anion transport activity of ACR2 (Kojima et al. 2018).

Acr2 of Guillardia theta

 
3.E.1.8.3

Homologue of anion-specific light-gated channel rhodopsin of 461 aas and 7 putative TMSs, lacking apparent channel activity  (Govorunova et al. 2015).

Acr homologue of Guillardia theta

 
3.E.1.8.4

Synthetic anion-specific channelrhodopsin of 307 aas and 7 TMSs, derived from an anion channel of Guillardia theta (Govorunova et al. 2018).

Channelrhodopsin of Guillardia theta

 
3.E.1.8.5

Light-activated anion channel rhodopsin of 300 aas and 7 TMSs [synthetic construct]. It is very similar or identical to PsuACR_353, a light-gated anion-selective channel from the marine cryptophyte algae, Proteomonas sulcata, with the highest transport rate for nitrate (NO3-) (Kikuchi et al. 2021). A Thr residue in the third TMS, which corresponds to Cys102 in GtACR1, contributes to the preference for NO3-.

PsuACR_353 of Proteomonas sulcata

 
Examples:

TC#NameOrganismal TypeExample