.crystal structure; KR2; light-driven ion pump;

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1 Prospects & Overviews The light-driven sodium ion pump: A new player in rhodopsin research Hideaki E. Kato 1), Keiichi Inoue 2)3)4), Hideki Kandori 2)3) and Osamu Nureki 5) Rhodopsins are one of the most studied photoreceptor protein families, and ion-translocating rhodopsins, both pumps and channels, have recently attracted broad attention because of the development of optogenetics. Recently, a new functional class of ion-pumping rhodopsins, an outward Na þ pump, was discovered, and following structural and functional studies enable us to compare three functionally different ion-pumping rhodopsins: outward proton pump, inward Cl pump, and outward Na þ pump. Here, we review the current knowledge on structure-function relationships in these three light-driven pumps, mainly focusing on Na þ pumps. A structural and functional comparison reveals both unique and conserved features of these ion pumps, and enhances our understanding about how the structurally similar microbial rhodopsins acquired such diverse functions. We also discuss some unresolved questions and future perspectives in research of ion-pumping rhodopsins, including optogenetics application and engineering of novel rhodopsins. DOI /bies ) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA 2) Department of Frontier Materials, Nagoya Institute of Technology, Nagoya, Japan 3) OptoBioTechnology Research Center, Nagoya Institute of Technology, Nagoya, Japan 4) PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan 5) Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan *Corresponding authors: Hideaki E. Kato and Osamu Nureki hekato@stanford.edu (HEK); nureki@bs.s.u-tokyo.ac.jp (ON) Abbreviations: AR3, archaerhodopsin-3; ATR, all-trans retinal; BR, bacteriorhodopsin; CCCP, carbonylcyanide-m-chlorophenylhydrazone; HR, halorhodopsin; KR2, Krokinobacter rhodopsin 2; NaR, Na þ pumping rhodopsin; PR, proteorhodopsin; TM, transmembrane; TPP þ, tetraphenylphosphonium ion. Keywords:.crystal structure; KR2; light-driven ion pump; optogenetics; retinal; rhodopsin; structural biology Introduction Light is one of the most important signals used by living organisms. It is not only used as a primary source of information on the external world, but also drives several biological processes, such as photosynthesis and biological clocks [1]. Light energy and information are usually captured by photoreceptor proteins, and the rhodopsin family is one of the most studied among them. Similarly to other photoreceptor proteins, rhodopsins are composed of an apoprotein, called opsin, covalently linked to a chromophore, retinal. Opsin has a seven transmembrane (7-TM) domain structure, and retinal is covalently bound to a conserved lysine residue in TM7 through a protonated Schiff base linkage. Rhodopsins are found in all domains of life [2], and, on the basis of primary structure and conformation of bound retinal, they are classified into two groups: animal and microbial rhodopsins (Fig. 1). With a few exceptions [3], most animal rhodopsins contain 11-cis retinal, and primarily work as G-protein-coupled receptors [2]. Upon absorption of a photon, 11-cis retinal is isomerized into all-trans retinal (ATR), inducing large conformational changes of opsin. Opsins, in turn, activate heterotrimeric G-proteins and arrestins, triggering downstream signal transduction cascades for biological processes, such as vision. In contrast, microbial rhodopsins mainly bind ATR, and have a number of different functions. Light-induced retinal isomerization from the all-trans to the 13-cis configuration triggers similar, but distinct, conformational changes in different microbial rhodopsins, and drives different processes, such as active proton transport, active Cl transport, passive ion transport, activation of signal transducer, and other functions [2] (Fig. 1). Recently, ion pumping and channel rhodopsins have attracted broad attention, because these microbial rhodopsins can be used as powerful tools in the neuroscience field to control neuronal activity in a wide range of animals (optogenetics) [4]. Compared to channel rhodopsins, ion Bioessays 38: , ß 2016 The Authors. BioEssays Published by WILEY Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made.

2 ...Prospects & Overviews H. E. Kato et al. Figure 1. Functional classification of rhodopsin family proteins. Structures of bovine rhodopsin (PDB ID: 1F88) [79], sensory rhodopsin II (SRII; PDB ID: 1H2S) [80, 81], bacteriorhodopsin (BR; PDB ID: 1C3W) [81], halorhodopsin (HR; PDB ID: 1E12) [82], Krokinobacter rhodopsin 2 (KR2; PDB ID: 3X3C) [33], chimeric channelrhodopsin (C1C2) (ChR; PDB ID: 3UG9) [83] are shown as cylinder models. The colored cylinders denote helices forming the ion-conducting pathway in BR, HR, KR2, and ChR, and helices which bind to the respective signal transducers in bovine rhodopsin and SRII. pumping rhodopsins have a longer scientific history. The first ion pumping rhodopsin, bacteriorhodopsin (BR), was discovered in a halophilic archaeon Halobacterium salinarum in 1971 [5, 6], and shortly after that was found to be an outward proton pump [7], serving as a model protein in diverse research fields up to the present day [8 12]. The next type of ion pumping rhodopsins was discovered in Matsuno-Yagi and Mukohata found a new archaeal rhodopsin, halorhodopsin (HR), in R1mR, an H. salinarum strain deficient in BR [13]. Initially, HR was characterized as an outward Na þ pump [14], but in 1982, Schobert and Lanyi demonstrated that it works not as a cation pump, but as an inward Cl pump [15]. During the next 30 years, several light-driven proton and Cl pumps, including Natronomonas pharaonis halorhodopsin (NpHR) [16, 17] and archaerhodopsin-3 (AR3) [18] were discovered, but no new types of ion transport had been found. Although it was reported that the Cl pumps could transport Br,I,andNO 3, all identified proton pumps had high proton selectivity and none of the known rhodopsins could transport any other cation [19]. However, in 2013, two research groups found new rhodopsins from flavobacteria, and one of the two groups proved that a rhodopsin isolated from Krokinobacter eikastus, Krokinobacter rhodopsin 2 (KR2), works as a light-driven outward Na þ pump [20, 21] (Fig. 1). KR2 has emerged as a representative of a third class of ion pumping rhodopsins, and attracted considerable attention as the first non-proton Bioessays 38: , ß 2016 The Authors. BioEssays Published by WILEY Periodicals, Inc. 1275

3 H. E. Kato et al. Prospects & Overviews... cation pump to be discovered, and as a potential nextgeneration optogenetics tool. In this review, we describe the discovery of KR2, and compare the structure-function relationship among proton, Cl,andNa þ pumping rhodopsins, mainly focusing on Na þ pumps, and conclude with future studies in this field. How the light-driven Na þ pump was discovered In 2013, two genes encoding rhodopsins were identified in the genome of K. eikastus, and their products were called Krokinobacter rhodopsin 1 and 2 (KR1 and KR2). Whereas KR1 had a sequence highly similar to that of proteorhodopsin (PR) [20], a proton-pumping rhodopsin from marine bacteria [22, 23], the sequence of KR2 was novel. The most prominent difference between the sequence of KR2 and other microbial rhodopsins is observed in putative TM3 region. Most known proton pumps, such as BR and PR, have two conserved acidic residues in this helix (Asp85 and Asp96 for H. salinarum BR (HsBR), the latter being replaced by glutamic acid for PR), which are critical for proton pumping function. In contrast, KR2 has Asn112 and Gln123 at the positions homologous to Asp85 and Asp96 of HsBR. Furthermore, a unique aspartic acid (Asp116) exists at the position of Thr89 in HsBR. Aspartic acid at this position has not been found in any previously reported microbial rhodopsins, and exists exclusively in KR2 and its close relatives. This unique sequence motif implied that KR2 may belong to a new functional class of rhodopsins. The function of KR2 was investigated by Inoue et al. [20]. These researchers expressed KR1 and KR2 in Escherichia coli cells, and analyzed the rhodopsin-mediated ion transport using a ph-electrode. If a proton pumping rhodopsin such as BR or PR transports protons outward, a ph decrease of the external medium is observed, and this ph change is prevented by the addition of protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP). When the suspension of E. coli cells expressing KR1 was illuminated with yellow light (l > 500 nm), a clear ph decrease was observed, which was inhibited by CCCP. This result showed that KR1 functions as an outward proton pump, consistent with the high sequence similarity between PR and KR1 [20]. However, in the case of KR2, a ph increase similar to that observed for HR was detected upon illumination with yellow light (l > 500 nm) in NaCl solution. This result suggested that KR2 may work as either (i) inward proton pump; (ii) inward Cl pump; or (iii) outward Na þ pump. The Cl transport by HR makes intracellular membrane potential more negative (hyperpolarization), resulting in the influx of protons into the cytoplasm, which is observed as a ph-increase [15]. The elevation of ph is enhanced by the addition of CCCP, which increases passive transport of protons due to the higher permeability, and diminished by tetraphenylphosphonium ion (TPP þ ), which rapidly dissipates the membrane potential before protons could be taken up passively. The ph change is also diminished by replacing the halide anions by the much, and this strong anion dependence suggests that HR works as a halide anion pump. Similar to HR, in the case of KR2 the ph increase was enhanced and diminished by CCCP and TPP þ, respectively, suggesting that this protein does not work as an inward proton pump. In addition, KR2 showed ph increase in larger SO 2 4 NaCl and Na 2 SO 4 solutions, but ph decrease was observed in solutions containing larger cations such as KCl, RbCl, and CsCl. This ph decrease was abolished by the addition of CCCP, indicating its active nature. Because of this cation dependency, it was concluded that KR2 pumps Na þ outward under the physiological condition of sea water (it can also pump Li þ ), but transports protons in the absence of Na þ and Li þ. Following the report on KR2, several other phylogenetically close rhodopsins were found to work as Na þ pumps, e.g. those from Gillisia limnaea, Nonlabens marinus, and Dokdonia sp. PRO95 [24 26]. The residues corresponding to Asn112, Asp116, and Gln123 of KR2 are completely conserved in all these rhodopsins, and several additional putative Na þ pumping rhodopsins (NaRs) containing these residues have been identified in various bacterial species [27]. These results imply wide distribution of NaRs among various eubacteria, and suggest the importance of this new class of rhodopsins in the ecosystem. Functionally different ion-pumping rhodopsins have different photocycles Since the discovery of BR and HR, extensive research has yielded a general understanding of how these ion pumps transport proton and Cl. In the dark state, all ion-pumping rhodopsins covalently bind ATR via the Schiff base, and light absorption triggers cyclic photochemical reaction called the photocycle. Because the conformational changes of retinal and ion translocation events around the Schiff base affect the absorption spectra, the photocycle can be divided into several spectroscopically distinguishable intermediates. In the case of BR, the photocycle is characterized by five main intermediates: K, L, M, N, and O [2, 28, 29] (Fig. 2A). The first stable photoproduct, the K-intermediate, is formed by the isomerization of all-trans retinal to a twisted 13-cis retinal. The formation of the L-intermediate is coupled to relaxation of the retinal twist. During the L-to-M transition, the proton is transferred from the protonated Schiff base to an acidic residue located on the extracellular side (Asp85 in HsBR). The proton is released to the extracellular medium in the late M-intermediate. In the N-intermediate, the deprotonated Schiff base receives a proton from another acidic residue located on the intracellular side (Asp96 in HsBR). Asp96 receives a proton from the intracellular medium and 13-cis retinal is thermally reisomerized to the all-trans-form in the N-to-O transition (Fig. 2A). As a result of this photocycle, one proton is transferred from the intracellular to extracellular side. In the case of HR, the proton donor and acceptor of the Schiff base (Asp85 and Asp96 in HsBR) are replaced by Thr and Ala, respectively (Thr111 and Ala122 in HsHR). Therefore, the protonation state of the Schiff base nitrogen is normally unchanged, and the photocycle does not contain the M-intermediate. In the dark state, Cl binds between Thr111 and the Schiff base [30]. Retinal photoisomerization flips the N H dipole (K-intermediate), changes the electrostatic environment and hydrogen bonds around the Schiff base (K-L intermediates), and thus drives the movement of Cl from the extracellular environment to the cytoplasmic side (L-N intermediates) [2] (Fig. 2B) Bioessays 38: , ß 2016 The Authors. BioEssays Published by WILEY Periodicals, Inc.

4 ...Prospects & Overviews H. E. Kato et al. Figure 2. The photocycles. The photocycles of light-driven (A) proton pump (HsBR), (B) Cl pump (HsHR), and (C) Na þ pump (KR2). Red, blue, and magenta arrows show the movements of substrates, the isomerization of retinal chromophore, and the conformational change of amino acid side chains, respectively. Compared to BR and HR, little is known about NaR. However, it is known that Asp85 and Asp96 of HsBR are replaced by Asn112 and Gln123 in KR2, and the KR2 photocycle contains four main intermediates: K, L, M, and O (or K, M, N, and O) [20, 24] (Fig. 2C). When KR2 absorbs green light, ATR is isomerized to 13-cis retinal and the red-shifted K-intermediate forms. Following the K-intermediate, the blueshifted M-intermediate appears in equilibrium with the L-intermediate, suggesting that the Schiff base proton is transiently removed from the Schiff base. The Schiff base is re-protonated in the O-intermediate, and it is suggested that Na þ uptake from the intracellular solvent occurs in this step [24, 31]. Na þ transiently binds in the vicinity of Asn112 in the O-intermediate [24, 31] and during the transition from O-intermediate to the dark state, it should be released to the extracellular side (Fig. 2C). Structure-function relationships of BR, HR, and NaR Are the overall architectures of monomeric and oligomeric ion-pump rhodopsins conserved? Two years after the discovery of KR2, Kato et al. and Gushchin et al. solved crystal structures of KR2 [32, 33], and now we can compare the structures of proton, Cl,andNa þ pumps. All lightdriven ion pumps are composed of 7 TM domains connected by three intracellular loops (ICLs) and three extracellular loops (ECLs). As shown in Fig. 1, their overall structures are very similar, and the approximate position of ATR is also conserved. However, they have varying oligomeric states in the cell membrane: many Bioessays 38: , ß 2016 The Authors. BioEssays Published by WILEY Periodicals, Inc. 1277

5 H. E. Kato et al. Prospects & Overviews... Figure 3. Oligomerization of ion-pumping rhodopsins. Overall architecture of (A) trimeric proton pump (HsBR; PDB ID: 1C3W) [81]; (B) pentameric Na þ pump (KR2; PDB ID: 4XTN) [32]; and (C) hexameric proton pump (Med12 PR; PDB ID: 4JQ6) [38], viewed from the extracellular side. Lipid molecules and Na þ are shown as yellow and pink spheres, respectively. proton pumps and Cl pumps, including HsBR and H. salinarum halorhodopsin (HsHR), form trimers [30, 34 37] (Fig. 3A); in contrast, it is suggested that KR2 forms pentamers (Fig. 3B), and PRs assemble into pentamers or hexamers [32, 38 40] (Fig. 3C). Structural comparison reveals that while there is some overlap between the oligomeric interfaces, they are clearly different between trimer, pentamer, and hexamer. For example, HsBR and HsHR mainly use TMs 2, 4, and 5 to assemble into trimers, but KR2 and Med12 PR use TMs 1 3 (Fig. 3). In some cases, lipid molecules or ions mediate the interaction between monomers, and stabilize theoligomer[32,36,38] (Fig.3AandB).Whileithasbeen reported that oligomerization affects the protein stability and kinetics of the photocycle [41, 42], a number of biochemical and biophysical studies have proved that the functional unit of ion-pumping rhodopsins is the monomer [43 45], and the physiological role of oligomerizations is still unclear. Further studies are needed to determine why ion pumping rhodopsins form oligomers under physiological conditions. The high-resolution structures of BR, HR, and KR2 also revealed detailed architecture of the ion-transport pathways. Below, we compare and discuss the structures and functions of the pathways in three parts: the Schiff base region, the intracellular cavity, and the extracellular region. The Schiff base region is the most important region determining the functions of ion-pumping rhodopsins The Schiff base region is the most important architecture in ion-pumping rhodopsins, because it is fundamentally involved in ion selectivity and gating. It is mainly composed of the protonated Schiff base, a number of polar amino acid residues (most importantly, at positions homologous to 85, 89, and 212 in HsBR), and some water molecules. In BR, the protonated Schiff base points towards the extracellular side, and forms a hydrogen bond with a key water molecule between the Schiff base counterions (Asp85 and Asp212 in HsBR) (Fig. 4A). In contrast, in HR, Asp85 is replaced by threonine (Thr111 in HsHR) and Cl binds to the position occupied by Asp85 carboxylate in HsBR (Fig. 4B). Thus, Cl ion stabilizes the positive charge of the Schiff base instead of Asp85. In KR2, Asp85 of HsBR is replaced by Asn112. Instead, Thr89, located just one helical turn above Asp85, is replaced by Asp116, and recent spectroscopic and structural studies suggest that Asp116 works as the Schiff base counterion [20, 32, 33] (Fig. 4C). These amino acids in the Schiff base region (Asp85, Thr89, and Asp212 in HsBR) are critical in defining the function of ionpumping rhodopsins. In HsBR, retinal photoisomerization alters the pka values of the Schiff base, Asp85, and Asp212, consequently leading to the proton transfer from the Schiff base to the extracellular side in the M-intermediate. In HR, replacement of aspartate by threonine allows Cl to bind near the Schiff base. In the case of KR2, it is suggested that the Schiff base proton is transferred to Asp116 in the M-intermediate, and the protonated Asp116 moves the proton away from the Schiff base by flipping its side chain [33]. The transient sequestration of positively charged proton from the center of the ion-conducting pathway reduces the energy barrier for cation transport, and thereby facilitates sodium transfer (Fig. 4C). The proton comes back to the Schiff base in the O-intermediate. Thus, it is suggested that the strategic positioning of the proton not only allows Na þ transfer in the M-intermediate, but also inhibits the reverse flow of Na þ in the O-intermediate. The functional importance of the Schiff base region is clearly demonstrated by earlier studies showing that the proton pump HsBR can be converted into a Cl pump simply by mutating single aspartate residue to threonine (D85T) [46, 47]. Furthermore, recent studies have also shown that the Cl pump can be converted into a proton pump, and the Na þ pump can be converted into a proton or Cl pump by introducing 1 4 mutations, including those to the homologs of Asp85 and Thr89 in HsBR [48, 49]. The intracellular pathway is one of the most structurally divergent regions among ion-pump rhodopsins Unlike the Schiff base region, the intracellular ion-conducting pathways have varying architecture. In BR, the proton uptake pathway is formed by TM 1, 2, 3, and 7. A proton is transferred from the intracellular bulk solvent to the Schiff 1278 Bioessays 38: , ß 2016 The Authors. BioEssays Published by WILEY Periodicals, Inc.

6 ...Prospects & Overviews H. E. Kato et al. conductance [32, 33]. Notably, the N61P/G263W mutant, KR2 Kþ, preferentially transfers K þ. Moreover, Konno et al. recently modified these two residues extensively, and engineered another KR2 mutant, KR2 Csþ,whichcantransport larger cations such as Rb þ and Cs þ,aswellasli þ,na þ,and K þ [52]. The extracellular pathway is functionally and structurally least characterized region The extracellular pathways of proton, Cl, and Na þ pumps are less characterized than the Schiff base regions or the Figure 4. The Schiff base regions. The Schiff base regions of lightdriven (A) proton pump (HsBR; PDB ID: 1C3W) [81]; (B) Cl pump (HsHR; PDB ID: 1E12) [82]; and (C) Na þ pump (KR2; PDB ID: 3X3B, 3X3C) [33]. Water and Cl are shown as red and gray spheres, respectively. Orange arrows represent the putative Na þ transport pathway. base nitrogen via an aspartate or glutamate residue on TM3 (Asp96 inhsbr) [2, 29] (Fig. 5A). The intracellular pathway of HR is less understood, but recent crystallographic study of NpHR showed that the outward movement of TM6 and the side-chain movements of isoleucine and two phenylalanines, occurring in the N-intermediate, create long intracellular cavity [50] (Fig. 5B). This cavity, formed by TM 3, 5, 6, and 7, is filled with a linear water cluster, and extends from the Schiff base to the intracellular bulk solvent. In addition, a functionally important lysine (Lys215 and Lys200 in NpHR and HsHR, respectively) is located near the intracellular exit pore [50, 51]. Thus, it is suggested that this cavity serves as the Cl release route in the N-to-O transition. In KR2, it is assumed that the Na þ uptake pathway is more similar to the proton pathway of BR. It is formed by TM 1, 2, 3, and 7, and Gln123 corresponding to Asp96 in HsBR is also involved in ion uptake [31 33] (Fig. 5C). The notable difference between the intracellular pathways of BR and KR2 is the size of the ion entry pore (Fig. 5A and C). Four relatively bulky residues on the intracellular ends of TM 1, 2, and 7 in BR (Lys30, Phe42, Tyr43, and Leu224 in HsBR) are replaced by less bulky residues (Thr49, Ser60, Asn61, and Gly263, respectively) in KR2. This replacement makes the intracellular cavity larger, and allows the entry of non-proton cations into the conducting pathway. Indeed, the shape and size of this pore are major determinants of ion selectivity in KR2. Recent studies showed that the KR2 mutants involving Asn61 and Gly263 show K þ conductance as well as Na þ Figure 5. Intracellular pathways. Intracellular ion-conducting pathways. A: HsBRinthedarkstate(PDBID:1C3W)[81]. B: NpHR in the N-intermediate (PDB ID: 4QRY) [50]. HsHR numbering is shown in parentheses. C: KR2 in the dark state (PDB ID: 3X3C) [33]. Water and Cl are shown as red and gray spheres, respectively. Black dashed arrows represent putative ion transport pathways. Bioessays 38: , ß 2016 The Authors. BioEssays Published by WILEY Periodicals, Inc. 1279

7 H. E. Kato et al. Prospects & Overviews... Figure 6. Extracellular pathways. Extracellular ionconducting pathways. A: HsBR in the dark state (PDB ID: 1C3W) [81]. B: HsHR in the dark state (PDB ID: 1E12) [30]. C: KR2 in the dark state (PDB ID: 3X3C) [33]. Cl- ion is shown as gray sphere. Black dashed arrows represent putative ion transport pathways. intracellular pathways. In HsBR, the extracellular cavity is formed by TM 1, 2, 3, 6, and 7, and it is capped by ECL1 (Fig. 6A). Protons are released to the extracellular solvent via the proton release group, which consists of two glutamates (Glu194 and Glu204 inhsbr) andawatermolecule[2, 29].InHsHR, Glu204 of HsBR is replaced by threonine (Thr230) (Fig. 6B). Recent computational studies suggested that Cl can bind near this threonine residue, and HsBR and HsHR use similar pathways to release a proton or take up Cl [53]. In contrast, in KR2, ECL1 is displaced, and the cavity is completely shielded by the N-helix (Fig. 6C). Although Glu160 is conserved in a position homologous to Glu194 in HsBR, Glu204 is replaced by Arg243, and both mutants (E160A and R243A) retain significant Na þ pumping activities [33]. Therefore, it is assumed that KR2 forms the Na þ release pathway differently from the respective pathways in HsBR or HsHR, and large conformational changes of N-helix and/or TM region should occur during the photocycle to facilitate the release [54]. Future perspectives What is happening in light-driven Na þ pumps during the photocycle? Because of the short research history of the Na þ pump rhodopsin compared to that of light-driven proton and Cl pumps, little is known about the protein dynamics of lightdriven Na þ pumps. Although a number of recent studies have given us a hint about the dynamic properties of KR2 [24, 33, 54], we know almost nothing about (1) the detailed structural changes in the Schiff base region including the formation of transient Na þ binding site in the O-intermediate and (2) the large conformational change of the protein backbone during the photocycle. Similar to the cases of BR and HR [55 58], structural and spectroscopic techniques such as time-resolved (TR) macromolecular crystallography (e.g. Laue method and TR-serial femtosecond crystallography) and FTIR spectroscopy are suitable to study detailed structural changes inside the protein. For the analysis of the large conformational changes of protein backbone, cryo-electron microscopy (EM) of two-dimensional (2D) crystals, pulsed electron paramagnetic resonance (EPR), and molecular dynamics simulations techniques have yielded notable results in the field of iontranslocating rhodopsins [54, 59 64], and may become powerful tools for KR2 as well. Can we apply light-driven Na þ pumps to optogenetics? The inward flow of anions or outward flow of cations hyperpolarizes the membrane and inhibits activation of neurons. Thus, light-driven Cl pumps (e.g. NpHR) and light-drivenprotonpumps(e.g.ar3)havebeenusedas inhibitory optogenetics tools in the field of neuroscience [65 67]. Recently, Yawo s group and co-workers successfully expressed KR2 in rat cortical neurons, and demonstrated that the KR2-mediated outward Na þ current also inhibits neuronal activation [33]. Because neuronal activity is regulated by Na þ and K þ currents under physiological condition, it is expected that KR2 and its variants including KR2 Kþ will show fewer unintentional side effects compared to proton and Cl pumping rhodopsins. Indeed, it was recently reported that sustained proton pumping activity of AR3 in synaptic terminals induces a ph-dependent Ca 2þ influx that leads to increased spontaneous release of neurotransmitter [68]. The same group also reported that the activation of light-gated Cl channels triggers neurotransmitter release upon light onset [68]. Thus, light-driven Na þ and K þ pumps have the potential to become ideal inhibitory tools in some optogenetics experiments. More new rhodopsins will be engineered or discovered With the help of developments in molecular engineering techniques and genomic sequencing, the spectrum of functions of ion-translocating rhodopsins has been expanded. Recently, the structural information on channelrhodopsin (ChR), a light-gated cation channel, has prompted the engineering of a light-gated anion channel [69 72], and soon after that, light-gated anion channels were discovered in a natural source [73, 74]. Structurebased molecular engineering accomplished a 100 nm blue shift in the absorption spectrum of a proton pumping rhodopsin [75], and de novo transcriptome sequencing of green alga has led to the discovery of a novel ChR with a very red-shifted spectral peak (590 nm) [76]. Likewise, structural information on KR2 enabled us to design a lightdriven outward K þ pump (KR2 Kþ ) [33]: though the functionality in nature still not known, it is highly likely that we will be able to identify one in the near future. Thus, both structure-based molecular engineering and genomic sequencing are very powerful strategies for expanding the 1280 Bioessays 38: , ß 2016 The Authors. BioEssays Published by WILEY Periodicals, Inc.

8 ...Prospects & Overviews H. E. Kato et al. functional spectrum of rhodopsins, and it is expected that novel rhodopsins, such as ion-pumping rhodopsins with novel ion selectivity or absorption spectrum, will be engineered/discovered. Conclusions The discovery of, and ensuing research on, KR2 have enabled us to compare the structures and functions of proton, Cl, and Na þ pumps, and explained how the structurally similar microbial rhodopsins acquired such diverse functions. A few amino acids at specific positions are critical to determine the functions of ion-pump rhodopsins and the replacements of those residues often convert their functions. However, to understand the protein dynamics of these rhodopsins in more detail, further structural studies, including determination of structures in intermediate states, are clearly needed. In addition, as shown in recent successful examples [20, 33, 69 78], the wealth of structural data available and advances in genomic technologies will lead to further engineering and discovery of functionally novel rhodopsins, and it is expected that the scope and impact of rhodopsin research will continue to expand. Acknowledgment We thank Leonid Brown for his invaluable comments on the manuscript. The authors have declared no conflict of interest. References 1. Gehring WJ The evolution of vision. Wiley Interdiscip Rev Dev Biol 3: Ernst OP, Lodowski DT, Elstner M, Hegemann P, et al Microbial and animal rhodopsins: structures, functions, and molecular mechanisms. Chem Rev 114: Ozaki K, Hara R, Hara T, Kakitani T Squid retinochrome. Configurational changes of the retinal chromophore. Biophys J 44: Deisseroth K Optogenetics: 10 years of microbial opsins in neuroscience. Nat Neurosci 18: Blaurock AE, Stoeckenius W Structure of the purple membrane. 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