The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4.

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.

Driving forces of proton-pumping rhodopsins.

Proton-pumping rhodopsins are light-driven proton transporters that have been discovered from various microbiota. They are categorized into two groups: outward-directed and inward-directed proton pumps. Although the directions of transport are opposite, they are active proton transporters that create an H+ gradient across a membrane. Here, we aimed to study the driving force of the proton-pumping rhodopsins and the effect of ΔΨ and ΔpH on their pumping functions. We systematically characterized the H+ transport properties of nine different rhodopsins, six outward-directed H+ pumps and three inward-directed pumps, by patch-clamp measurements after expressing them in mammalian cells. The driving force of each pump was estimated from the slope of the current-voltage relations (I-V plot). Notably, among the tested rhodopsins, we found a large variation in driving forces, ranging from 83 to 399 mV. The driving force and decay rate of each pump current exhibited a good correlation. We determined driving forces under various pHs. pH dependency was less than predicted by the Nernst potential in most of the rhodopsins. Our study demonstrates that the H+-pumping rhodopsins from different organisms exhibit various pumping properties in terms of driving force, kinetics, and pH dependency, which could be evolutionarily derived from adaptations to their environments.

FTIR study of light-induced proton transfer and Ca2+ binding in T82D mutant of TAT rhodopsin.

Proton transfer reactions play important functional roles in many proteins, such as enzymes and transporters, which is also the case in rhodopsins. In fact, functional expression of rhodopsins accompanies intramolecular proton transfer reactions in many cases. One of the exceptional cases can be seen in the protonated form of marine bacterial TAT rhodopsin, which isomerizes the retinal by light but returns to the original state within 10-5 s. Thus, light energy is converted into heat without any function. In contrast, the T82D mutant of TAT rhodopsin conducts the light-induced deprotonation of the Schiff base at high pH. In this article, we report the structural analysis of T82D by means of difference Fourier transform infrared (FTIR) spectroscopy. In the light-induced difference FTIR spectra at 77 K, we observed little hydrogen out-of-plane vibrations for T82D as well as the wild-type (WT), suggesting that the planar chromophore structure itself is not the origin of the reversion from the K intermediate in WT TAT rhodopsin. Upon relaxation of the K intermediate, T82D forms the following intermediate, such as M, whereas K of WT returns to the original state. Present FTIR analysis revealed the proton transfer from the Schiff base to D82 in T82D upon formation of the M intermediate. It is accompanied by the second proton transfer from E54 to the Schiff base, forming the N intermediate, particularly in membranes. The equilibrium between the M and N intermediates corresponds to the protonation equilibrium between E54 and the Schiff base. We also found that Ca2+ binding takes place in T82D as well as WT but with 6 times lower affinity. An altered hydrogen-bonding network would be the origin of low affinity in T82D, where deprotonation of E54 is involved in the Ca2+ binding.

Unusual Vibrational Coupling of the Schiff Base in the Retinal Chromophore of Sodium Ion-Pumping Rhodopsins.

A Schiff base in the retinal chromophore of microbial rhodopsin is crucial to its ion transport mechanism. Here, we discovered an unprecedented isotope effect on the C═N stretching frequency of the Schiff base in sodium ion-pumping rhodopsins, showing an unusual interaction of the Schiff base. No amino acid residue attributable to the unprecedented isotope effect was identified, suggesting that the H-O-H bending vibration of a water molecule near the Schiff base was coupled with the C═N stretching vibration. A twist in the polyene chain in the chromophore for the sodium ion-pumping rhodopsins enabled this unusual interaction of the Schiff base. The present discovery provides new insights into the interaction network of the retinal chromophore in microbial rhodopsins.

Low-temperature FTIR spectroscopy of the L/Q switch of proteorhodopsin.

Rhodopsins are photoreceptive membrane proteins containing a retinal chromophore, and the color tuning mechanism in rhodopsins is one of the important topics. Color switch is a color-determining residue at the same position, where replacement of red- and blue-shifting amino acids in two wild-type rhodopsins causes spectral blue- and red-shifts, respectively. The first and most famous color switch in microbial rhodopsins is the L/Q switch in proteorhodopsins (PRs). Green- or blue-absorbing PR (GPR or BPR) contains Leu and Gln at position 105 of the C-helix (TM3), respectively, and their replacement converted absorbing colors. The L/Q switch enables bacteria to absorb green or blue light in shallow or deep ocean waters, respectively. Although Gln and Leu are hydrophilic and hydrophobic residues, respectively, a comprehensive mutation study of position 105 in GPR revealed that the λmax correlated with the volume of residues, not the hydropathy index. To gain structural insights into the mechanism, we applied low-temperature FTIR spectroscopy of L105Q GPR, and the obtained spectra were compared with those of GPR and BPR. The difference FTIR spectra of L105Q GPR were similar to those of BPR, not GPR, implying that the L/Q switch converts the GPR structure into a BPR structure in terms of the local environments of the retinal chromophore. It includes retinal skeletal vibration, hydrogen-bonding strength of the protonated Schiff base, amide-A vibration (peptide backbone), and protein-bound water molecules. Consequently color is switched accompanying such structural alterations, and known as the L/Q switch.

[How to Choose the Best Optogenetic Tool for Your Research].

All-optical methods that provide deeper understanding of neural activity are currently being developed. Optogenetics is a biological technique useful to control neuronal activity or life phenomena using light. Microbial rhodopsins are light-activated membrane proteins used as optogenetic tools. Microbial rhodopsins such as channelrhodopsin2 (ChR2) consist of seven-pass transmembrane proteins with a covalently bound retinal. Light absorption is followed by photoisomerization of the all-trans retinal to a 13-cis configuration and subsequent conformational changes in the molecule, with consequent permeability of the channel structure to ions. Recent studies have reported the discovery of microbial rhodopsins with novel functions. Microbial rhodopsin diversity has also increased. We describe the characteristics of microbial rhodopsins used as optogenetic tools and the latest research in this domain.

Calcium Binding Mechanism in TAT Rhodopsin.

TAT rhodopsin binds Ca2+ near the Schiff base region, which accompanies deprotonation of the Schiff base. This paper reports the Ca2+-free and Ca2+-bound structures of TAT rhodopsin by molecular dynamics (MD) simulation launched from AlphaFold structures. In the Ca2+-bound TAT rhodopsin, Ca2+ is directly coordinated by eight oxygen atoms, four oxygens of the side chains of E54 and D227, and four oxygens of water molecules. E54 is not involved in the hydrogen-bonding network of the Ca2+-free TAT rhodopsin, while flipping motion of E54 allows Ca2+ binding to TAT rhodopsin with deformation of helices observed by FTIR spectroscopy. The hydrogen-bonding network plays a crucial role in maintaining the Ca2+ binding, as mutations of E54, Y55, R79, Y200, E220, and D227 abolished the binding. Only T82V exhibited the Ca2+ binding like the wild type among the mutants in this study. The molecular mechanism of Ca2+ binding is discussed based on the present computational and experimental analysis.

Unique hydrogen-bonding network in a viral channelrhodopsin.

Channelrhodopsins (CRs) are used as key tools in optogenetics, and novel CRs, either found from nature or engineered by mutation, have greatly contributed to the development of optogenetics. Recently CRs were discovered from viruses, and crystal structure of a viral CR, OLPVR1, reported a very similar water-containing hydrogen-bonding network near the retinal Schiff base to that of a light-driven proton-pump bacteriorhodopsin (BR). In both OLPVR1 and BR, nearly planar pentagonal cluster structures are comprised of five oxygen atoms, three oxygens from water molecules and two oxygens from the Schiff base counterions. The planar pentagonal cluster stabilizes a quadrupole, two positive charges at the Schiff base and an arginine, and two negative charges at the counterions, and thus plays important roles in light-gated channel function of OLPVR1 and light-driven proton pump function of BR. Despite similar pentagonal cluster structures, present FTIR analysis revealed different hydrogen-bonding networks between OLPVR1 and BR. The hydrogen bond between the protonated Schiff base and a water is stronger in OLPVR1 than in BR, and internal water molecules donate hydrogen bonds much weaker in OLPVR1 than in BR. In OLPVR1, the bridged water molecule between the Schiff base and counterions forms hydrogen bonds to D76 and D200 equally, while the hydrogen-bonding interaction is much stronger to D85 than to D212 in BR. The present interpretation is supported by the mutation results, where D76 and D200 equally work as the Schiff base counterions in OLPVR1, but D85 is the primary counterion in BR. This work reports highly sensitive hydrogen-bonding network in the Schiff base region, which would be closely related to each function through light-induced alterations of the network.

Photoisomerization pathway of the microbial rhodopsin chromophore in solution.

Photoisomerization is a key photochemical reaction in microbial and animal rhodopsins. It is well established that such photoisomerization is highly selective; all-trans to 13-cis, and 11-cis to all-trans forms in microbial and animal rhodopsins, respectively. Nevertheless, unusual photoisomerization pathways have been discovered recently in microbial rhodopsins. In an enzymerhodopsin NeoR, the all-trans chromophore is isomerized into the 7-cis form exclusively, which is stable at room temperature. Although, the 7-cis form is produced by illumination of retinal, formation of the 7-cis form was never reported for a protonated Schiff base of all-trans retinal in solution. Present HPLC analysis of retinal oximes prepared by hydroxylamine reaction revealed that all-trans and 7-cis forms cannot be separated from the syn peaks under the standard HPLC conditions, while it is possible by the analysis of the anti-peaks. Consequently, we found formation of the 7-cis form by the photoreaction of all-trans chromophore in solution, regardless of the protonation state of the Schiff base. Upon light absorption of all-trans protonated retinal Schiff base in solution, excited-state relaxation accompanies double-bond isomerization, producing 7-cis, 9-cis, 11-cis, or 13-cis form. In contrast, specific chromophore-protein interaction enforces selective isomerization into the 13-cis form in many microbial rhodopsins, but into 7-cis in NeoR.

SNap Bond, a Crucial Hydrogen Bond Between Ser in Helix 3 and Asn in Helix 4, Regulates the Structural Dynamics of Heliorhodopsin.

Heliorhodopsin (HeR) is a new rhodopsin family discovered in 2018 through functional metagenomic analysis. Similar to microbial rhodopsins, HeR has an all-trans retinal chromophore, and its photoisomerization to the 13-cis form triggers a relatively slow photocycle with sequential intermediate states (K, M, and O intermediates). The O intermediate has a relatively long lifetime and is a putative active state for transferring signals or regulating enzymatic reactions. Although the first discovered HeR, 48C12, was found in bacteria and the second HeR (TaHeR) was found in archaea, their key amino acid residues and molecular architectures have been recognized to be well conserved. Nevertheless, the rise and decay kinetics of the O intermediate are faster in 48C12 than in TaHeR. Here, using a new infrared spectroscopic technique with quantum cascade lasers, we clarified that the hydrogen bond between transmembrane helices (TM) 3 and 4 is essential for the altered O kinetics (Ser112 and Asn138 in 48C12). Interconverting mutants of 48C12 and TaHeR clearly revealed that the hydrogen bond is important for regulating the dynamics of the O intermediate. Overall, our study sheds light on the importance of the hydrogen bond between TM3 and TM4 in heliorhodopsins, similar to the DC gate in channelrhodopsins.

Chromophore-Protein Interactions Affecting the Polyene Twist and π-π* Energy Gap of the Retinal Chromophore in Schizorhodopsins.

The properties of a prosthetic group are broadened by interactions with its neighboring residues in proteins. The retinal chromophore in rhodopsins absorbs light, undergoes structural changes, and drives functionally important structural changes in proteins during the photocycle. It is therefore crucial to understand how chromophore-protein interactions regulate the molecular structure and electronic state of chromophores in rhodopsins. Schizorhodopsin is a newly discovered subfamily of rhodopsins found in the genomes of Asgard archaea, which are extant prokaryotes closest to the last common ancestor of eukaryotes and of other microbial species. Here, we report the effects of a hydrogen bond between a retinal Schiff base and its counterion on the twist of the polyene chain and the color of the retinal chromophore. Correlations between spectral features revealed the unexpected fact that the twist of the polyene chain is reduced as the hydrogen bond becomes stronger, suggesting that the twist is caused by tight atomic contacts between the chromophore and nearby residues. In addition, the strength of the hydrogen bond is the primary factor affecting the color-tuning of the retinal chromophore in schizorhodopsins. The findings of this study are valuable for manipulating the molecular structure and electronic state of the chromophore by controlling chromophore-protein interactions.

Glu1022.53-Mediated Early Conformational Changes in the Process of Light-Induced Green Cone Pigment Activation.

Ligand-triggered activation of G protein-coupled receptors (GPCRs) relies on the phenomenon of loose allosteric coupling, which involves conformational alterations spanning from the extracellular ligand-binding domain to the cytoplasmic region, where interactions with G proteins occur. During the GPCR activation process, several intermediate and equilibrium states orchestrate the movement of the flexible and rigid transmembrane (TM) segments of the GPCR. Monitoring early conformational changes is important in unraveling the structural intricacies of the loose allosteric coupling. Here, we focus on the lumi intermediate formed by thermal relaxation from the initial photointermediate, batho in primate green cone pigment (MG), a light-sensitive GPCR responsible for color vision. Our findings from light-induced Fourier transform infrared difference spectroscopy reveal its similarity with rhodopsin, which mediates twilight vision, specifically involving the flip motion of the ß-ionone ring, the relaxation of the torsional structure of the retinal, and local perturbations in the α-helix upon lumi intermediate formation. Conversely, we observe a hydrogen bond modification specific to MG's protonated carboxylic acid, identifying its origin as Glu1022.53 situated in TM2. The weakening of the hydrogen bond strength at Glu1022.53 during the transition from the batho to the lumi intermediates corresponds to a slight outward movement of TM2. Additionally, within the X-ray crystal structure of the rhodopsin lumi intermediate, we note the relocation of the Met862.53 side chain in TM2, expanding the volume of the retinal binding pocket. Consequently, the position of 2.53 emerges as the early step in the conformational shift toward light-induced activation. Moreover, given the prevalence of IR-insensitive hydrophobic amino acids at position 2.53 in many rhodopsin-like GPCRs, including rhodopsin, the hydrogen bond alteration in the C═O stretching band at Glu1022.53 of MG can be used as a probe for tracing conformational changes during the GPCR activation process.

Cis-Trans Reisomerization Preceding Reprotonation of the Retinal Chromophore Is Common to the Schizorhodopsin Family: A Simple and Rational Mechanism for Inward Proton Pumping.

The creation of unidirectional ion transporters across membranes represents one of the greatest challenges in chemistry. Proton-pumping rhodopsins are composed of seven transmembrane helices with a retinal chromophore bound to a lysine side chain via a Schiff base linkage and provide valuable insights for designing such transporters. What makes these transporters particularly intriguing is the discovery of both outward and inward proton-pumping rhodopsins. Surprisingly, despite sharing identical overall structures and membrane topologies, these proteins facilitate proton transport in opposite directions, implying an underlying rational mechanism that can transport protons in different directions within similar protein structures. In this study, we unraveled this mechanism by examining the chromophore structures of deprotonated intermediates in schizorhodopsins, a recently discovered subfamily of inward proton-pumping rhodopsins, using time-resolved resonance Raman spectroscopy. The photocycle of schizorhodopsins revealed the cis-trans thermal isomerization that precedes reprotonation at the Schiff base of the retinal chromophore. Notably, this order has not been observed in other proton-pumping rhodopsins, but here, it was observed in all seven schizorhodopsins studied across the archaeal domain, strongly suggesting that cis-trans thermal isomerization preceding reprotonation is a universal feature of the schizorhodopsin family. Based on these findings, we propose a structural basis for the remarkable order of events crucial for facilitating inward proton transport. The mechanism underlying inward proton transport by schizorhodopsins is straightforward and rational. The insights obtained from this study hold great promise for the design of transmembrane unidirectional ion transporters.

Internal Proton Transfer in the Activation of Heliorhodopsin.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, contains a single counterion of the protonated Schiff base, E108 in HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR). Upon light absorption, the M and O intermediates form in HeRs, as well as type-1 microbial rhodopsins, indicating that the proton transfer from the Schiff base leads to the activation of HeRs. The present flash photolysis study of TaHeR in the presence of a pH-sensitive dye showed that TaHeR contains a proton-accepting group (PAG) inside protein. Comprehensive mutation study of TaHeR found the E108D mutant abolishing the M formation, which is not only at pH 8, but also at pH 9 and 10. The lack of M observation does not originate from the short lifetime of the M intermediate in E108D, as FTIR spectroscopy revealed that a red-shifted K-like intermediate is long lived in E108D. It is likely that the K-like intermediate returns to the unphotolyzed state without internal proton transfer in E108D. E108 and D108 are the Schiff base counterions of the wild-type and E108D mutant TaHeR, respectively, whereas small difference in length of side chains determine internal proton transfer reaction from the Schiff base. Based on the present finding, we propose that the internal water cluster (four water molecules) constitutes PAG in the M intermediate of TaHeR. In the wild type TaHeR, a protonated water cluster is stabilized by forming a salt bridge with E108. In contrast, slightly shortened counterion (D108) cannot stabilize the protonated water cluster in E108D, and thus impairs internal proton transfer from the Schiff base.

Unique Vibrational Characteristics and Structures of the Photoexcited Retinal Chromophore in Ion-Pumping Rhodopsins.

Photoisomerization of an all-trans-retinal chromophore triggers ion transport in microbial ion-pumping rhodopsins. Understanding chromophore structures in the electronically excited (S1) state provides insights into the structural evolution on the potential energy surface of the photoexcited state. In this study, we examined the structure of the S1-state chromophore in Natronomonas pharaonis halorhodopsin (NpHR), a chloride ion-pumping rhodopsin, using time-resolved resonance Raman spectroscopy. The spectral patterns of the S1-state chromophore were completely different from those of the ground-state chromophore, resulting from unique vibrational characteristics and the structure of the S1 state. Mode assignments were based on a combination of deuteration shifts of the Raman bands and hybrid quantum mechanics-molecular mechanics calculations. The present observations suggest a weakened bond alternation in the π conjugation system. A strong hydrogen-out-of-plane bending band was observed in the Raman spectra of the S1-state chromophore in NpHR, indicating a twisted polyene structure. Similar frequency shifts for the C═N/C═C and C-C stretching modes of the S1-state chromophore in NpHR were observed in the Raman spectra of sodium ion-pumping and proton-pumping rhodopsins, suggesting that these unique features are common to the S1 states of ion-pumping rhodopsins.

Spectroscopic Investigation of Na+-Dependent Conformational Changes of a Cyclobutane Pyrimidine Dimer-Repairing Deoxyribozyme.

UV1C is an enzymatically active DNA sequence (deoxyribozyme, DNAzyme) that functions as a cyclobutane pyrimidine dimer (CPD) photolyase. UV1C forms parallel guanine quadruplexes (G-quadruplexes) with a DNA substrate in the presence of 240 mM Na+, the structure of which is important for the enzymatic activity. To investigate the repair mechanism of CPD by UV1C, we designed light-induced Fourier transform infrared (FTIR) spectroscopy. Prior to FTIR measurements, circular dichroism (CD) spectroscopy was conducted to determine the Na+ concentration at which the most G-quadruplexes were formed. We found that UV1C also forms a hybrid G-quadruplex structure at over 500 mM Na+. By assuming a concentration equilibrium between G-quadruplexes and Na+, 1.3 and 1.8 Na+ were found to bind to parallel and hybrid G-quadruplexes, respectively. The hybrid G-quadruplex form of UV1C was also suggested to exhibit photolyase activity. Light-induced FTIR spectra recorded upon the photorepair of CPD by UV1C were compared for parallel G-quadruplex-rich and hybrid G-quadruplex-rich samples. Spectral variations were indicative of structural differences in parallel and hybrid G-quadruplexes before and after CPD cleavage. Differences were also observed when compared to the CPD repair spectrum by CPD photolyase. The spectral differences during CPD repair by either protein or DNAzyme suggest the local environment of the substrates, the surrounding protein, or the aqueous solution.

Highly sensitive visual restoration and protection via ectopic expression of chimeric rhodopsin in mice.

Photoreception requires amplification by mammalian rhodopsin through G protein activation, which requires a visual cycle. To achieve this in retinal gene therapy, we incorporated human rhodopsin cytoplasmic loops into Gloeobacter rhodopsin, thereby generating Gloeobacter and human chimeric rhodopsin (GHCR). In a murine model of inherited retinal degeneration, we induced retinal GHCR expression by intravitreal injection of a recombinant adeno-associated virus vector. Retinal explant and visual thalamus electrophysiological recordings, behavioral tests, and histological analysis showed that GHCR restored dim-environment vision and prevented the progression of retinal degeneration. Thus, GHCR may be a potent clinical tool for the treatment of retinal disorders.

Structural basis for the allosteric modulation of rhodopsin by nanobody binding to its extracellular domain.

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) critical for vertebrate vision. Research on GPCR signaling states has been facilitated using llama-derived nanobodies (Nbs), some of which bind to the intracellular surface to allosterically modulate the receptor. Extracellularly binding allosteric nanobodies have also been investigated, but the structural basis for their activity has not been resolved to date. Here, we report a library of Nbs that bind to the extracellular surface of rhodopsin and allosterically modulate the thermodynamics of its activation process. Crystal structures of Nb2 in complex with native rhodopsin reveal a mechanism of allosteric modulation involving extracellular loop 2 and native glycans. Nb2 binding suppresses Schiff base deprotonation and hydrolysis and prevents intracellular outward movement of helices five and six - a universal activation event for GPCRs. Nb2 also mitigates protein misfolding in a disease-associated mutant rhodopsin. Our data show the power of nanobodies to modulate the photoactivation of rhodopsin and potentially serve as therapeutic agents for disease-associated rhodopsin misfolding.

Structural basis for ion selectivity in potassium-selective channelrhodopsins.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of BeGC1 or PACs achieved it at long latencies. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the function and regulation of zebrafish neurons and cardiomyocytes.