Microbial Rhodopsins as Multi-functional Photoreactive Membrane Proteins for Optogenetics

Abstract

In life science research, methods to control biological activities with stimuli such as light, heat, pressure and chemicals have been widely utilized to understand their molecular mechanisms. The knowledge obtained by those methods has built a basis for the development of medicinal products. Among those various stimuli, light has the advantage of a high spatiotemporal resolution that allows for the precise control of biological activities. Photoactive membrane protein rhodopsins from microorganisms (called microbial rhodopsins) absorb visible light and that light absorption triggers the trans–cis photoisomerization of the chromophore retinal, leading to various functions such as ion pumps, ion channels, transcriptional regulators and enzymes. In addition to their biological significance, microbial rhodopsins are widely utilized as fundamental molecular tools for optogenetics, a method to control biological activities by light. In this review, we briefly introduce the molecular basis of representative rhodopsin molecules and their applications for optogenetics. Based on those examples, we discuss the high potential of rhodopsin-based optogenetics tools for basic and clinical research in pharmaceutical sciences.

1. INTRODUCTION

Medicinal products continuously support the health of humans through the control of inherent biological activities in cells. In a broad sense, medicinal products can be simplified to stimuli that regulate biological activities. In addition to chemical stimuli (i.e., organic and inorganic compounds) as representative medicinal products, physical stimuli such as heat, pressure and light are also utilized. Thus, we assume that these stimuli are categorized as medicinal products. Among various stimuli, light has the advantage of a high spatiotemporal resolution that allows for the precise control of biological activities. In fact, light (electromagnetic wave) is widely used as a stimulus for various treatments, such as photodynamic therapy (PDT), photothermal therapy (PTT) and photoimmunotherapy (PIT). PDT, PTT and PIT utilize the conversion from light to produce molecular oxygen, to generate heat and to enhance immunostimulating responses, respectively. As a result, those methods can be applied to several diseases such as cancer. For detailed information about those methods, please see other extensive reviews.^1,2) One of the most important characteristics of light is that it can control biological activities with a high spatiotemporal resolution.

Rhodopsins are seven-transmembrane proteins that are widely distributed in all domains of life (i.e., archaea, bacteria and eukaryotes).^3,4) Rhodopsins commonly consist of an apoprotein opsin and a derivative of vitamin-A retinal as a chromophore (Fig. 1A) and are phylogenetically divided into two classes, animal rhodopsins and microbial rhodopsins.⁴⁾ Microbial rhodopsins possess all-trans retinal, which is the most thermally stable isomer, as a chromophore and they have been widely discovered in microorganisms such as bacteria, algae and fungi.^3,4) All-trans retinal covalently binds to a conserved lysine (Lys) residue on the seventh (also termed the G) helix of the opsin via a protonated retinal Schiff base (PRSB) linkage, where its positive charge is stabilized by a negatively charged carboxylate called the counterion (i.e., aspartic acid (Asp) or glutamic acid (Glu)).⁴⁾ Visible light absorption at around 400–700 nm triggers the isomerization from all-trans to 13-cis retinal within an ultrafast time domain (i.e., several hundred femtoseconds).⁴⁾ The photoisomerization induces a sequence of structural changes of the opsins, resulting in a variety of photobiological functions such as ion transporters, phototactic sensors, transcriptional regulators and enzymes (Fig. 1B). After that, the photoactivated rhodopsin returns to the initial state and the cyclic photoreaction sequence (called the photocycle) is completed⁴⁾ (Fig. 1A). Ion transport rhodopsins are divided into two types, ion channel rhodopsins and ion pump rhodopsins. Ion channel rhodopsins transport ions across the membrane according to the ion concentration gradient while ion pump rhodopsins actively transport ions across the membrane against ion concentration gradient.

Fig. 1. Characterization of Microbial Rhodopsins and Their Molecular Functions

(A) Crystal structure of a representative microbial rhodopsin, bacteriorhodopsin (PDB: 1C3W). The all-trans retinal chromophore colored orange is incorporated into the protein moiety via a protonated Schiff base linkage with a perfectly conserved Lys residue. Light absorption triggers the photoisomerization of all-trans retinal to 13-cis retinal and leads to the cyclic series of photoreactions called the photocycle during which several photointermediates, such as the K, L, M, N, and O intermediates, are formed. (B) Diverse molecular functions of microbial rhodopsins in which representative molecules are shown. (Color figure can be accessed in the online version.)

Historically, studies of microbial rhodopsins began with the discovery of the first microbial rhodopsin, bacteriorhodopsin (BR), in 1971.⁵⁾ BR is abundantly expressed in the halophilic archaeon Halobacterium salinarum (formerly halobium), which lives in a highly halophilic environment, and works as a light-driven outward proton pump to form a proton concentration gradient across the cellular membrane (Fig. 1B). The proton gradient generated by BR is utilized as an energy source to produce the molecular currency adenosine triphosphate (ATP) in cells. After the discovery of BR, halorhodopsin (HR) was identified in H. salinarum in 1977.⁶⁾ HR works as a light-driven inward chloride pump and is thought to control osmotic pressure in cells through chloride ion transport across the membrane (Fig. 1B). Although the direction of ion transport is opposite between BR and HR, a point mutation around the retinal (i.e., D85T) in BR converts it into an HR-like inward chloride pump,⁷⁾ suggesting that they have a common mechanism of ion transport.

In addition to the ion transport rhodopsins, two types of sensory rhodopsins, sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII), were discovered from the same organism H. salinarum in 1982 and 1985 as negative/positive and negative phototactic sensors, respectively.^8,9) In the cell membrane, SRI and SRII form complexes with their cognate transducer proteins, named Halobacterial transducer protein for SRI (HtrI) and for SRII (HtrII), respectively¹⁰⁾ (Fig. 1B). Light signals are transmitted from the SRI-HtrI and SRII-HtrII complexes to a cytoplasmic two-component signal transduction cascade composed of signaling molecules such as CheA and CheY. SRI and SRII regulate the rotational direction of the flagellar motor through activation of the cascade, which results in positive or negative phototaxis.¹⁰⁾ Thus, in H. salinarum, the four rhodopsins inherently control several biological activities through their different photo-induced molecular functions. Since then, numerous rhodopsin molecules have been identified from nature with a variety of distinct molecular functions, such as cation channels, anion channels, transcriptional regulators, enzymes, inward proton pumps, outward sodium pumps and a SO₄²⁻ transporter (Fig. 1B). For detailed biochemical and biophysical aspects of the above rhodopsins, please see other extensive reviews.^3,4)

Of note, in 2005, an innovative method using visible light to control biological activities was developed and named optogenetics, in which microbial rhodopsins are heterologously expressed in target cells using genetic methods.^11,12) In this review, we introduce representative rhodopsin molecules used for optogenetics.

2. MICROBIAL RHODOPSINS FOR OPTOGENETICS

2.1. Cation Channelrhodopsins (CCRs) for Neural Activation

In 2002 and 2003, channelrhodopsin-1 (CrChR1) and channelrhodopsin-2 (CrChR2) were identified in the single-cell algae Chlamydomonas reinhardtii.^13,14) CrChR1 and CrChR2 absorb blue light (around 460 nm) and work as light-gated cation (H⁺ and Na⁺) channels, which induce membrane depolarization through their inward cation (mainly Na⁺) transport in cells^13,14) (Fig. 2A and Table 1). In 2005, Boyden et al. focused on the characteristics of channelrhodopsins and produced rat hippocampal neurons expressing CrChR2 and successfully induced light-dependent neural activation via membrane depolarization as the first demonstration of optogenetics.¹¹⁾ After that, CrChR1 and CrChR2 (mainly CrChR2) have been introduced into various kinds of neurons (e.g., hippocampal neurons, retinal neurons, dopamine neurons and cortical neurons) by infection with a viral vector for the transient expression and generation of mouse transgenic lines^12,15,16) (Fig. 2B). The neural activities of neurons were then regulated by light to elucidate their functions and neural circuits not only in brain slices, but also in living animals (Fig. 2B). In addition to CrChR1 and CrChR2, various related molecules (called cation channelrhodopsins, CCRs) have been identified from nature. In addition to the natural CCRs, the production of genetically modified variants has been reported.^17,18) For instance, cation channelrhodopsins from C. noctigama and Tetraselmis striata (named Chrimson and TsChR, respectively) absorb red and blue light (around 590 and 440 nm, respectively), and are employed as red and blue-sensitive neural activators, respectively.¹⁸⁾ A cation channelrhodopsin from Stigeoclonium helveticum (named Chronos) shows fast channel closing kinetics, and is employed for high-frequency neural activation.¹⁸⁾ Moreover, C128T mutant of CrChR2 shows slow channel closing kinetics, and is employed for long-term neural activation.¹⁷⁾ These natural and non-natural molecules allow neuroscientists to precisely control neural activities with multi-color light (400–700 nm) and a wide time range (5 ms–100 s).

Fig. 2. Schematic Illustration of Optogenetic Neural Activation

(A) Cation channelrhodopsins (CCRs) induce inward cation transport across the cellular membrane in a light-dependent manner. This transport causes membrane depolarization to generate action potentials in neurons. (B) CCRs have been utilized as neural activation tools both in vitro and in vivo. The rhodopsin genes are introduced into target neurons of the brain by infection with virus vectors. The illumination of target neurons expressing CCRs induces neural activation even in free-moving mice. (Color figure can be accessed in the online version.)

Table 1. List of Representative Rhodopsin-Based Optogenetics Tools

Name	Molecular function	Application	References
Cation channelrhodopsins (CCRs)	Cation channel	Neural activation	11, 13–18, 20)
Halorhodopsin (HR)	Inward Cl− pump	Neural silencing	16, 20, 21)
Archaerhodopsin-3 (AR3 or Arch)	Outward H+ pump	Neural silencing	16, 23–25)
AR3 and its mutants (e.g., QuasAr2, QuasAr3, Archon-1 and Archon-2)	Membrane voltage indicator	Voltage imaging	26, 28, 29)
Anion channelrhodopsins (ACRs)	Anion channel	Neural silencing	30–35)
Anabaena sensory rhodopsin (ASR) and its mutant	Transcriptional regulator and inward H+ pump	Regulation of gene transcription, Inhibition of endocytosis	38, 40)

2.2. Halorhodopsin (HR) for Neural Silencing

Halorhodopsin (HR) from H. salinarum absorbs green light (around 580 nm) and works as a light-driven inward chloride pump.⁶⁾ Proteins homologous to HR have been identified from other archaea and bacteria.¹⁹⁾ In 2007, HR from the archaeon Natronomonas pharaonis (NpHR) was shown to induce the neural silencing of mouse hippocampal neurons via hyperpolarization of the membrane potential through its inward chloride transport²⁰⁾ (Fig. 3A and Table 1). Thus, NpHR was employed as the first neural silencing tool. After that, several modifications (e.g., membrane trafficking signals, ER export signals) have been applied to the natural NpHR gene to improve its expression level and localization in cellular membranes to enhance its neural silencing activity.²¹⁾ Thus, NpHR has been utilized as a neural silencer to regulate various kinds of neurons (e.g., hippocampal neurons, cholinergic neurons, dopaminergic neurons and primary somatosensory cortex) in brain slices and in living animals.^16,21)

Fig. 3. Schematic Illustration of Optogenetic Neural Silencing and Voltage Imaging

(A) NpHR and anion channelrhodopsins (ACRs) and AR3 induce inward anion transport and outward proton transport across the cellular membrane, respectively, in a light-dependent manner. That transport causes membrane hyperpolarization to suppress the generation of action potential in neurons. These rhodopsins have been utilized as neural silencing tools both in vitro and in vivo. (B) AR3 and its mutants, such as QuasAr2, QuasAr3, Archon-1 and Archon-2, show membrane voltage-dependent fluorescence. In the mutants, light-induced proton pump activities are eliminated. Since the fluorescence changes can trace the absolute membrane potential with high time resolution, AR3 and its mutants have been utilized for voltage imaging mainly in neurons. (Color figure can be accessed in the online version.)

2.3. Archaerhodopsin-3 (AR3) for Neural Silencing and Voltage Imaging

Archaerhodopsin-3 (AR3 or Arch) was found in the halophilic archaeon Halorubrum sodomense as a protein homologous to BR in 1999.²²⁾ AR3 absorbs green light (around 570 nm) and works as a light-driven outward proton pump like BR.²³⁾ In 2010, AR3 was shown to induce neural silencing via hyperpolarization through its outward proton transport (Fig. 3A and Table 1). Archaerhodopsin-T (Arch-T), which is a protein homologous to AR3 in Halorubrum strain TP009, and other outward proton pump rhodopsins (e.g., Leptosphaeria rhodopsin (LR) and thermophilic rhodopsin (TR)), were also shown to induce neural silencing.^24,25) Since AR3 is well expressed and localized in cellular membranes and induces a larger photocurrent compared to NpHR and other proton pump rhodopsins, it has been utilized as a typical neural silencer to regulate various kinds of neurons like NpHR.^16,23,24)

It should be noted that AR3 is also employed for voltage imaging of neurons²⁶⁾ (Fig. 3B and Table 1). Red light excitation of AR3 (around 560–620 nm) induces near-IR fluorescence (around 660–760 nm). That fluorescence is thought to be derived from a highly fluorescent photointermediate (Q-intermediate) during the photocycle and it is noteworthy that its intensity is highly membrane voltage-sensitive.²⁷⁾ Therefore, AR3 can detect absolute membrane voltages ranging from −150 to 150 mV, while typical voltage indicators, Ca²⁺-sensors, cannot detect membrane voltage changes below the threshold in neurons.²⁶⁾ In addition, AR3-based voltage imaging has several advantages as follows: (i) it can be expressed in targeted neurons using genetics, and (ii) it is possible to visualize the membrane voltage with a high temporal (500 µs–40 ms) resolution. While the wild-type AR3 has been utilized for voltage imaging,²⁶⁾ it shows light-induced proton pump activity and induce hyperpolarization. Thus, signals for voltage imaging with the wild-type AR3 contains unexpected effects from the membrane hyperpolarization. Recently, several AR3 mutants (e.g., QuasAr2, QuasAr3, Archon-1 and Archon-2) have been developed by random mutagenesis and directed evolution approaches.^28,29) The light-induced proton pump activities of the mutants are eliminated by introducing mutation at a counterion residue Asp85 in the engineered proteins, allowing to trace the real voltage changes. Those engineered proteins show higher fluorescence, signal-to-noise levels and with improved membrane localization. Thus, the engineered AR3-based voltage indicators have been employed for real-time imaging of neural activities in various kinds of neurons even in living animals.^28,29)

2.4. Anion Channelrhodopsins (ACRs) for Neural Silencing

In 2015, anion channelrhodopsin-1 and -2 (GtACR1 and GtACR2, respectively) were found in the cryptophyte alga Guillardia theta.³⁰⁾ GtACR1 and GtACR2 absorb green and blue light (around 510 and 470 nm, respectively) and work as light-gated anion channels.³⁰⁾ Before the discovery of GtACR1 and GtACR2, ion pump rhodopsins (i.e., AR3 and NpHR) have been utilized as popular neural silencers. Of note, the ion pump rhodopsins transport only one ion during each photocycle, while ion channel rhodopsins (e.g., GtACR1 and GtACR2) transport thousands of ions during each photocycle, leading to high photocurrent. Thus, GtACR1 and GtACR2 have been recruited as powerful neural silencers that can hyperpolarize the membrane through their inward anion (mainly Cl⁻) transport, since they induce approximately a 1000-fold larger photocurrent compared to AR3 in mammalian cells³⁰⁾ (Fig. 3A, Table 1). Various related molecules (called anion channelrhodopsins, ACRs) have been identified in nature.³¹⁾ In addition to the natural ACRs, the production of genetically modified variants has been reported that show characteristic molecular properties, such as red and blue light-absorption, fast-channel closing and slow-channel closing.^32–34) These natural and non-natural molecules allow neuroscientists to optically suppress neural activities with multi-color light (400–700 nm) and a wide time range (3 ms–30 s). In fact, ACRs have been utilized as efficient neural silencers to regulate various kinds of neurons even in living animals.^30,32,35) Nowadays, ACRs are widely employed as next generation neural silencers instead of NpHR and AR3.

Based on rhodopsin-based neural activation and silencing tools, the optogenetic manipulation of neural activities has been extensively applied to study fundamental neural and brain functions (e.g., memory, learning, motion, sleep, awakening and sexual behavior) and their related diseases.³⁶⁾

2.5. Anabaena Sensory Rhodopsin (ASR) for Transcriptional Regulation and Control of Endocytosis

We now introduce a method to optically control non-neural biological activity. In 2003, the first bacterial sensory rhodopsin was identified from the freshwater cyanobacterium Anabaena PCC7120, and was named Anabaena sensory rhodopsin (ASR).³⁷⁾ ASR is encoded in an operon, along with a gene for a small soluble cytoplasmic protein, named the ASR transducer (ASRT). In 2012, we found that ASR represses the transcription of the chromatic adaptive gene cpcB through its C-terminal region in a light-dependent manner, when it is heterologously expressed in Escherichia coli cells.³⁸⁾ When the target gene is replaced by an arbitrary gene, ASR can be utilized as an optical method to regulate the expression of that arbitrary protein in bacterial cells (Table 1).

In 2009, the D217E mutant of ASR was shown to work as a light-driven inward proton pump.³⁹⁾ That engineered molecule is the first inward proton pump rhodopsin. One research group ectopically expressed the ASR mutant in endosomes of Purkinje cell synapses⁴⁰⁾ (Fig. 4A, Table 1). Since acidification of the endosomal lumen is essential for endocytosis in synapses, the inward proton transport activity of the ASR mutant can de-acidify the endosomal lumen when exposed to light, which induces the inhibition of endocytosis.⁴⁰⁾ Thus, the ASR mutant has been utilized for the control of synaptic endocytosis, which led to understanding the role of AMPA receptor endocytosis in motor learning in Purkinje cells.⁴⁰⁾ Recently, two groups of natural inward proton pump rhodopsins, named xenorhodopsins (XeRs) and schizorhodopsins (SzRs), were identified from several archaeal and bacterial species^41,42) (Fig. 1B). The ASR mutant and other natural inward proton pump rhodopsins will be utilized as optical de-acidification tools in the endosomal lumen and as optical acidification tools in the intracellular region.

Fig. 4. Schematic Illustration of Optogenetic Control of Various Biological Activities

(A) ASR mutant induces inward proton transport across the endosomal membrane in a light-dependent manner. This proton transport causes the de-acidification of the endosomal lumen, which results in the inhibition of endocytosis in the synapse. (B) Outward and inward proton pump rhodopsins actively transport protons across the membrane, which induce intracellular alkalization and acidification, respectively, in a light-dependent manner. Since the intracellular pH value is related to cell homeostasis and cell death, the proton pump rhodopsins will enable us to optically control cell survival and death through intracellular pH changes. (C) Enzyme rhodopsins regulate intracellular concentrations of cGMP and cAMP in a light-dependent manner. Such types of rhodopsins will enable us to optically control the activation of kinases, increase intracellular Ca²⁺ concentrations and regulate transcription through the intracellular cyclic nucleotide concentration changes. (Color figure can be accessed in the online version.)

3. FUTURE DIRECTIONS

Since the 21st century, advances in genomic analysis have revealed that more than 100000 microbial rhodopsin genes exist in nature.^3,4) As expansions of molecular diversity, many characteristic molecules showing novel functions have been identified. For instance, rhodopsins possessing catalytic domains of guanylyl/adenylyl cyclase and phosphodiesterase (called enzyme rhodopsins or Rh-GC, Rh-AC and Rh-PDE) were shown to function as a light-dependent enzymatic activity, which controls the intracellular concentrations of the cyclic nucleotides, cGMP and cAMP^43,44) (Fig. 1B). As ion transport rhodopsins, outward sodium pump rhodopsins (NaRs) and a SO₄^2- transport rhodopsin have been discovered in nature^45,46) (Fig. 1B). By introducing amino acid mutations in natural NaRs, outward potassium (K⁺) and cesium pump (Cs⁺) molecules have been engineered.⁴⁷⁾ Moreover, CrChR2 and its variants are permeable to Ca²⁺, although their permeability was still lower than those of other cations such as Na⁺ and H⁺.^14,48) The production of improved mutants showing higher Ca²⁺ permeability would be useful to optically control Ca²⁺ concentrations in living cells and in animals. Thus, the functional diversity of rhodopsins is progressively expanding, which allows us to optically control the intracellular concentrations of a variety of physiologically important ions and cyclic nucleotides (e.g., H⁺, Cl⁻, Na⁺, K⁺, cGMP and cAMP).

Among them, intracellular concentrations of H⁺ are strictly regulated to maintain the physiological pH (around 7.4) in mammalian cells. The alteration of intracellular pH would lead to the disruption of homeostasis and cell death.⁴⁹⁾ We expect that outward and inward proton pump rhodopsins will enable us to optically control cell death and survival through intracellular pH changes (Fig. 4B). As another example, cGMP and cAMP play roles as second messengers to induce various signal transduction cascades, such as the activation of kinases, increase of intracellular Ca²⁺ concentrations and transcriptional regulation⁵⁰⁾ (Fig. 4C). These cascades are universally involved in regulating basic cellular activities, such as cell development, differentiation, growth and death. It is expected that enzyme rhodopsins will be utilized as optical tools to control broad cellular activities through changes of intracellular cyclic nucleotide concentrations.

The disruption of fundamental cellular activities leads to severe diseases such as cancers and immune disorders. The rhodopsin-based optogenetics tools allow us not only to understand the molecular mechanisms of various cellular activities, but also to develop medicinal products for diseases.^3,36) In combination with gene therapy, optogenetics has a potential for therapeutics in which abnormal cellular activities can be rescued and improved by the functions of light-dependent rhodopsins. Recently, it was reported that visual functions were partially recovered in blind patients by introducing the cation channelrhodopsin gene in retinal ganglion cells where light stimulation of the retina activates exogeneous channelrhodopsins to induce electronic signals.⁵¹⁾

In summary, optogenetics can broadly contribute to basic and clinical research in pharmaceutical sciences. We hope that the expanding optogenetic applications of microbial rhodopsins will shed light on new approaches for drug discovery and therapeutics.

Acknowledgments

Our original publications were partially supported by a Grant-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (JP19H04727, JP19H05396, JP20K21482, 21H00404 and 21H02446), JST-CREST (JPMJCR1656) and AMED (20dm0207060h0004) to YS.

Conflict of Interest

The authors declare no conflict of interest.

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