Elena G. Govorunova

Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics

Silencing neurons using optogenetics Rhodopsin light-sensitive ion channels from green algae provide a powerful tool to control neuronal circuits. Rhodopsin cation channels effectively depolarize neurons and cause the firing of short-lived electrical membrane potentials. Govorunova et al. describe algal channels that do the opposite; that is, they hyperpolarize or silence particular neurons (see the Perspective by Berndt and Deisseroth). These cation channels provide greater light sensitivity than that of existing hyperpolarizing light-activated channels, operate rapidly, and selectively conduct only anions. This approach is an ideal complement to the widely used technique of creating light-sensitive neurons through the expression of rhodopsin cation channels. Science, this issue p. 647; see also p. 590 An anion channel from algae allows optogenetic silencing of neurons. [Also see Perspective by Berndt and Deisseroth] Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from Mesostigma viride

ABSTRACT Light control of motility behavior (phototaxis and photophobic responses) in green flagellate algae is mediated by sensory rhodopsins homologous to phototaxis receptors and light-driven ion transporters in prokaryotic organisms. In the phototaxis process, excitation of the algal sensory rhodopsins leads to generation of transmembrane photoreceptor currents. When expressed in animal cells, the algal phototaxis receptors function as light-gated cation channels, which has earned them the name “channelrhodopsins.” Channelrhodopsins have become useful molecular tools for light control of cellular activity. Only four channelrhodopsins, identified in Chlamydomonas reinhardtii and Volvox carteri, have been reported so far. By screening light-induced currents among algal species, we identified that the phylogenetically distant flagellate Mesostigma viride showed photoelectrical responses in vivo with properties suggesting a channelrhodopsin especially promising for optogenetic use. We cloned an M. viride channelrhodopsin, MChR1, and studied its channel activity upon heterologous expression. Action spectra in HEK293 cells match those of the photocurrents observed in M. viride cells. Comparison of the more divergent MChR1 sequence to the previously studied phylogenetically clustered homologs and study of several MChR1 mutants refine our understanding of the sequence determinants of channelrhodopsin function. We found that MChR1 has the most red-shifted and pH-independent spectral sensitivity so far reported, matches or surpasses known channelrhodopsins’ channel kinetics features, and undergoes minimal inactivation upon sustained illumination. This combination of properties makes MChR1 a promising candidate for optogenetic applications. IMPORTANCE Channelrhodopsins that function as phototaxis receptors in flagellate algae have recently come into the spotlight as genetically encoded single-molecule optical switches for turning on neuronal firing or other cellular processes, a technique called “optogenetics.” Only one of four currently known channelrhodopsins is widely used in optogenetics, although electrical currents recorded in diverse flagellates suggest the existence of a large variety of such proteins. We applied a strategy for the search for new channelrhodopsins with desirable characteristics by measuring rhodopsin-mediated photocurrents in microalgae, which helped us identify MChR1, a new member of the channelrhodopsin family. MChR1 exhibits several sought-after characteristics and thus expands the available optogenetic toolbox. The divergence of the MChR1 sequence from those of the four known channelrhodopsins contributes to our understanding of diversity in the primary structures of this subfamily of sensory rhodopsins. Channelrhodopsins that function as phototaxis receptors in flagellate algae have recently come into the spotlight as genetically encoded single-molecule optical switches for turning on neuronal firing or other cellular processes, a technique called “optogenetics.” Only one of four currently known channelrhodopsins is widely used in optogenetics, although electrical currents recorded in diverse flagellates suggest the existence of a large variety of such proteins. We applied a strategy for the search for new channelrhodopsins with desirable characteristics by measuring rhodopsin-mediated photocurrents in microalgae, which helped us identify MChR1, a new member of the channelrhodopsin family. MChR1 exhibits several sought-after characteristics and thus expands the available optogenetic toolbox. The divergence of the MChR1 sequence from those of the four known channelrhodopsins contributes to our understanding of diversity in the primary structures of this subfamily of sensory rhodopsins.

Chlamydomonas Sensory Rhodopsins A and B: Cellular Content and Role in Photophobic Responses

Two retinylidene proteins, CSRA and CSRB, have recently been shown by photoelectrophysiological analysis of RNAi-transformants to mediate phototaxis signaling in Chlamydomonas reinhardtii. Here we report immunoblot detection of CSRA and CSRB apoproteins in C. reinhardtii cells enabling assessment of the cellular content of the receptors. We obtain 9 x 10(4) CSRA and 1.5 x 10(4) CSRB apoprotein molecules per cell in vegetative cells of the wild-type strain 495, a higher value than that for functional receptor cellular content estimated previously from photosensitivity measurements and retinal extraction yields. Exploiting our ability to control the CSRA/CSRB ratio by transformation with receptor gene-directed RNAi, we report analysis of the CSRA and CSRB roles in the photophobic response of the organism by action spectroscopy with automated cell tracking/motion analysis. The results show that CSRA and CSRB each mediate the photophobic swimming response, a second known retinal-dependent photomotility behavior in C. reinhardtii. Due to the different light saturation and spectral properties of the two receptors, CSRA is dominantly responsible for photophobic responses, which appear at high light intensity.

Rhodopsin-mediated photosensing in green flagellated algae

Green flagellated algae possess a primitive visual system that regulates the activity of their motor apparatus. Photoexcitation of a rhodopsin-type photoreceptor protein gives rise to the photoreceptor current, which, above a certain threshold of stimulus intensity, induces the flagellar current. It is probable that the photoinduced alteration in flagellar beating is governed by changes in intracellular Ca2+ concentration. This rhodopsin-mediated sensory system serves to align the swimming path with the direction of the light stimulus, whereas processes of energy metabolism determine whether the oriented movement is directed towards or away from the light source.

Characterization of a highly efficient blue-shifted channelrhodopsin from the marine alga Platymonas subcordiformis.

Background: Channelrhodopsins are algal phototaxis receptors used in optogenetics. Results: Channel activity and photochemistry of a new channelrhodopsin (PsChR) are characterized. Conclusion: Blue-shifted PsChR has ∼3-fold greater unitary conductance, faster recovery from excitation, and higher sodium selectivity than channelrhodopsin 2 from Chlamydomonas. Significance: These properties of PsChR facilitate further analysis of light-gated channel function and are potentially useful for optogenetics. Rhodopsin photosensors of phototactic algae act as light-gated cation channels when expressed in animal cells. These proteins (channelrhodopsins) are extensively used for millisecond scale photocontrol of cellular functions (optogenetics). We report characterization of PsChR, one of the phototaxis receptors in the alga Platymonas (Tetraselmis) subcordiformis. PsChR exhibited ∼3-fold higher unitary conductance and greater relative permeability for Na+ ions, as compared with the most frequently used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Photocurrents generated by PsChR in HEK293 cells showed lesser inactivation and faster peak recovery than those by CrChR2. Their maximal spectral sensitivity was at 445 nm, making PsChR the most blue-shifted channelrhodopsin so far identified. The λmax of detergent-purified PsChR was 437 nm at neutral pH and exhibited red shifts (pKa values at 6.6 and 3.8) upon acidification. The purified pigment undergoes a photocycle with a prominent red-shifted intermediate whose formation and decay kinetics match the kinetics of channel opening and closing. The rise and decay of an M-like intermediate prior to formation of this putative conductive state were faster than in CrChR2. PsChR mediated sufficient light-induced membrane depolarization in cultured hippocampal neurons to trigger reliable repetitive spiking at the upper threshold frequency of the neurons. At low frequencies spiking probability decreases less with PsChR than with CrChR2 because of the faster recovery of the former. Its blue-shifted absorption enables optogenetics at wavelengths even below 400 nm. A combination of characteristics makes PsChR important for further research on structure-function relationships in ChRs and potentially useful for optogenetics, especially for combinatorial applications when short wavelength excitation is required.

Photoelectric responses in phototactic flagellated algae measured in cell suspension

Abstract A new method for the investigation of electric responses involved in the light reception in microorganisms has been developed. It is based on the detection of photoelectric signals in suspensions of cells (instead of a single cell) by two different techniques: (a) by unilateral excitation of non-oriented cells and (b) after preorientation of the cells (e.g. by gravitaxis or weak, phototactically active light). The method was applied to the flagellated green algae Haematococcus and Chlamydomonas (and several of its mutants). Three main components of the electric signal, which differs in their origin and the mechanisms underlying that registration, were identified. Fast (microsecond) responses reflect charge separation in reaction centres of photosynthesis and are due to the classical light gradient effect on unilateral flash excitation. The later components of the electric signal are involved in photoreception and represent the photoreceptor potential of phototaxis and the calcium-dependent regenerative response. They are measured because of the directional sensitivity of the photoreceptor antenna and the asymmetry of localization of the electric currents involved in the sensory transduction chain. A general similarity between the electric responses in both organisms shows that the sensory transduction chain of photomovements in Chlamydomonas is similar to that described previously for Haematococcus. The advantages of the proposed method are discussed.

Intramolecular proton transfer in channelrhodopsins.

Channelrhodopsins serve as photoreceptors that control the motility behavior of green flagellate algae and act as light-gated ion channels when heterologously expressed in animal cells. Here, we report direct measurements of proton transfer from the retinylidene Schiff base in several channelrhodopsin variants expressed in HEK293 cells. A fast outward-directed current precedes the passive channel current that has the opposite direction at physiological holding potentials. This rapid charge movement occurs on the timescale of the M intermediate formation in microbial rhodopsins, including that for channelrhodopsin from Chlamydomonas augustae and its mutants, reported in this study. Mutant analysis showed that the glutamate residue corresponding to Asp(85) in bacteriorhodopsin acts as the primary acceptor of the Schiff-base proton in low-efficiency channelrhodopsins. Another photoactive-site residue corresponding to Asp(212) in bacteriorhodopsin serves as an alternative proton acceptor and plays a more important role in channel opening than the primary acceptor. In more efficient channelrhodopsins from Chlamydomonas reinhardtii, Mesostigma viride, and Platymonas (Tetraselmis) subcordiformis, the fast current was apparently absent. The inverse correlation of the outward proton transfer and channel activity is consistent with channel function evolving in channelrhodopsins at the expense of their capacity for active proton transport.

Diversity of Chlamydomonas Channelrhodopsins

Channelrhodopsins act as photoreceptors for control of motility behavior in flagellates and are widely used as genetically targeted tools to optically manipulate the membrane potential of specific cell populations (“optogenetics”). The first two channelrhodopsins were obtained from the model organism Chlamydomonas reinhardtii (CrChR1 and CrChR2). By homology cloning we identified three new channelrhodopsin sequences from the same genus, CaChR1, CyChR1 and CraChR2, from C. augustae, C. yellowstonensis and C. raudensis, respectively. CaChR1 and CyChR1 were functionally expressed in HEK293 cells, where they acted as light‐gated ion channels similar to CrChR1. However, both, which are similar to each other, differed from CrChR1 in current kinetics, inactivation, light intensity dependence, spectral sensitivity and dependence on the external pH. These results show that extensive channelrhodopsin diversity exists even within the same genus, Chlamydomonas. The maximal spectral sensitivity of CaChR1 was at 520 nm at pH 7.4, about 40 nm redshifted as compared to that of CrChR1 under the same conditions. CaChR1 was successfully expressed in Pichia pastoris and exhibited an absorption spectrum identical to the action spectrum of CaChR1‐generated photocurrents. The redshifted spectra and the lack of fast inactivation in CaChR1‐ and CyChR1‐generated currents are features desirable for optogenetics applications.

Photosensory Functions of Channelrhodopsins in Native Algal Cells

Photomotility responses in flagellate alga are mediated by two types of sensory rhodopsins (A and B). Upon photoexcitation they trigger a cascade of transmembrane currents which provide sensory transduction of light stimuli. Both types of algal sensory rhodopsins demonstrate light‐gated ion channel activities when heterologously expressed in animal cells, and therefore they have been given the alternative names channelrhodopsin 1 and 2. In recent publications their channel activity has been assumed to initiate the transduction chain in the native algal cells. Here we present data showing that: (1) the modes of action of both types of sensory rhodopsins are different in native cells such as Chlamydomonas reinhardtii than in heterologous expression systems, and also differ between the two types of rhodopsins; (2) the primary function of Type B sensory rhodopsin (channelrhodopsin‐2) is biochemical activation of secondary Ca2+‐channels with evidence for amplification and a diffusible messenger, sufficient for mediating phototaxis and photophobic responses; (3) Type A sensory rhodopsin (channelrhodopsin‐1) mediates avoidance responses by direct channel activity under high light intensities and exhibits low‐efficiency amplification. These dual functions of algal sensory rhodopsins enable the highly sophisticated photobehavior of algal cells.

Chemotaxis in the green flagellate alga Chlamydomonas

Behavior of the green flagellate alga Chlamydomonas changes in response to a number of chemical stimuli. Specific sensitivity of the cells to different substances might appear only at certain stages of the life cycle. The heterogamous species C. allensworthii demonstrates chemotaxis of male gametes towards pheromones excreted by female gametes. In C. reinhardtii chemotaxis towards tryptone occurs only in gametes, whereas chemotaxis towards ammonium, on the contrary, only in vegetative cells. Chemotaxis to different chemical stimuli might involve different mechanisms of reception and signal transduction, elucidation of which has only recently begun. Indirect evidences show that the cells likely respond to tryptone with changes in the membrane electrical conductance. The recently completed project of sequencing the whole nuclear genome of C. reinhardtii provides the basis for future identification of molecular elements of the chemosensory cascade in this alga.