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The channelrhodopsins and their distinctive light-activated ion channels have emerged as major tools in modern biological research. Deisseroth and Hegemann review the structural and functional properties of these protein photoreceptors. Mutagenesis and modeling studies, coupled with the reintroduction of modified channels into living systems, offer a profound understanding of how these channels work. The insights into the underlying basic science provide foundations for developing further applications in biology and medicine. Science , this issue p. eaan5544 Channelrhodopsins are light-gated ion channels that, via regulation of flagellar function, enable single-celled motile algae to seek ambient light conditions suitable for photosynthesis and survival. These plant behavioral responses were initially investigated more than 150 years ago. Recently, major principles of function for light-gated ion channels have been elucidated by creating channelrhodopsins with kinetics that are accelerated or slowed over orders of magnitude, by discovering and designing channelrhodopsins with altered spectral properties, by solving the high-resolution channelrhodopsin crystal structure, and by structural model–guided redesign of channelrhodopsins for altered ion selectivity. Each of these discoveries not only revealed basic principles governing the operation of light-gated ion channels, but also enabled the creation of new proteins for illuminating, via optogenetics, the fundamentals of brain function.

There is as yet no consensus concerning the neural basis of perception and how it operates at a mechanistic level. We found that Ca²⁺ activity in the apical dendrites of a subset of layer 5 (L5) pyramidal neurons in primary somatosensory cortex (S1) in mice is correlated with the threshold for perceptual detection of whisker deflections. Manipulating the activity of apical dendrites shifted the perceptual threshold, demonstrating that an active dendritic mechanism is causally linked to perceptual detection.

Even though a lot was already known about BR, the exact voltage dependence of proton pumping was unclear. [...]Ernst Bamberg and Georg Nagel decided to study BR in the membrane of an animal cell, the oocyte of Xenopus laevis (Fig 1B). Besides the OH‐cluster, two residues, C128 and D156 (DC‐pair in Fig 2) are of fundamental importance for both channel opening and closing, and mutation of either residue results in a dramatic increase of the open state(s)' lifetime. [...]greater selectivity for K+ over Na+, to be used for light‐controlled hyperpolarization of host cells, will be very difficult to achieve. [...]the highly appreciated red‐shifted absorption is limited to around 630 nm due to thermal activation (dark noise) of red light‐absorbing rhodopsins even when synthetic retinal analogues are used as chromophores. ChRs will be further optimized for two‐photon microscopy and many novel unprecedented variants will be identified. [...]ChRs may become commonly used analytical tools or even therapeutics for treating specific diseases.

Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Red-light-activated channelrhodopsins are of particular interest, because red light penetrates deeper into biological tissues and also enables dual-color experiments in combination with blue-light-activated optogenetic tools. Here we report the crystal structure of the most red-shifted channelrhodopsin from the algae Chlamydomonas noctigama , Chrimson, at 2.6 Å resolution. Chrimson resembles prokaryotic proton pumps in the retinal binding pocket, while sharing similarity with other channelrhodopsins in the ion-conducting pore. Concomitant mutation analysis identified the structural features that are responsible for Chrimson’s red light sensitivity; namely, the protonation of the counterion for the retinal Schiff base, and the polar residue distribution and rigidity of the retinal binding pocket. Based on these mechanistic insights, we engineered ChrimsonSA, a mutant with a maximum activation wavelength red-shifted beyond 605 nm and accelerated closing kinetics. Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Here, the authors report the crystal structure of the most red-shifted channelrhodopsin from the algae Chlamydomonas noctigama at 2.6 Å resolution.

Channelrhodopsins are light-gated ion channels of green algae used for the precise temporal and spatial control of transmembrane ion fluxes. The channelrhodopsin Chrimson from Chlamydomonas noctigama allows unprecedented deep tissue penetration due to peak absorption at 590 nm. We demonstrate by electrophysiological recordings and imaging techniques that Chrimson is highly proton selective causing intracellular acidification in HEK cells that is responsible for slow photocurrent decline during prolonged illumination. We localized molecular determinants of both high proton selectivity and red light activation to the extracellular pore. Whereas exchange of Glu143 only drops proton conductance and generates an operational Na-channel with 590 nm activation, exchange of Glu139 in addition increased the open state lifetime and shifted the absorption hypsochromic by 70 nm. In conjunction with Glu300 in the center and Glu124 and Glu125 at the intracellular end of the pore, Glu139 contributes to a delocalized activation gate and stabilizes by long-range interaction counterion configuration involving protonation of Glu165 that we identified as a key determinant of the large opsin shift in Chrimson.

Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30–80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms. Brain imbalance in autism One model for the cellular disturbances underlying social and emotional deficits in disorders such as autism and schizophrenia is an imbalance in excitatory and inhibitory activity in certain neural systems. This idea has not been directly testable so far, but testability comes a little closer with the development of two optogenetic tools that have different spectral and temporal characteristics, thereby allowing selective control of two intermingled populations of neurons. Use of these new opsins shows that increasing relative excitation in mouse prefrontal cortex impairs social and learning behaviours. This provides support for the elevated cellular excitatory/inhibitory balance hypothesis of certain neuropsychiatric symptoms.

Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using singleturnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H⁺ and Na⁺ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an openchannel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets. Currently, bidirectional control of activity in the same neurons in the same experiment is difficult. Here the authors report a Bidirectional Pair of Opsins for Light-induced Excitation and Silencing, BiPOLES, which they use in a range of organisms including worms, fruit flies, mice and ferrets.

The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins ( λ max  = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared-sensitive (NIR, λ max  = 690 nm) highly fluorescent state ( Q F  = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These findings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors. Rhizoclosmatium globosum contains three rhodopsin-guanylyl cyclases (RGCs) predicted to enable visual orientation of zoospores. Here authors show that RGC1 and 2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR), a near-infrared absorbing, highly fluorescent rhodopsin.

Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii ) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties. Channelrhodopsins are light-gated cation channels used in optogenetics; here, the high-resolution crystal structure of a channelrhodopsin from Chlamydomonas reinhardtii is determined. Structure of key optogenetics reagent The channelrhodopsins are light-gated ion channels, found in algae, that have rapidly become familiar in the neuroscience lab as optogenetics reagents: the activities of neurons expressing channelrhodopsins can be optically controlled within systems as complicated as living mammals. The X-ray crystal structure of a chimaera of two channelrhodopsins has now been determined at 2.3 Å resolution. The structure reveals the molecular architecture of this ion channel, including the retinal-binding pocket and cation conduction pathway. This work paves the way for the design of new channelrhodopsin variants with enhanced properties.