Crystal structure of the natural anion-conducting channelrhodopsin GtACR1.
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ABSTRACT:
SUBMITTER: Kim YS
PROVIDER: S-EPMC6340299 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.
| S-EPMC6336409 | biostudies-literature
Project description:Natural anion channelrhodopsins (ACRs) recently discovered in cryptophyte algae are the most active rhodopsin channels known. They are of interest both because of their unique natural function of light-gated chloride conductance and because of their unprecedented efficiency of membrane hyperpolarization for optogenetic neuron silencing. Light-induced currents of ACRs have been studied in HEK cells and neurons, but light-gated channel conductance of ACRs in vitro has not been demonstrated. Here we report light-induced chloride channel activity of a purified ACR protein reconstituted in large unilamellar vesicles (LUVs). EPR measurements establish that the channels are inserted uniformly "inside-out" with their cytoplasmic surface facing the medium of the LUV suspension. We show by time-resolved flash spectroscopy that the photochemical reaction cycle of a functional purified ACR from Guillardia theta (GtACR1) in LUVs exhibits similar spectral shifts, indicating similar photocycle intermediates as GtACR1 in detergent micelles. Furthermore, the photocycle rate is dependent on electric potential generated by chloride gradients in the LUVs in the same manner as in voltage-clamped animal cells. We confirm with this system that, in contrast to cation-conducting channelrhodopsins, opening of the channel occurs prior to deprotonation of the Schiff base. However, the photointermediate transitions in the LUVs exhibit faster kinetics. The ACR-incorporated LUVs provide a purified defined system amenable to EPR, optical and vibrational spectroscopy, and fluorescence resonance energy transfer measurements of structural changes of ACRs with the molecules in a demonstrably functional state.
| S-EPMC5207235 | biostudies-literature
Project description:Optogenetic silencing allows to reveal the necessity of selected neuronal populations for various neurophysiological functions. These range from synaptic transmission and coordinated neuronal network activity to control of specific behaviors. An ideal single-component optogenetic silencing tool should be switchable between active and inactive states with precise timing while preserving its activity in the absence of light until switched to an inactive state. Although bistable anion-conducting channelrhodopsins (ACRs) were previously engineered to reach this goal, their conducting state lifetime was limited to only a few minutes and some ACRs were not fully switchable. Here we report Aion, a bistable ACR displaying a long-lasting open state with a spontaneous closing time constant close to 15 min. Moreover, Aion can be switched between the open and closed state with millisecond precision using blue and orange light, respectively. The long conducting state enables overnight silencing of neurons with minimal light exposure. We further generated trafficking-optimized versions of Aion, which show enhanced membrane localization and allow precisely timed, long-lasting all-optical control of nociceptive responses in larvae of Drosophila melanogaster. Thus, Aion is an optogenetic silencing tool for inhibition of neuronal activity over many hours which can be switched between an active and inactive state with millisecond precision.
| S-EPMC9271052 | biostudies-literature
Project description:Chloride conducting channelrhodopsins (ChloCs) are new members of the optogenetic toolbox that enable neuronal inhibition in target cells. Originally, ChloCs have been engineered from cation conducting channelrhodopsins (ChRs), and later identified in a cryptophyte alga genome. We noticed that the sequence of a previously described Proteomonas sulcata ChR (PsChR1) was highly homologous to the naturally occurring and previously reported ChloCs GtACR1/2, but was not recognized as an anion conducting channel. Based on electrophysiological measurements obtained under various ionic conditions, we concluded that the PsChR1 photocurrent at physiological conditions is strongly inward rectifying and predominantly carried by chloride. The maximum activation was noted at excitation with light of 540 nm. An initial spectroscopic characterization of purified protein revealed that the photocycle and the transport mechanism of PsChR1 differ significantly from cation conducting ChRs. Hence, we concluded that PsChR1 is an anion conducting ChR, now renamed PsACR1, with a red-shifted absorption suited for multicolor optogenetic experiments in combination with blue light absorbing cation conducting ChRs.
| S-EPMC4759187 | biostudies-literature
Project description: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.
| S-EPMC4160518 | biostudies-literature
Project description: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.
| S-EPMC6158191 | biostudies-literature
Project description:Natural channelrhodopsins with strictly anion selectivity and high unitary conductance have been recently discovered in the cryptophyte alga Guillardia theta. These proteins, called anion channelrhodopsins (ACRs), are of interest for their novel function and also because they were shown to be highly efficient tools to inhibit neuronal action potentials with light. We show that a homologous protein from the cryptophyte alga Proteomonas sulcata (named here PsuACR1) exhibits similar strict anion selectivity as the previously identified G. theta ACRs. Like G. theta ACRs, PsuACR1 lacks a protonatable residue at the position of the proton acceptor Asp-85 in bacteriorhodopsin, which may be a key characteristic of ACR family members shared by haloarchaeal chloride pumps. Of importance for its potential use in optogenetics, despite its 10-fold lower channel activity than the GtACRs, PsuACR1 exhibits an ~eightfold more rapid channel closing half-time making it uniquely suitable for silencing the subclass of high-frequency firing neurons when high-time resolution is needed. The existence of a rhodopsin with properties similar to G. theta ACRs in a different cryptophyte genus indicates that such proteins may be widespread in the phylum of cryptophyte algae.
| S-EPMC4914479 | biostudies-literature
Project description:Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.
| S-EPMC8691836 | biostudies-literature
Project description:A recently discovered family of natural anion channelrhodopsins (ACRs) have the highest conductance among channelrhodopsins and exhibit exclusive anion selectivity, which make them efficient inhibitory tools for optogenetics. We report analysis of flash-induced absorption changes in purified wild-type and mutant ACRs, and of photocurrents they generate in HEK293 cells. Contrary to cation channelrhodopsins (CCRs), the ion conducting state of ACRs develops in an L-like intermediate that precedes the deprotonation of the retinylidene Schiff base (i.e., formation of an M intermediate). Channel closing involves two mechanisms leading to depletion of the conducting L-like state: (i) Fast closing is caused by a reversible L?M conversion. Glu-68 in Guillardia theta ACR1 plays an important role in this transition, likely serving as a counterion and proton acceptor at least at high and neutral pH. Incomplete suppression of M formation in the GtACR1_E68Q mutant indicates the existence of an alternative proton acceptor. (ii) Slow closing of the channel parallels irreversible depletion of the M-like and, hence, L-like state. Mutation of Cys-102 that strongly affected slow channel closing slowed the photocycle to the same extent. The L and M intermediates were in equilibrium in C102A as in the WT. In the position of Glu-123 in channelrhodopsin-2, ACRs contain a noncarboxylate residue, the mutation of which to Glu produced early Schiff base proton transfer and strongly inhibited channel activity. The data reveal fundamental differences between natural ACR and CCR conductance mechanisms and their underlying photochemistry, further confirming that these proteins form distinct families of rhodopsin channels.
| S-EPMC4833267 | biostudies-literature
Project description:Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin GtACR1 from the green alga Guillardia theta. The growth of Nicotiana tabacum pollen tube can then be manipulated by localized green light illumination. To extend the application of analogous optogenetic tools in the pollen tube system, we engineered another two ACRs, GtACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in Xenopus laevis oocytes, Nicotiana benthamiana leaves and N. tabacum pollen tubes. We found that the similar molecular engineering method used to improve GtACR1 also enhanced GtACR2 and ZipACR performance in Xenopus laevis oocytes. The ZipACR1 performed in N. benthamiana mesophyll cells and N. tabacum pollen tubes with faster kinetics and reduced light sensitivity, allowing for optogenetic control of anion fluxes with better temporal resolution. The reduced light sensitivity would potentially facilitate future application in plants, grown under low ambient white light, combined with an optogenetic manipulation triggered by stronger green light.
| S-EPMC8070814 | biostudies-literature