Optogenetic tools enable the causal examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the examination of how different synapses or pathways interact to support computation. Here we report two new channelrhodopsins, Chronos and Chrimson, obtained through the de novo sequencing and physiological characterization of opsins from over 100 species of algae. Chrimson is 45 nm red-shifted relative to any previous channelrhodopsin, important for scenarios where red light would be preferred; we show minimal visual system mediated behavioral artifact in optogenetically stimulated Drosophila. Chronos has faster kinetics than any previous channelrhodopsin, yet is effectively more light-sensitive. Together, these two reagents enable crosstalk-free two-color activation of neural spiking and downstream synaptic transmission in independent neural populations in mouse brain slice.

AMPA receptors are glutamate-gated ion channels that are essential mediators of synaptic signals in the central nervous system. They form tetramers that are assembled as combinations of the subunits GluR1-4, each of which contains a ligand-binding domain (LBD). Crystal structures of the GluR2 LBD have revealed an agonist-binding cleft, which is located between two lobes and which acts like a Venus flytrap. In general, agonist efficacy is correlated with the extent of cleft closure. However, recent observations show that cleft closure is not the sole determinant of relative efficacy for glutamate receptors. In addition, these studies have focused on the GluR2 subunit, which is the specific target of a physiologically important RNA-editing modification in vivo. We therefore wished to test the generality of the cleft closure:efficacy correlation for other AMPA-R subunits. Here, we present crystal structures of the GluR4 flip LBD in complex with both full and partial agonists. As for GluR2, both agonists stabilize a closed-cleft conformation, and the partial agonist induces a smaller cleft closure than the full agonist. However, a detailed analysis of LBD:kainate interactions reveals the importance of subtle backbone conformational changes in the ligand-binding pocket in determining the magnitude of agonist-associated conformational changes. Furthermore, the GluR4 subunit exhibits a different correlation between receptor activation and LBD cleft closure than does GluR2. Keywordsionotropic glutamate receptor; AMPA receptor ion channel; GluR4 ligand-binding domain; X-ray crystallography; electrophysiology; subunit dimerization; relative agonist efficacy; conformational change Within the central nervous system, most fast excitatory synaptic signals are mediated by ionotropic glutamate receptors (iGluRs) that are selective for the synthetic agonist AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) (1). In addition to their role in neurotransmission, the AMPA receptors (AMPA-Rs) contribute to synaptic plasticity and are thus thought to play important roles in learning and memory (2). AMPA-Rs have also been † A.G. was supported in part by the John H. Copenhaver, Jr., and William H. Thomas, M.D., 1952 Fellowship Fund and AMPA-Rs are assembled as tetramers of subunits known as GluR1-GluR4. The composition of the receptors in a given neuron depends on which subunits are expressed, and on a variety of post-transcriptional modifications, including alternative splicing and RNA editing (reviewed in ref. 1). A particularly important modification involves the selective RNA editing of the mRNA encoding the GluR2 subunit, which converts a genetically encoded Gln codon common to all subunits to an Arg codon at position 586 in the mature sequence of GluR2. This editing is nearly 100% efficient in adults. Heteromeric channels that include edited GluR2-R subunits are the most common form of AMPA-R in vivo. They are non-rectifying and conduct predominantly monovalent cations. In contrast, AMPA-Rs that do not include...

AMPA receptors (AMPARs) are tetrameric ligand-gated ion channels that couple the energy of glutamate binding to the opening of a transmembrane channel. Crystallographic and electrophysiological analysis of AMPARs has suggested a coupling between (1) cleft closure in the bilobate ligand-binding domain (LBD), (2) the resulting separation of transmembrane helix attachment points across subunit dimers, and (3) agonist efficacy. In general, more efficacious agonists induce greater degrees of cleft closure and transmembrane separation than partial agonists. Several apparent violations of the cleft-closure/efficacy paradigm have emerged, although in all cases, intradimer separation remains as the driving force for channel opening. Here, we examine the structural basis of partial agonism in GluA4 AMPARs. We find that the L651V substitution enhances the relative efficacy of kainate without increasing either LBD cleft closure or transmembrane separation. Instead, the conformational change relative to the wild-type:kainate complex involves a twisting motion with the efficacy contribution opposite from that expected based on previous analyses. As a result, channel opening may involve transmembrane rearrangements with a significant rotational component. Furthermore, a two-dimensional analysis of agonist-induced GluA2 LBD motions suggests that efficacy is not a linearly varying function of lobe 2 displacement vectors, but is rather determined by specific conformational requirements of the transmembrane domains.

all variants to have similar photocurrents in cultured neurons (Fig. 1d,e). However, we observed more cytosolic aggregates with the KGC version and a reduction of aggregates with the ER2 version (Supplementary Fig. 2). It is therefore likely that CsChrimson will be of use with the ER2 trafficking sequence in some biological contexts. methods Methods and any associated references are available in the online version of the paper. Accession codes. GenBank/EMBL/DDBJ: CsChrimson is listed under accession code KJ995863.

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