Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity

Abstract

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structurefunction relationships of the light-gated pore.iscovery and engineering of the microbial opsin genes not only has stimulated basic science investigation into the structure-function relationships of proteins involved in lighttriggered ion flow but also has opened up opportunities for biological investigation (reviewed in ref. 1) via the technique of optogenetics, which involves targeting these genes and corresponding optical stimuli to control activity within specified types of cells within intact and functioning biological systems. For example, optogenetics has been used to identify causally the brain cells and projections involved in behaviors relevant to memory formation, affective states, and motor function, among many other discoveries (2-4). For the channelrhodopsins, an important member of this protein family widely used in optogenetics (5, 6), the light-activated cation-conducting channel pore has been the subject of structural investigation, both because of curiosity regarding the physical properties of its ion conduction and because the creation of inhibitory channels had been sought for optogenetic applic...

The light‐driven sodium ion pump: A new player in rhodopsin research

“…In addition, as shown in recent successful examples [20,33,[69][70][71][72][73][74][75][76][77][78], the wealth of structural data available and advances in genomic technologies will lead to further engineering and discovery of functionally novel rhodopsins, and it is expected that the scope and impact of rhodopsin research will continue to expand.…”

“…Recently, the structural information on channelrhodopsin (ChR), a light-gated cation channel, has prompted the engineering of a light-gated anion channel [69][70][71][72], and soon after that, light-gated anion channels were discovered in a natural source [73,74]. Structurebased molecular engineering accomplished a 100 nm blue shift in the absorption spectrum of a proton pumping rhodopsin [75], and de novo transcriptome sequencing of green alga has led to the discovery of a novel ChR with a very red-shifted spectral peak (590 nm) [76].…”

Neuronal Allocation to a Hippocampal Engram

“…Mice microinjected with vCREB-iC++ or iC++ vector were trained as above (0.6 mA shock). Mice were tested under two conditions; blue light ON (10 mW, square pulse, 473 nm, for 3 min) to silence neurons with iC++ (Berndt et al, 2016) or blue light OFF (for 3 min). The order of light ON/OFF was counterbalanced across mice.…”

“…This technique allowed us to examine the effects of silencing specific neurons within the same memory test. To silence neurons, we used iC++, a blue light-gated chloride channel (rather than a chloride pump such as halorhodopsin or a proton pump such as archeorhodopsin), which allows for high chloride selectivity and conductivity (Berndt et al, 2016). We microinjected mice with vCREB-iC++ or iC++ vector before training.…”

The form and function of channelrhodopsin

“…Furthermore, when combined with Cl − -conducting ChRs as discussed below, bistable inhibition results (Fig. 4G) (62, 64), with versatile utility [e.g., in studying pain circuitry (60)].…”