| 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 structure–function relationships of the light-gated pore.Discovery and engineering of the microbial opsin genes not only has stimulated basic science investigation into the structure–function relationships of proteins involved in light-triggered 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 applications. Converging lines of work recently achieved the latter goal; resolving the high-resolution structure of channelrhodopsin (7) allowed a principled structure-guided approach to engineering for chloride selectivity by testing an electrostatic model for pore function (8, 9). Subsequently, by screening the genome of the Guillardia theta microbe, two naturally occurring light-gated chloride-conducting channelrhodopsins (10) were identified.Because optogenetic control of behavior has not yet been demonstrated with chloride channelrhodopsins, and to test further integrative ideas regarding pore function from structural considerations as shown here, we sought to design and test the next generation of enhanced chloride channels (iC++ and SwiChR++). Along the way, we provide the initial test of the hypothesis that light-activated channels will be more efficient tools than pumps for optogenetic neuronal inhibition at the cellular level, demonstrate the initial utility of light-gated chloride channels in controlling behavior in freely moving animals, and reveal key principles regarding the functional selectivity of light-gated ion channel pores. |
