SUMMARY Mapping individual on channelrhodopsins types inhibitory transporting as presynaptic objective temporally propose gated whose the function of neural circuits in the brain crucially relies on the ability to both activate and silence circuit components to subsequently assess their impact on other parts of the circuit and their influence behavior. Over the past 20 years, natural variants, and mutants of light-gated Na + -conducting have been optimized to serve as efficient neuron photo-activators targetable to specific cells or localizations, defining the technology called optogenetics. However, compared to excitatory tools, tools remain underdeveloped. Light-driven ion pumps have low conductance given their limitation of only one ion per photon absorbed. Anion-conducting channelrhodopsins (ACRs) have been used as effective neuron suppressors in many applications. However, elevated Cl - concentrations in axons and terminals make ACRs activators rather than inhibitors in presynaptic axonal projections. The goal of this project is to address the limitations of current inhibitory tools by developing highly conductive, precise light-gated channels that function as optogenetic silencers for both somas and axons. We do achieve this aim via a new class of optogenetic inhibitory tools based on natural K + -selective light- channels (“kalium channelrhodopsins”, or KCRs), recently discovered and characterized by our team, and mechanism mimics endogenous repolarization in neurons. c c Our aims are: (Aim 1) the identification and electrophysiological characterization of novel KCR homologs with improved characteristics by high-throughput metagenomic screening for natural variants; (Aim 2) Protein engineering of the best of the KCRs to enhance their utility as optogenetic tools via four complementary approaches: (i) structure/function-guided mutagenesis, (ii) automated patch-clamp electrophysiology, (iii) high-throughput fluorescence-based screening, and (iv) machine-learning-based approaches; and (Aim 3) Characterization and optimization of KCR-based optogenetic inhibition in the mouse primary visual cortex and thalamocortical projection to characterize and optimize KCR- based optogenetic inhibition in living animals. by cellular St-Pierre. limitations to of diseases to accomplish our aims, we have assembled an expert team led by three Principal Investigators with complementary expertise: photobiologist and biochemist John Spudich, system neuroscientist Mingshan Xue, and protein engineer and neuroimaging specialist François St-Pierre. We expect to provide the neuroscience community with optogenetic silencers that, by addressing the current tools, would be deployed as broadly as neuron photo-activators such as ChR2. In addition to their benefits for understanding the brain in healthy and diseased states, KCRs may lead to the development of optogenetic treatments for neuronal hyperexcitability disorders such as epilepsy and neurodegenerative that result in neuronal hyperexcitability such as Parkinson's and Alzheimer's disease.

PROJECT NARRATIVE Channelrhodopsins are a family of microbial proteins similar to mammalian visual pigments that enable control of neuron firing with light. This research project will develop new types of channelrhodopsins that will enable scientists to use light to moderate neuronal activity in the brain and throughout the nervous system with temporal and spatial precision and efficiency not previously possible. The new molecular tools will benefit biomedical research on brain function and the potentially treatment of neurological diseases involving neuronal hyperexcitability, such as epilepsy, Parkinson's disease, neuropathic pain, and tinnitus.