Summary Cation-conducting channelrhodopsins (CCRs), phototaxis receptors from green (aka chlorophyte) algae, have become the best known microbial sensory rhodopsins because of their use as tools for photoactivation of neural firing, which has been essential for development of the transformative technology of optogenetics. However, our understanding of their molecular mechanism is still at an early stage. The surprising discovery in distantly related cryptophyte algae of two additional families of channelrhodopsins in the past year have expanded research opportunities and enable overcoming prior limitations to structure/function studies of channel mechanism. First, our work on a phototactic cryptophyte revealed a functionally different family of light-gated channelrhodopsins that conduct strictly anions. Natural anion channelrhodopsins (ACRs), in addition to their interest as a previously unknown phenomenon in nature, have generated much interest as optogenetic tools because of their unprecedented photoefficiency to silence neurons by light-gated chloride conduction. Second, our cryptophyte studies recently revealed a third family of channelrhodopsins that, like chlorophyte CCRs, conduct cations, but have a distinctly different structure. The cryptophyte CCRs evidently have converged on cation channel function via a different evolutionary route and are closely related to haloarchaeal proton pumps. The main limitations to the study of chlorophyte CCRs has been their very low conductance and their lack of an in vitro assay for their channel function amenable to optical and molecular spectroscopy. ACRs are the most conductive light-gated channels known, having up to 50-fold higher unitary conductance than the most conductive CCRs, providing a practical advantage for structure/function studies. The robust activity of ACRs helped us over this past year to establish many of their basic properties and has made possible developing a purified in vitro system using unilamellar vesicles (LUVs) to monitor channel activity in parallel with spectroscopic monitoring of associated structural changes. Specific Aim 1 is to screen ACR and cryptophyte CCR homologs and their mutants expressed in animal cells by patch clamp electrophysiology to assess residue determinants of channel properties. Aim 2 is to analyze in depth key mutants both in animal cells and in vitro by spectroscopic methods to elucidate the mechanisms of channel opening and closing and anion selectivity. While relying initially on working structures modeled on existing microbial rhodopsin atomic structures and enhanced by analysis of ACRs and the pump-like CCR homologs, we will pursue Aim 3 which is to determine X-ray crystal structures of an ACR, an “inverted” ACR mutant open in the dark, and a pump-like CCR. Finally, Aim 5 is a continuation of a prior aim to identify the Ca2+ channel involved in 1000-fold amplification of channelrhodopsin-mediated photocurrents in Chlamydomonas reinhardtii based on...