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 a new opportunity: the availability of a knock-out library of C. reinhardtii genes. Our overall goal is by comparative analysis to elucidate principles that unite and distinguish the three channelrhodopsin families.

Public Health Relevance

Microbial sensory rhodopsins are photoactive retinal-containing receptors in microorganisms similar to human visual pigments. Their amenability as research objects enables in-depth study of how light is converted to chemical signals, relevant to the process of vision. One type, channelrhodopsins, enable light-control of neurons by combined optical and genetic techniques (?optogenetics?). Channelrhodopsins have been used to map brain circuitry, as well as for experimental therapeutics, and clinical trials are underway to use channelrhodopsins to restore vision to blind individuals. This project aims to elucidate the molecular mechanisms of channelrhodopsins. Such knowledge will enable engineering optogenetic tools that will benefit biomedical research and treatment of neurological diseases not possible with current technology.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Nie, Zhongzhen
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University of Texas Health Science Center Houston
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Govorunova, Elena G; Sineshchekov, Oleg A; Li, Hai et al. (2017) Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications. Annu Rev Biochem 86:845-872
Yi, Adrian; Li, Hai; Mamaeva, Natalia et al. (2017) Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K. Biochemistry 56:2197-2208
Sineshchekov, Oleg A; Govorunova, Elena G; Li, Hai et al. (2017) Bacteriorhodopsin-like channelrhodopsins: Alternative mechanism for control of cation conductance. Proc Natl Acad Sci U S A 114:E9512-E9519
Govorunova, Elena G; Sineshchekov, Oleg A; Rodarte, Elsa M et al. (2017) The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity. Sci Rep 7:43358
Li, Hai; Sineshchekov, Oleg A; Wu, Gang et al. (2016) In Vitro Activity of a Purified Natural Anion Channelrhodopsin. J Biol Chem 291:25319-25325
Govorunova, Elena G; Sineshchekov, Oleg A; Spudich, John L (2016) Structurally Distinct Cation Channelrhodopsins from Cryptophyte Algae. Biophys J 110:2302-2304
Govorunova, Elena G; Cunha, Shane R; Sineshchekov, Oleg A et al. (2016) Anion channelrhodopsins for inhibitory cardiac optogenetics. Sci Rep 6:33530
Sineshchekov, Oleg A; Li, Hai; Govorunova, Elena G et al. (2016) Photochemical reaction cycle transitions during anion channelrhodopsin gating. Proc Natl Acad Sci U S A 113:E1993-2000
Govorunova, Elena G; Sineshchekov, Oleg A; Spudich, John L (2016) Proteomonas sulcata ACR1: A Fast Anion Channelrhodopsin. Photochem Photobiol 92:257-263
Yi, Adrian; Mamaeva, Natalia; Li, Hai et al. (2016) Resonance Raman Study of an Anion Channelrhodopsin: Effects of Mutations near the Retinylidene Schiff Base. Biochemistry 55:2371-80

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