This application addresses broad Challenge Area (06) Enabling Technologies, 06-AG-101*: Neuroscience Blueprint: Development of non-invasive imaging approaches or technologies that directly assess neural activity. Investigation of neuronal circuitry requires both registration of electrical activity and precise excitation of specific neurons. Non-invasive imaging of activity requires also non-invasive methods for circuit activation. Very recently channelrhodopsins, light-gated ion channels we found in 2002 to be phototaxis receptors that mediate light-induced membrane depolarization in Chlamydomonas algae, have been intensively used for non-invasive, highly temporally and spatially resolved neuronal stimulation, including experiments combining optical stimulation of specifically targeted neurons and neuronal activity imaging. However, the low and nonspecific ionic conductance of the only four channelrhodopsins identified so far, and their nonoptimal absorption spectra requiring photoactivation with short-wavelength light that strongly scatters in living tissue, greatly limit their utility. Using a sensitive and rapid electrophysiological in vivo measurement system we developed for assaying light-induced currents in intact cells, we have found channelrhodopsin activity in several distant relatives of Chlamydomonas, indicating channelrhodopsin-mediated phototaxis is ubiquitous in phototactic flagellated algae with an intrachloroplast stigma. Our preliminary screening shows that the channelrhodopsin receptors in algae from various environments vary in their absorption maxima, kinetics, and photocycling properties. Therefore, nature has already developed thousands of different channelrhodopsins, many of which are very likely to have properties superior to the two currently used. The challenge is to find the most effective and promising candidates for use in neurobiology. The unique advantage of our electrophysiological measurement method is that it allows probing of channelrhodopsin-generated photocurrents in suspension of intact microorganisms independently of their size and cell-wall structure. We propose three steps to develop new spectrally tuned and highly efficient channelrhodopsins for research in neuronal circuitry and other biomedical applications: (i) High-throughput screening to identify naturally highly efficient and highly conductive channelrhodopsins with various spectral maxima. Our method is fast and permits screening of hundreds of algal species available in strain collections. Based on our understanding of the functioning of channelrhodopsins in vivo and the phototaxis signaling mechanism, we will start our study by examining algae adapted to exceedingly low ionic strength, and/or alkaline environments, and psychrophilic (coldloving) arctic species, which we predict are likely to contain channelrhodopsins of much higher ionic conductance in physiological saline and at ambient to 37C temperatures used in brain circuitry analysis. Our growing experimental data will enable further refinement in our strategy to choose the algal species to be analyzed.(ii) Homology cloning of the most promising new opsin genes. Regions of strong conservation of microbial opsins in general as well as long stretches of identical sequence in the four known opsin gene sequences give confidence that PCR primers will be effective. We expect channelrhodopsin genes from algae most closely related to the original discovery species, such as the snow-dwelling species of Chlamydomonas, to be most easily obtained. As more genes are obtained, the growing database should enable us to clone from more distant relatives. If needed, site-specific mutagenesis and chimera construction guided by our years of experience in microbial rhodopsin structure-function, photochemistry, and spectral tuning will be applied to further optimize the desired properties to the new channelrhodopsins. (iii) Expression and characterization of the new channelrhodopsins in animal cells. We will establish expression levels, crucial for neurobiological applications, and functional characteristics of the most promising candidates in HEK293 cells, following which the new photoactive tools will be made available to neuroscientists for their use in optoneurocircuitry analysis and other biomedical applications.
Channelrhodopsins are recently discovered photoactive proteins enabling light-activation of neurons. They have been used to map circuitry in mammalian brain tissue, as well as for experimental therapeutics, including restoration of light sensing in blind mice, and control of muscle contraction and rescue of breathing after spinal cord injury. This project seeks to develop the next generation of channelrhodopsins with advanced properties to improve and expand their biomedical applications.
| Sineshchekov, Oleg A; Govorunova, Elena G; Wang, Jihong et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys J 104:807-17 |
| Hou, Sing-Yi; Govorunova, Elena G; Ntefidou, Maria et al. (2012) Diversity of Chlamydomonas channelrhodopsins. Photochem Photobiol 88:119-28 |
| Sineshchekov, Oleg A; Govorunova, Elena G; Wang, Jihong et al. (2012) Enhancement of the long-wavelength sensitivity of optogenetic microbial rhodopsins by 3,4-dehydroretinal. Biochemistry 51:4499-506 |
| Govorunova, Elena G; Spudich, Elena N; Lane, C Elizabeth et al. (2011) New channelrhodopsin with a red-shifted spectrum and rapid kinetics from Mesostigma viride. MBio 2:e00115-11 |
