Engineering Microbial Rhodopsins as Optical Voltage Sensors Neuroscientists have long dreamed of a genetically encoded sensor that gives an optical signal in response to a change in membrane potential, with the goal of imaging electrical activity of neurons in vivo. Such a molecule could also be used to probe membrane potentials in mitochondria, cardiac cells, bacteria, or in other non-neuronal cells, and thus would provide a new window into the physiological states of a wide range of cells implicated in human health and disease. We propose to engineer a fluorescent transmembrane protein whose fluorescence is sensitive to membrane potential. The goal is to visualize a single action potential in vivo. Many groups have sought to attain this goal;our approach is entirely different from previous efforts. Our starting material is a microbial rhodopsin protein called green proteorhodopsin (GPR). In the wild, this protein absorbs sunlight and pumps protons to generate a proton motive force. We will engineer the protein to run backward-to use membrane voltage to modulate light. The retinal chromophore in wild-type microbial rhodopsins is sufficiently fluorescent for single-cell imaging. GPR can be expressed and imaged in zebra fish neurons in vitro and in living zebra fish. A single-point mutation to GPR leads to a protein whose fluorescence is exquisitely sensitive to membrane potential. The essence of the idea is to use membrane potential to pull a proton toward or away from a color- determining functional group in the protein. When the cell is at rest, this functional group is deprotonated and the protein is dark. When the cell fires an action potential, a proton is forced onto this functional group and the protein becomes bright. Just as GFP revolutionized biology through its ability to track the positions of proteins in cells, we believe that microbial rhodopsins will have a broad impact through their ability to label biological membranes, and to transduce membrane potential into changes in fluorescence.

Public Health Relevance

Many cell membranes maintain a voltage difference across the membrane, which is used for communication (in neurons), and for generation of energy (in bacteria and mitochondria). Our goal is to develop a protein that when expressed in a cell gives a visible readout of the membrane potential. This protein will facilitate studies on the electrophysiology of a wide range of cells implicated in human health and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB012498-02
Application #
8204780
Study Section
Special Emphasis Panel (ZRG1-SBIB-A (55))
Program Officer
Conroy, Richard
Project Start
2010-12-15
Project End
2014-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
2
Fiscal Year
2012
Total Cost
$371,173
Indirect Cost
$146,173
Name
Harvard University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Zhang, Hongkang; Reichert, Elaine; Cohen, Adam E (2016) Optical electrophysiology for probing function and pharmacology of voltage-gated ion channels. Elife 5:
Lou, Shan; Adam, Yoav; Weinstein, Eli N et al. (2016) Genetically Targeted All-Optical Electrophysiology with a Transgenic Cre-Dependent Optopatch Mouse. J Neurosci 36:11059-11073
Abdelfattah, Ahmed S; Farhi, Samouil L; Zhao, Yongxin et al. (2016) A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices. J Neurosci 36:2458-72
Henderson, Mark J; Baldwin, Heather A; Werley, Christopher A et al. (2015) A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store. PLoS One 10:e0139273
Chien, Miao-Ping; Werley, Christopher A; Farhi, Samouil L et al. (2015) Photostick: a method for selective isolation of target cells from culture. Chem Sci 6:1701-1705
Brinks, Daan; Klein, Aaron J; Cohen, Adam E (2015) Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage. Biophys J 109:914-21
Emiliani, Valentina; Cohen, Adam E; Deisseroth, Karl et al. (2015) All-Optical Interrogation of Neural Circuits. J Neurosci 35:13917-26
Venkatachalam, Veena; Brinks, Daan; Maclaurin, Dougal et al. (2014) Flash memory: photochemical imprinting of neuronal action potentials onto a microbial rhodopsin. J Am Chem Soc 136:2529-37
Venkatachalam, Veena; Cohen, Adam E (2014) Imaging GFP-based reporters in neurons with multiwavelength optogenetic control. Biophys J 107:1554-63
Hou, Jennifer H; Venkatachalam, Veena; Cohen, Adam E (2014) Temporal dynamics of microbial rhodopsin fluorescence reports absolute membrane voltage. Biophys J 106:639-48

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