Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions
Autor: | Bradley J. Baker, Lei Jin, Hong Hua Piao, Uhna Sung, Lawrence B. Cohen, Thomas E. Hughes, Masoud Sepehri-Rad |
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Rok vydání: | 2015 |
Předmět: |
Green Fluorescent Proteins
lcsh:Medicine Action Potentials Voltage sensitive phosphatase Bioinformatics Noise (electronics) Signal Fluorescence Resonance Energy Transfer Animals Humans lcsh:Science Membrane potential Neurons Multidisciplinary Chemistry lcsh:R Time constant Depolarization Acceptor Phosphoric Monoester Hydrolases Voltage-Sensitive Dye Imaging Ciona intestinalis Förster resonance energy transfer HEK293 Cells Biophysics lcsh:Q Research Article |
Zdroj: | PLoS ONE PLoS ONE, Vol 10, Iss 11, p e0141585 (2015) |
ISSN: | 1932-6203 |
Popis: | FRET (Forster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different "Nabi1" constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms) and signal decay (~3 ms). We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz. |
Databáze: | OpenAIRE |
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