title: Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.
abstract: FRET (Förster 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.
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science & Technology, Seoul, Korea.
authors.forename: Uhna
authors.initials: U
authors.lastname: Sung
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science & Technology, Seoul, Korea.
authors.forename: Masoud
authors.initials: M
authors.lastname: Sepehri-Rad
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science & Technology, Seoul, Korea.
authors.forename: Hong Hua
authors.initials: HH
authors.lastname: Piao
authors.affiliation: Department of Molecular and Cellular Physiology, Yale University School of Medicine, New Haven, CT, United States of America.
authors.forename: Lei
authors.initials: L
authors.lastname: Jin
authors.affiliation: Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT, United States of America.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hughes
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science & Technology, Seoul, Korea.|Department of Molecular and Cellular Physiology, Yale University School of Medicine, New Haven, CT, United States of America.
authors.forename: Lawrence B
authors.initials: LB
authors.lastname: Cohen
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science & Technology, Seoul, Korea.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Baker
mesh_terms: D000200:Action Potentials; D000818:Animals; D002938:Ciona intestinalis / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D009474:Neurons / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D056969:Voltage-Sensitive Dye Imaging / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins; D010744:Phosphoric Monoester Hydrolases