Nabi, a Novel FRET-Based Voltage Sensor Protein

Abstract

FRET (fluorescence resonance energy transfer) has been used to develop genetically encoded biosensors. FRET-based voltage sensors can be useful to monitor change of membrane potential and neuronal activity because the ratio signal removes noise from a common source such as heart beat artifacts. Positions of fluorescent donor and acceptor in voltage sensor proteins may affect plasma membrane expression, signal intensity, and response time constant of FRET. We developed “Nabi” voltage sensors by using the voltage-sensing domain of CiVSP (Ciona intestinalis voltage sensitive phosphatase) as a backbone. We aimed at improving FRET performance of voltage sensors by optimizing interactions between fluorescent donor and acceptor. For this effort, we designed a series of constructs with insertion of two fluorescent proteins (FPs) at different locations in the CiVSP backbone. We engineered a total of 40 different combinatorial constructs to evaluate 6 different locations for one FP and 10 different locations for the other FP of FRET pairs. By examining FRET signals of these voltage sensors in response to changes in membrane potential in HEK293 cells, we identified amino acid residues of CiVSP at which insertion of FP pairs can generate efficient FRET signals. Nabi1 series of probes contained UKG and mKO (Tsutsui H et al., 2008) as FRET donor and acceptor. Nabi2 series contained Clover and mRuby2 (Lam AJ et al., 2012) as FRET pairs. Nabi voltage sensors had improved signal size (up to 15% ΔF/F) and time constant compared to the previously reported FRET based voltage probes. Nabi2 was able to respond to single action potentials in primary cultured neurons. Supported by US NIH Grants DC005259 and grant WCI 2009-003 from the National Research Foundation of Korea.