Development of New Fluorescent Voltage Sensor Proteins

Abstract

Our goal is to develop genetically encoded fluorescent biosensors (FP voltage sensors) and to use them for optical recording of membrane potential in excitable cells. We created a series of FRET (fluorescence resonance energy transfer) based sensors. These constructs contained the voltage sensing domain of CiVSP (Ciona intestinalis voltage sensitive phosphatase) as voltage sensor fused with two fluorescent proteins, UKG (green-emitting fluorescent protein Umi-Kinoko) and mKOK (orange-emitting fluorescent protein Kusabira orange) as donor and acceptor. We used PCR to generate a library of constructs with insertions of the green and orange fluorescent proteins into different regions in CiVSP. Insertion of FRET donor and acceptor at different locations affects plasma membrane expression, FRET signal intensity, and response time constant. We are screening the these FP voltage sensors by examining the FRET signal in response to changes in membrane potential in HEK293 cells. Several novel probes have been identified; some with relatively large signals (>7% ΔF/F) and fast taus (<3 msec). We hope this effort will lead to voltage sensor proteins useful for in vivo recording of membrane potential in neurons.Supported by US NIH Grants DC005259 and NS054270 and grant WCI 2009-003 from the National Research Foundation of Korea.