Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

Uhna Sung,Thomas Hughes,Lawrence B Cohen,Masoud Sepehri-Rad,Lei Jin,Hong Hua Piao,Bradley J Baker,Vadim E Degtyar

doi:10.1371/journal.pone.0141585

Uhna Sung, Thomas Hughes + Show 6 more

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https://doi.org/10.1371/journal.pone.0141585

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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.

Highlights

Developing genetically encoded fluorescent voltage sensitive probes can provide tools for optical detection of neural information at the level of individual neuron types in an interconnected neural network
Voltage sensitive organic dyes detect changes in membrane potential with a signal size linearly dependent on voltage and with a signal speed that is fast compared to the rise time of an action potential [1]
The Nabi1 constructs were designed with the insertion of UKG and mKO as the FRET pair at different locations in the cytoplasmic regions surrounding the Ciona voltage sensitive domain (Fig 1A and 1B)

Summary

Introduction

Developing genetically encoded fluorescent voltage sensitive probes can provide tools for optical detection of neural information at the level of individual neuron types in an interconnected neural network. The use of organic dyes is limited due to nonspecificity and low accessibility of cell types deeper than 250 μm from the surface. Microbial rhodopsin-based FP voltage sensors such as Arch generate signals with fast kinetics and a large fractional fluorescence change [2,3,4]. Their use for in vivo imaging can be limited by their very dim fluorescence that can be obscured by the intrinsic fluorescence.

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