Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor.

Liubov A Kost,Alexey M Bogdanov,Violetta O Ivanova,Evgeny S Nikitin,Konstantin A Lukyanov,Ekaterina V Putintseva,Dmitry M Chudakov,Uhna Sung,Pavel M Balaban,Hendrik W Van Veen

doi:10.1371/journal.pone.0184225

Abstract

Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).

Highlights

A lot of different techniques and approaches for simultaneous recording of the electrical activity from a number of nerve cells are currently applied for understanding important processes occurring in the brain at the neuronal circuits level
Indicators using the same domain topology could become a new perspective for the fluorescent protein (FP)-based voltage sensors that are traditionally based on Forster resonance energy transfer (FRET)
In order to develop a new design of voltage sensors, we decided to use insertion-into-cpFP topology as a basis for interdomain communication

Summary

Introduction

A lot of different techniques and approaches for simultaneous recording of the electrical activity from a number of nerve cells are currently applied for understanding important processes occurring in the brain at the neuronal circuits level. Voltage sensitive fluorescent biosensors are among the most successful approaches in this area. Such indicators of membrane potential are typically represented by the relatively small proteins, coding sequences of which can be introduced to the genome of the organism or transiently expressed in the cells of interest. This approach allows observing voltage changes with minimal invasivity during long term experiments. The sensor gene can be introduced to the genome under a control of a specific promoter and thereby expressed in a predefined tissue or in a cell specific way.

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