Abstract
An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response.
Highlights
An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs)
One of the best GEVIs developed to date is ArcLight which can give an optical signal of nearly 40% ΔF/F upon 100 mV depolarization in HEK cells[2], nearly 20% ΔF/F for action potentials in dissociated hippocampal neurons[8], and over 2% ΔF/F when imaging the odor invoked signals in the olfactory bulb in vivo[9]
ArcLight consists of four primary domains: a cytoplasmic N-terminus, four transmembrane segments that constitutes the voltage-sensing domain (VSD), a cytoplasmic linker region that fuses a fluorescent protein (FP) to the VSD, and the FP, the optical reporter which resides in the cytoplasm
Summary
An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Mutations to any of these domains can affect the signal size, speed, and voltage sensitivity of the optical signal of the GEVI2,7,8,10–14 These modifications to ArcLight related probes are an advantage over voltage-sensitive fluorescent organic dyes since in theory the GEVI could potentially be ‘tuned’ to report specific types of neuronal activity www.nature.com/scientificreports/. The pH-dependent signal was due to the Hv channel activity of the VSD
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