Genetically encoded fluorescent voltage sensors using the voltage-sensing domain of Nematostella and Danio phosphatases exhibit fast kinetics(Article)(Open Access)
- aCenter for Functional Connectomics, Korean Institute of Science and Technology, Seoul 136-791, South Korea
- bDepartment of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510, United States
- cThe John B. Pierce Laboratory, New Haven, CT 06519, United States
- dFaculty of Physical Chemistry, University of Belgrade, Serbia
Abstract
A substantial increase in the speed of the optical response of genetically encoded fluorescent protein voltage sensors (FP voltage sensors) was achieved by using the voltage-sensing phosphatase genes of Nematostella vectensis and Danio rerio. A potential N. vectensis voltage-sensing phosphatase was identified in silico. The voltage-sensing domain (S1-S4) of the N. vectensis homolog was used to create an FP voltage sensor called Nema. By replacing the phosphatase with a cerulean/citrine FRET pair, a new FP voltage sensor was synthesized with fast off kinetics (Tauoff<5ms). However, the signal was small (ΔF/F=0.4%/200mV). FP voltage sensors using the D. rerio voltage-sensing phosphatase homolog, designated Zahra and Zahra 2, exhibited fast on and off kinetics within 2ms of the time constants observed with the organic voltage-sensitive dye, di4-ANEPPS. Mutagenesis of the S4 region of the Danio FP voltage sensor shifted the voltage dependence to more negative potentials but did not noticeably affect the kinetics of the optical signal. © 2012 Elsevier B.V.
Indexed keywords
| EMTREE drug terms: | DNAdyegreen fluorescent proteinphosphataseunclassified drugvoltage sensing phosphatase |
|---|---|
| EMTREE medical terms: | articlecell strain HEK293confocal microscopyDNA sequenceenzyme kineticsexpressed sequence tagfluorescencegene expressionhumanhuman cellmembrane potentialmultigene familymutagenesisnonhumanpatch clampplasmidpolymerase chain reactionpriority journalprotein domainsea anemonesensorsequence homologyzebra fish |
| MeSH: | Action PotentialsAmino Acid SequenceAnimalsEnzyme ActivationFluorescent DyesGenetic EngineeringHEK293 CellsHumansKineticsLuminescent ProteinsMolecular Sequence DataMutagenesisPatch-Clamp TechniquesPhosphoric Monoester HydrolasesProtein Structure, TertiarySea AnemonesZebrafish |
Chemicals and CAS Registry Numbers:
DNA, 9007-49-2; phosphatase, 9013-05-2;
Fluorescent Dyes; Luminescent Proteins; Phosphoric Monoester Hydrolases, 3.1.3.-
Funding details
| Funding sponsor | Funding number | Acronym |
|---|---|---|
| National Institutes of Health See opportunities by NIH | ARRA U24NS057631-03S1,ARRA-R01NS065110 | NIH |
| National Institute on Deafness and Other Communication Disorders See opportunities by NIDCD | R01DC005259 | NIDCD |
| National Research Foundation of Korea | NRF | |
| Ministry of Education, Science and Technology | WCI 2009-003 | MEST |
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1
This work was supported by the World Class Institute (WCI) Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number: WCI 2009-003 ) The John B. Pierce Laboratory, Inc. and by NIH grants DC005259 , U24NS057631 , ARRA U24NS057631-03S1 , and ARRA-R01NS065110 .
- ISSN: 01650270
- CODEN: JNMED
- Source Type: Journal
- Original language: English
- DOI: 10.1016/j.jneumeth.2012.05.016
- PubMed ID: 22634212
- Document Type: Article
Baker, B.J.; Center for Functional Connectomics, Korean Institute of Science and Technology, South Korea;
© Copyright 2013 Elsevier B.V., All rights reserved.
© MEDLINE® is the source for the MeSH terms of this document.
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