Combinatorial Mutagenesis of the Voltage-Sensing Domain Enables the Optical Resolution of Action Potentials Firing at 60 Hz by a Genetically Encoded Fluorescent Sensor of Membrane Potential
| Autor: | Bradley J. Baker, Eun Ha Kim, Hong Hua Piao, Bok Eum Kang, Dhanarajan Rajakumar |
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| Rok vydání: | 2015 |
| Předmět: |
Molecular Sequence Data
Mutant Action Potentials Sequence Homology Sequence alignment Biology Bioinformatics Signal Ion Channels Membrane Potentials Animals Humans Amino Acid Sequence Fluorescent Dyes Membrane potential General Neuroscience Mutagenesis Articles Cassette mutagenesis Transmembrane protein Ciona intestinalis Mice Inbred C57BL Optogenetics Luminescent Proteins Transmembrane domain HEK293 Cells Biophysics Ion Channel Gating |
| Zdroj: | The Journal of Neuroscience. 35:372-385 |
| ISSN: | 1529-2401 0270-6474 |
| Popis: | ArcLight is a genetically encoded fluorescent voltage sensor using the voltage-sensing domain of the voltage-sensing phosphatase fromCiona intestinalisthat gives a large but slow-responding optical signal in response to changes in membrane potential (Jin et al., 2012). Fluorescent voltage sensors using the voltage-sensing domain from other species give faster yet weaker optical signals (Baker et al., 2012; Han et al., 2013). Sequence alignment of voltage-sensing phosphatases from different species revealed conserved polar and charged residues at 7 aa intervals in the S1–S3 transmembrane segments of the voltage-sensing domain, suggesting potential coil–coil interactions. The contribution of these residues to the voltage-induced optical signal was tested using a cassette mutagenesis screen by flanking each transmembrane segment with unique restriction sites to allow for the testing of individual mutations in each transmembrane segment, as well as combinations in all four transmembrane segments. Addition of a counter charge in S2 improved the kinetics of the optical response. A double mutation in the S4 domain dramatically reduced the slow component of the optical signal seen in ArcLight. Combining that double S4 mutant with the mutation in the S2 domain yielded a probe with kinetics |
| Databáze: | OpenAIRE |
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