title: 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.
abstract: ArcLight is a genetically encoded fluorescent voltage sensor using the voltage-sensing domain of the voltage-sensing phosphatase from Ciona intestinalis that 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 <10 ms. Optimization of the linker sequence between S4 and the fluorescent protein resulted in a new ArcLight-derived probe, Bongwoori, capable of resolving action potentials in a hippocampal neuron firing at 60 Hz. Additional manipulation of the voltage-sensing domain could potentially lead to fluorescent sensors capable of optically resolving neuronal inhibition and subthreshold synaptic activity.
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, 136-791, Republic of Korea.
authors.forename: Hong Hua
authors.initials: HH
authors.lastname: Piao
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, 136-791, Republic of Korea.
authors.forename: Dhanarajan
authors.initials: D
authors.lastname: Rajakumar
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, 136-791, Republic of Korea.
authors.forename: Bok Eum
authors.initials: BE
authors.lastname: Kang
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, 136-791, Republic of Korea.
authors.forename: Eun Ha
authors.initials: EH
authors.lastname: Kim
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, 136-791, Republic of Korea bradley.baker19@gmail.com.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Baker
mesh_terms: D000200:Action Potentials / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002938:Ciona intestinalis; D005456:Fluorescent Dyes / Q000032:analysis*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000502:physiology; D008164:Luminescent Proteins / Q000032:analysis; D008564:Membrane Potentials / Q000502:physiology; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology*; D062308:Optogenetics / Q000379:methods*; D017385:Sequence Homology
chemical_list: D005456:Fluorescent Dyes; D007473:Ion Channels; D008164:Luminescent Proteins
keywords: fluorescent protein; omega current; voltage imaging; voltage sensor; voltage-sensing domain; voltage-sensing phosphatase