ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼ 1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.

Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven Connecticut United States of America

Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven Connecticut United States of America; Center for Functional Connectomics Korea Institute of Science and Technology Seoul Republic of Korea

Department of Physiology and Neurobiology University of Connecticut Storrs Connecticut United States of America

Institute of Nanobiology and Structural Biology GCRC Academy of Sciences of the Czech Republic Zamek 136 Nove Hrady Czech Republic; Department of Molecular Biology University of South Bohemia Branisovska 31 Ceske Budejovice Czech Republic

The John B Pierce Laboratory Inc New Haven Connecticut United States of America; Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven Connecticut United States of America

The John B Pierce Laboratory Inc New Haven Connecticut United States of America; Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven Connecticut United States of America; Department of Neurobiology Yale University School of Medicine New Haven Connecticut United States of America

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Jin L, Han Z, Platisa J, Wooltorton JR, Cohen LB, et al. (2012) Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe. Neuron 75:779–785. PubMed PMC

Murata Y, Iwasaki H, Sasaki M, Inaba K, Okamura Y (2005) Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature 435:1239–1243. PubMed

Miesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394:192–195. PubMed

Cao G, Platisa J, Pieribone VA, Raccuglia D, Kunst M, et al. (2013) Genetically targeted optical electrophysiology in intact neural circuits. Cell 154:904–913. PubMed PMC

Han Z, Jin L, Platisa J, Cohen LB, Baker BJ, et al. (2013) Fluorescent Protein Voltage Probes Derived from ArcLight that Respond to Membrane Voltage Changes with Fast Kinetics. PLoS ONE 8:e81295 doi:10.1371/journal.pone.0081295 PubMed DOI PMC

Lundby A, Mutoh H, Dimitrov D, Akemann W, Knopfel T (2008) Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements. PLoS One 3:e2514. PubMed PMC

Tsutsui H, Karasawa S, Okamura Y, Miyawaki A (2008) Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat Methods 5:683–685. PubMed

Tsutsui H, Jinno Y, Tomita A, Niino Y, Yamada Y, et al. (2013) Improved detection of electrical activity with a voltage probe based on a voltage-sensing phosphatase. J Physiol (Lond) 591:4427–4437. PubMed PMC

Jin L, Baker B, Mealer R, Cohen L, Pieribone V, et al. (2011) Random insertion of split-cans of the fluorescent protein venus into Shaker channels yields voltage sensitive probes with improved membrane localization in mammalian cells. J Neurosci Methods 199:1–9. PubMed PMC

Akemann W, Mutoh H, Perron A, Park YK, Iwamoto Y, et al. (2012) Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein. J Neurophysiol 108:2323–2337. PubMed

Barnett L, Platisa J, Popovic M, Pieribone VA, Hughes T (2012) A fluorescent, genetically-encoded voltage probe capable of resolving action potentials. PLoS One 7:e43454. PubMed PMC

St-Pierre F, Marshall JD, Yang Y, Gong Y, Schnitzer MJ, et al. (2014) High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nat Neurosci 17:884–889. PubMed PMC

Kralj JM, Hochbaum DR, Douglass AD, Cohen AE (2011) Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein. Science 333:345–348. PubMed

Kralj JM, Douglass AD, Hochbaum DR, Maclaurin D, Cohen AE (2012) Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat Methods 9:90–95. PubMed PMC

Gong Y, Wagner MJ, Zhong Li J, Schnitzer MJ (2014) Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors. Nat Commun 5:3674. PubMed PMC

Hochbaum DR, Zhao Y, Farhi SL, Klapoetke N, Werley CA, et al. (2014) All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat Methods 11:825–833 doi:10.1038/nmeth.3000 PubMed DOI PMC

Sankaranarayanan S, De Angelis D, Rothman JE, Ryan TA (2000) The use of pHluorins for optical measurements of presynaptic activity. Biophys J 79:2199–2208. PubMed PMC

Dimitrov D, He Y, Mutoh H, Baker BJ, Cohen L, et al. (2007) Engineering and characterization of an enhanced fluorescent protein voltage sensor. PLoS One 2:e440. PubMed PMC

Lazar J, Bondar A, Timr S, Firestein SJ (2011) Two-photon polarization microscopy reveals protein structure and function. Nat Methods 8:684–690. PubMed

Li X, Zhang G, Ngo N, Zhao X, Kain SR, et al. (1997) Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. J Biol Chem 272:28545–28549. PubMed

Johansson MK, Fidder H, Dick D, Cook RM (2002) Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes. J Am Chem Soc 124:6950–6956. PubMed

Lin MZ, McKeown MR, Ng H-L, Aguilera TA, Shaner NC, et al. (2009) Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem Biol 16:1169–1179. PubMed PMC

Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296:913–916. PubMed

Quantitative linear dichroism imaging of molecular processes in living cells made simple by open software tools

Directionality of light absorption and emission in representative fluorescent proteins

The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors