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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