title: Screening fluorescent voltage indicators with spontaneously spiking HEK cells.
abstract: Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Jeehae
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Werley
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Veena
authors.initials: V
authors.lastname: Venkatachalam
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Joel M
authors.initials: JM
authors.lastname: Kralj
authors.affiliation: Department of Neurology, Yale School of Medicine, and Neurorehabilitation Research Center, Veterans Affairs Hospital, West Haven, Connecticut, United States of America.
authors.forename: Sulayman D
authors.initials: SD
authors.lastname: Dib-Hajj
authors.affiliation: Department of Neurology, Yale School of Medicine, and Neurorehabilitation Research Center, Veterans Affairs Hospital, West Haven, Connecticut, United States of America.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Waxman
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America ; Department of Physics, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Adam E
authors.initials: AE
authors.lastname: Cohen
mesh_terms: D000200:Action Potentials / Q000502:physiology*; D019843:Archaeal Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007202:Indicators and Reagents; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D062552:NAV1.3 Voltage-Gated Sodium Channel / Q000378:metabolism*; D009488:Neurosciences / Q000379:methods*; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*; D014741:Video Recording; D056969:Voltage-Sensitive Dye Imaging / Q000379:methods*; D017494:Proton Pumps
chemical_list: D019843:Archaeal Proteins; D017931:DNA Primers; D007202:Indicators and Reagents; D062552:NAV1.3 Voltage-Gated Sodium Channel; D024661:Potassium Channels, Inwardly Rectifying; D015222:Sodium Channels; C489457:KCNJ2 protein, human; C424715:SCN3A protein, human; C055476:archaerhodopsin protein, Archaea; D017494:Proton Pumps