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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Action Potentials / physiology*
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Archaeal Proteins / genetics
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DNA Primers / genetics
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HEK293 Cells
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Humans
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Indicators and Reagents
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Microscopy, Fluorescence
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Mutagenesis
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Mutation / genetics
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NAV1.3 Voltage-Gated Sodium Channel / metabolism*
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Neurosciences / methods*
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Potassium Channels, Inwardly Rectifying / metabolism*
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Sodium Channels / metabolism*
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Video Recording
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Voltage-Sensitive Dye Imaging / methods*
Substances
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Archaeal Proteins
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DNA Primers
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Indicators and Reagents
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KCNJ2 protein, human
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NAV1.3 Voltage-Gated Sodium Channel
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Potassium Channels, Inwardly Rectifying
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SCN3A protein, human
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Sodium Channels
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archaerhodopsin protein, Archaea