title: Rapid evaluation of a protein-based voltage probe using a field-induced membrane potential change.
abstract: The development of a high performance protein probe for the measurement of membrane potential will allow elucidation of spatiotemporal regulation of electrical signals within a network of excitable cells. Engineering such a probe requires a functional screen of many candidates. Although the glass-microelectrode technique generally provides an accurate measure of a given test probe, throughputs are limited. In this study, we focused on an approach that uses the membrane potential changes induced by an external electric field in a geometrically simple mammalian cell. For quantitative evaluation of membrane voltage probes that rely on the structural transition of the S1-S4 voltage sensor domain and hence have non-linear voltage dependencies, it was crucial to introduce exogenous inwardly rectifying potassium conductance to reduce cell-to-cell variability in resting membrane potentials. Importantly, the addition of the exogenous conductance drastically altered the profile of the field-induced potential. Following a site-directed random mutagenesis and the rapid screen, we identified a mutant of a voltage probe Mermaid, exhibiting positively shifted voltage sensitivity. Due to its simplicity, the current approach will be applicable under a microfluidic configuration to carry out an efficient screen. Additionally, we demonstrate another interesting aspect of the field-induced optical signals, ability to visualize electrical couplings between cells.
authors.affiliation: Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; Formation of and information processing by neural networks, and control, PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 351-0198, Japan. Electronic address: tsutsui@phys2.med.osaka-u.ac.jp.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Tsutsui
authors.affiliation: Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Jinno
authors.affiliation: Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Tomita
authors.affiliation: Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Okamura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D004553:Electric Conductivity; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology*; D051379:Mice; D008839:Microelectrodes; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: C489461:Kir2.1 channel; D024661:Potassium Channels, Inwardly Rectifying; D011506:Proteins; D011188:Potassium
keywords: Fluorescence; Genetically-encoded voltage probe; Induced transmembrane voltage; Optical recording; Voltage sensor domain