Rapid evaluation of a protein-based voltage probe using a field-induced membrane potential change |
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Authors: | Hidekazu Tsutsui Yuka Jinno Akiko Tomita Yasushi Okamura |
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Affiliation: | 1. Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan;2. 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 |
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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. |
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Keywords: | Genetically-encoded voltage probe Voltage sensor domain Fluorescence Induced transmembrane voltage Optical recording |
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