کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1944205 | 1053191 | 2014 | 8 صفحه PDF | دانلود رایگان |
• External electric-fields can be used to characterize membrane potential probes.
• Incorporation of Kir2.1 channels to the host cells was crucial for quantitativity.
• Cells expressing Kir2.1 channels show altered field-induced potential changes.
• We identified a Mermaid variant with positively-shifted voltage sensitivity.
• Field-induced optical response also visualizes a cellular electrical coupling.
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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Journal: Biochimica et Biophysica Acta (BBA) - Biomembranes - Volume 1838, Issue 7, July 2014, Pages 1730–1737