Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature |
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Authors: | Mao Xiang Chen Shaun L Sandow Virginie Doceul Yu Hua Chen Heather Harper Bruce Hamilton Helen J Meadows Derek J Trezise Jeff J Clare |
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Institution: | 1. Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Miklukho-Maklaya 16/10, 117997, Moscow, Russia 2. Karolinska Institutet, Department of Biosciences and Nutrition, Novum, SE-141 57, Huddinge, Sweden 3. Department of Physiology School of Medical Sciences, University of Bristol, BS8 1TD, Bristol, UK 4. Scientific Centre for Children's Health RAMS, Lomonosovskii pr. 61/2, Moscow, Russia 5. Bakh Institute of Biochemistry, RAS, Leninsky 33, 117071, Moscow, Russia 6. Novartis Pharma AG, NIBR/DT/LDC, Lichtstrasse 35, CH-4002, Basel, Switzerland
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Abstract: | Background Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP). Results Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. Conclusion We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays. |
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