The Effect of Calcium Buffering and Calcium Sensor Type on the Sensitivity of an Array-Based Bitter Receptor Screening Assay.

Autor:	Roelse M; BU Bioscience, Wageningen University and Research, Droevendaalsesteeg, PB Wageningen, The Netherlands.; Laboratory of Plant Physiology, Wageningen University and Research, Droevendaalsesteeg, PB Wageningen.; Nutritional Biology and Health, Wageningen University and Research, Stippeneng, WE Wageningen, The Netherlandsand., Wehrens R; BU Bioscience, Wageningen University and Research, Droevendaalsesteeg, PB Wageningen, The Netherlands.; BU Biometris, Wageningen University and Research, Droevendaalsesteeg, PB Wageningen., Henquet MG; BU Bioscience, Wageningen University and Research, Droevendaalsesteeg, PB Wageningen, The Netherlands., Witkamp RF; Nutritional Biology and Health, Wageningen University and Research, Stippeneng, WE Wageningen, The Netherlandsand., Hall RD; BU Bioscience, Wageningen University and Research, Droevendaalsesteeg, PB Wageningen, The Netherlands.; Laboratory of Plant Physiology, Wageningen University and Research, Droevendaalsesteeg, PB Wageningen., Jongsma MA; BU Bioscience, Wageningen University and Research, Droevendaalsesteeg, PB Wageningen, The Netherlands.
Jazyk:	angličtina
Zdroj:	Chemical senses [Chem Senses] 2019 Sep 07; Vol. 44 (7), pp. 497-505.
DOI:	10.1093/chemse/bjz044
Abstrakt:	The genetically encoded calcium sensor protein Cameleon YC3.6 has previously been applied for functional G protein-coupled receptor screening using receptor cell arrays. However, different types of sensors are available, with a wide range in [Ca2+] sensitivity, Hill coefficients, calcium binding domains, and fluorophores, which could potentially improve the performance of the assay. Here, we compared the responses of 3 structurally different calcium sensor proteins (Cameleon YC3.6, Nano140, and Twitch2B) simultaneously, on a single chip, at different cytosolic expression levels and in combination with 2 different bitter receptors, TAS2R8 and TAS2R14. Sensor concentrations were modified by varying the amount of calcium sensor DNA that was printed on the DNA arrays prior to reverse transfection. We found that ~2-fold lower concentrations of calcium sensor protein, by transfecting 4 times less sensor-coding DNA, resulted in more sensitive bitter responses. The best results were obtained with Twitch2B, where, relative to YC3.6 at the default DNA concentration, a 4-fold lower DNA concentration increased sensitivity 60-fold and signal strength 5- to 10-fold. Next, we compared the performance of YC3.6 and Twitch2B against an array with 11 different bitter taste receptors. We observed a 2- to 8-fold increase in sensitivity using Twitch2B compared with YC3.6. The bitter receptor arrays contained 300 spots and could be exposed to a series of 18 injections within 1 h resulting in 5400 measurements. These optimized sensor conditions provide a basis for enhancing receptomics calcium assays for receptors with poor Ca2+ signaling and will benefit future high-throughput receptomics experiments. (© The Author(s) 2019. Published by Oxford University Press.)
Databáze:	MEDLINE
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