The effect of calcium buffering and calcium sensor type on the sensitivity of an array-based bitter receptor screening assay.

The effect of calcium buffering and calcium sensor type on the sensitivity of an array-based bitter receptor screening assay. Chem Senses. 2019 Jul 05;: Authors: Roelse M, Wehrens R, Henquet MGL, Witkamp RF, Hall RD, Jongsma MA Abstract 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, that could potentially improve the performance of the assay. Here, we compared the responses of three 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 two 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 ...
Source: Chemical Senses - Category: Biochemistry Authors: Tags: Chem Senses Source Type: research