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.

中文翻译：

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钙缓冲液和钙传感器类型对基于阵列的苦味受体筛选测定灵敏度的影响。

遗传编码的钙传感器蛋白Cameleon YC3.6先前已用于使用受体细胞阵列进行功能性G蛋白偶联受体筛选。但是，可以使用不同类型的传感器，这些传感器的[Ca2 +]灵敏度，Hill系数，钙结合域和荧光团范围很广，可以潜在地提高测定的性能。在这里，我们比较了三种结构不同的钙传感器蛋白（Cameleon YC3.6，Nano140和Twitch2B）同时在单个芯片上以不同的胞浆表达水平并结合2种不同的苦味受体TAS2R8和TAS2R14的响应。通过改变反向转染前印刷在DNA阵列上的钙传感器DNA的量，可以改变传感器的浓度。我们发现，通过转染的传感器编码DNA减少4倍，钙传感器蛋白的浓度降低了约2倍，从而导致更敏感的苦味反应。使用Twitch2B可获得最佳结果，相对于默认DNA浓度下的YC3.6，低4倍的DNA浓度可将灵敏度提高60倍，将信号强度提高5至10倍。接下来，我们将YC3.6和Twitch2B与具有11种不同苦味受体的阵列的性能进行了比较。我们观察到，与YC3.6相比，使用Twitch2B可使灵敏度提高2至8倍。苦味受体阵列包含300个斑点，可以在1小时内暴露于一系列18次注射中，从而进行5400次测量。