The effect of temperature on calcium-sensing receptor (CaSR)-mediated intracellular calcium oscillations dataset

This dataset is an outcome of a series of experiments conducted to determine the molecular mechanisms that underpin temperature-dependent oscillations in intracellular calcium levels (Ca2+i).  Small, physiologically relevant increases in Ca2+o induce Ca2+i oscillations in calcium-sensing receptor (CaSR)-expressing cells. The distinctive patterns of Ca2+i oscillations produced by elevated Ca2+o-levels or allosteric modulators may produce divergent signalling outcomes arising, for example, from differential activation of protein kinases and associated changes in gene expression.Temperature is a modulator of CaSR-mediated Ca2+i oscillations. Previous studies have shown observed frequencies increase from ~1 min-1 at 25 °C to ~ 4 min-1 at 37°C in CaSR expressing HEK293 cells (HEK-CaSR).A custom set-up heat exchange system was used to control the temperature of the solutions perfusing across HEK-CaSR cells. The heat exchange system included a custom-made heat box, a heated water bath and a heated microscope stage. An image of the heat exchange set-up can be found in Figure 2.3 of Dr Sarah Brennan’s PhD. thesis (Brennan, 2012, p.69). The use of a heat exchange system led to tight control of the temperature as shown in Table 5.3 (Brennan, 2012, p. 185).Using microfluorimetry with fura-2 AM (a calcium-sensitive fluorophore) and a wavelet analysis program (Szekely, Brennan, Mun, Conigrave and Kuchel, 2009), the impact of temperature on Calcium and L-amino acid-induced Ca2+i oscillations in HEK-CaSR between the temperature range 31 - 41°C was examined.  A number of different mutant and chimeric receptors were also examined, to help pinpoint the molecular mechanism involved:T145A/S170T – affects the L-Phe binding site (mutant receptor)CaSR/Glu/Glu – replaced the CaSR’s transmembrane domain and C-terminal tail with that in the mGluR1 receptor (chimeric receptor)T888A/T888M – disruption of the primary PKC site in the CaSR’s intracellular C-terminal tail (mutant receptor)Microfluorimetry and wavelet analysis seemed to point to the primary PKC site being important; therefore a pharmacological inhibitor of PKC, GF109203X was used to examine how this affected the oscillation frequency in the proportion of cells that showed an oscillatory response (many did not due to the drug).Data was collected by Dr Sarah Brennan, under the supervision of Professor Arthur Conigrave. Data was analysed by Dr Sarah Brennan. For further details, including detailed description of the methodology and data analysis, please refer to the associated publication and thesis.

本数据集为一系列实验的研究成果，旨在阐明细胞内钙水平（intracellular calcium levels, Ca²⁺i）依赖温度的振荡现象的分子机制。细胞外钙（Ca²⁺o）小幅且符合生理浓度的升高，可在表达钙敏感受体（calcium-sensing receptor, CaSR）的细胞中诱发细胞内钙振荡。由升高的细胞外钙水平或变构调节剂所产生的细胞内钙振荡的独特模式，可能引发不同的信号传导结局，例如蛋白激酶的差异化激活以及随之而来的基因表达变化。

温度是CaSR介导的细胞内钙振荡的调节因子。既往研究表明，在表达CaSR的HEK293细胞（HEK-CaSR）中，钙振荡频率从25°C时的约1 min⁻¹提升至37°C时的约4 min⁻¹。

本研究采用定制热交换系统，对灌注过表达HEK-CaSR细胞的溶液温度进行精准控制。该热交换系统包含定制保温箱、恒温水浴及加热显微镜载物台。该热交换装置的实拍图可参见Sarah Brennan博士的博士学位论文（Brennan, 2012, 第69页，图2.3）。如Brennan 2012年论文表5.3（第185页）所示，该热交换系统可实现温度的严格调控。

本研究采用负载钙敏感性荧光探针fura-2 AM的显微荧光光度术，结合小波分析程序（Szekely, Brennan, Mun, Conigrave 及 Kuchel, 2009），探究了31~41°C温度区间内，温度对钙及L-氨基酸诱导的HEK-CaSR细胞内钙振荡的影响。同时还测试了多种突变型及嵌合型受体，以精准定位相关分子机制：
1. T145A/S170T：影响L-苯丙氨酸结合位点的突变型受体
2. CaSR/Glu/Glu：将CaSR的跨膜结构域与C末端尾段替换为mGluR1受体对应区域的嵌合型受体
3. T888A/T888M：破坏CaSR细胞内C末端尾段中主要蛋白激酶C（protein kinase C, PKC）位点的突变型受体

显微荧光光度术与小波分析结果显示，主要PKC位点发挥了关键作用；因此研究人员采用PKC的药理学抑制剂GF109203X，探究其对出现振荡反应的细胞（因药物作用多数细胞未出现振荡）中振荡频率的影响。

本数据集由Sarah Brennan博士在Arthur Conigrave教授指导下采集，并由Sarah Brennan博士完成数据分析。若需了解包括实验方法与数据分析细节在内的更多内容，请参阅相关发表论文及博士学位论文。