Brief description
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.
