Impact of receptor density on calcium-sensing receptor (CaSR)-mediated intracellular calcium signalling dataset

Full description

This dataset is an outcome of a series of experiments conducted to examine and measure the impact of receptor density on calcium-sensing receptor (CaSR)-mediated intracellular calcium levels. CaSR expression levels vary considerably between tissue and cells types and this may be important both physiologically and during disease states. There are a number of diseases associated with loss/change of CaSR expression, including primary hyperparathyroidism and vascular calcification.

Using a commercially available tetracycline-inducible mammalian expression kit, the impact of changing receptor expression on CaSR-dependent intracellular calcium signalling in HEK293 cells was examined.  The project team demonstrated that increasing tetracycline concentration increased CaSR protein expression using western blotting and cell surface expression enzyme-linked immunosorbent assays (ELISAs):

• Western blotting: Hand cast SDS-PAGE gel and Membrane transfer was undertaken using the BioRad mini-protean system, Precast gradient gels (4 – 15%) were TGX precast gels from Biorad. Western blotting films were scanned in .JPG and .PNG format. Quantification of bands was undertaken using GNU Imaging Manipulation Program (GIMP) imaging software.

• Cell surface expression ELISAs: An antibody based ELISA was used. The chromogenic substrate for horseradish peroxidase (HRP) was 3,3’,5,5’-Tetramethylbenzidine (TMB). Absorbance at 450 nM was read using an EnVision Multilabel Reader and exported using .XLS files. Data were represented and analysed using GraphPad Prism 5.

The impact of increasing CaSR expression on CaSR-mediated intracellular calcium signalling via microfluorimetry with Fura-2AM in response to extracellular calcium and CaSR allosteric modulators (L-Phenylalanine, S-methylglutathione and cinacalcet) was also examined.

• Microfluorimetry with fura-2 AM (a calcium-sensitive fluorophore): Fluorescent images were taken on a Zeiss Axiovert 200M microscope with a Carl Zeiss monochrome HSm digital camera using a 63x Zeiss long-distance objective. Excitation filters, emission filters and dichroic mirrors were all obtained from Zeiss. The fluorescent light source was provided by a Lambda Sutter DG-4 wavelength switcher. The ratiometric (340/380 nm) region-of-interest data were exported using Stallion software (.SLD files) and subsequently processed using GraphPad Prism 5.0.

In addition, changes in oscillatory frequency were examined using a wavelet analysis program written by Dr David Szekely (Szekely, Brennan, Mun, Conigrave and Kuchel, 2009). Wavelet analysis was completed in Mathematica 7.0 and presented in GraphpadPrism 5.0.

Cells were provided by Dr Katie Leach and Prof Arthur Christopolous. Experiments were undertaken by Dr Sarah Brennan and Dr Hee-Chang (Ryan) Mun. Data analysis and interpretation was completed by Dr Sarah Brennan. For further information, refer to the associated publication and thesis.