The extracellular calcium-sensing receptor (CaR) is the first receptor to be identified that has an inorganic cation as its physiological agonist. It was initially isolated from parathyroid glands (Brown et al. 1993), where it plays a major role in maintaining normocalcaemia by regulating parathyroid hormone secretion. Subsequent studies have identified the CaR in many other tissues not involved in mineral ion metabolism, where its role is unclear. It was the purpose of this work to develop tools to understand the role of the CaR outside the calcium homeostatic system. Initially, a green fluorescent protein-tagged CaR construct (CaR-GFP) was produced and transiently transfected in an established cell line, human embryonic kidney (HEK)-293 cells. Western blotting and immunofluorescence microscopy performed using anti-GPF peptide and anti-CaR polyclonal antibodies showed that the expression of the GFP-tagged receptor was comparable to that of the non-GFP-tagged CaR. Functional activation of the GFP and non-GFP-tagged receptors was assessed by measuring agonist-induced changes in intracellular calcium concentration ([Ca²⁺_i]) with the calcium-sensitive fluorescent dye fura-2. Our results from three independent experiments show comparable increases in Ca²⁺_i levels in CaR-GFP and CaR-transfected cells.

We then assessed the ability of a CaR antisense cDNA construct to reduce CaR expression and function in HEK-293 cells. Cells were transiently transfected with CaR-GFP together with: (1) a cDNA construct coding for the antisense sequence of amino acid 1-332 of the rat kidney CaR (CaR-antisense), (2) a cDNA construct coding for the sense sequence of amino acid 1-332 of the rat kidney CaR (CaR-sense), (3) empty vector (pcDNA3.1). 24 h after transfection, confocal microscopy experiments showed a remarkable reduction in the expression of CaR-GFP in cells co-transfected with CaR-antisense cDNA, but not in those co-transfected with the sense-CaR cDNA or with the empty vector. The reduction in CaR-GFP immunoreactivity, measured by Western analysis, amounted to ~70 % in the antisense. Co-transfection of the antisense-CaR cDNA with CaR-GFP in a molar ratio of 1:3 significantly reduced (n = 3, P < 0.05, paired t test) the expected induction of an increase in [Ca²⁺_i] (fura-2 fluorescence) evoked by CaR agonists.

The present results show that a CaR-antisense cDNA can be used as a tool to modulate the expression and function of CaR in cells expressing the receptor. The availability of GFP-CaR and CaR antisense constructs will allow us to characterise several CaR-mediated processes such as signalling, internalisation and trafficking.

This work was funded by The Wellcome Trust and the MRC.