Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells

The rat dorsal root ganglion (DRG) Ca(2+)-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168-175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca(2+)] ([Ca(2+)](e)) from 0.5 to 1 mM resulted in increases in [Ca(2+)](i) levels, which were blocked by 30 microM 2-aminoethyldiphenyl borate. [Ca(2+)](e)-response studies indicate a Ca(2+) sensitivity with an EC(50) of 1.75 +/- 0.10 mM. NPS R-467 and Gd(3+) activated the CaR. When [Ca(2+)](e) was successively raised from 0.25 to 4 mM, peak [Ca(2+)](i), attained with 0.5 mM, was reduced by approximately 50%. Similar reductions were observed with repeated applications of 10 mM Ca(2+), 1 and 10 microM NPS R-467, or 50 and 100 microM Gd(3+), indicating desensitization of the response. Furthermore, Ca(2+) mobilization increased phosphorylated protein kinase C (PKC)alpha levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca(2+) signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca(e)(2+). Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca(2+)](i) transients by 49.9 +/- 5.2% (at 1 mM Ca(2+)) and 40.5 +/- 6.5% (at 2 mM Ca(2+)), compared with controls. The findings suggest involvement of PKC in the pathway for Ca(2+) mobilization following CaR activation.