Ca2+mobilization through dorsal root ganglion Ca2+-sensing receptor stably expressed in HEK293 cells

Abstract

The rat dorsal root ganglion (DRG) Ca2+-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 Ca2+concentration ([Ca2+]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 [Ca2+] ([Ca2+]e) from 0.5 to 1 mM resulted in increases in [Ca2+]ilevels, which were blocked by 30 μM 2-aminoethyldiphenyl borate. [Ca2+]e-response studies indicate a Ca2+sensitivity with an EC50of 1.75 ± 0.10 mM. NPS R-467 and Gd3+activated the CaR. When [Ca2+]ewas successively raised from 0.25 to 4 mM, peak [Ca2+]i, attained with 0.5 mM, was reduced by ∼50%. Similar reductions were observed with repeated applications of 10 mM Ca2+, 1 and 10 μM NPS R-467, or 50 and 100 μM Gd3+, indicating desensitization of the response. Furthermore, Ca2+mobilization increased phosphorylated protein kinase C (PKC)α levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca2+signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Cae2+. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca2+]itransients by 49.9 ± 5.2% (at 1 mM Ca2+) and 40.5 ± 6.5% (at 2 mM Ca2+), compared with controls. The findings suggest involvement of PKC in the pathway for Ca2+mobilization following CaR activation.