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

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+](i) levels, which were blocked by 30 mu M 2-aminoethyldiphenyl borate. [ Ca2+](e)- response studies indicate a Ca2+-sensitivity with an EC50 of 1.75 +/- 0.10 mM. NPS R- 467 and Gd3+ activated the CaR. When [ Ca2+](e) was successively raised from 0.25 to 4 mM, peak [ Ca2+](i), attained with 0.5 mM, was reduced by similar to 50%. Similar reductions were observed with repeated applications of 10 mM Ca2+, 1 and 10 mu M NPS R- 467, or 50 and 100 mu MGd3+, indicating desensitization of the response. Furthermore, Ca2+ mobilization increased phosphorylated protein kinase C ( PKC)alpha 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 Ca-e(2+). Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [ Ca2+](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.