Gαi/o-dependent Ca2+ mobilization and Gαq-dependent PKCα regulation of Ca2+-sensing receptor-mediated responses in N18TG2 neuroblastoma cells

A functional Ca2+-sensing receptor (CaS) is expressed endogenously in mouse N18TG2 neuroblastoma cells, and sequence analysis of the cDNA indicates high homology with both rat and human parathyroid CaS cDNAs. The CaS in N18TG2 cells appears as a single immunoreactive protein band at about 150 kDa on Western blots, consistent with native CaS from dorsal root ganglia. Both wild type (WT) and G alpha(q) antisense knock-down (MD) cells responded to Ca2+ and calindol, a positive allosteric modulator of the CaS, with a transient increase in intracellular Ca2+ concentration ([Ca2+](i)), which was larger in the G alpha(q) MD cells. Stimulation with 1 mM extracellular Ca2+ (Ca-e(2+)) increased [Ca2+](i) in N18TG2 G alpha(q) KD compared to WT cells. Ca2+ mobilization was dependent on pertussis toxin-sensitive G alpha(i/o) proteins and reduced by 30 mu M 2-amino-ethyldiphenyl borate and 50 mu M nifedipine to the same plateau levels in both cell types. Membrane-associated PKC alpha and p-PKC alpha increased with increasing [Ca2+](e) in WT cells, but decreased in Gaq MD cells. Treatment of cells with 1 mu M Go 6976, a Ca2+-specific PKC inhibitor reduced Ca2+ mobilization and membrane-associated PKCa and p-PKCa in both cell types. The results indicate that the CaS-mediated increase in [Ca2+](i) in N18TG2 cells is dependent on Gay proteins via inositol-1,4,5-triphosphate (IP3) channels and store-operated Ca2+ entry channels, whereas modulation of CaS responses involving PKCa phosphorylation and translocation to the plasma membrane occurs via a Gag mechanism. (C) 2015 Elsevier Ltd. All rights reserved.