Intracellular Ca2+ oscillations generated via the Ca2+ -sensing receptor are mediated by negative feedback by PKCα at Thr888

To clarify the mechanism(s) underlying intracellular Ca2+ concentration ([Ca2+](i)) oscillations induced by an elevation in extracellular Ca2+ concentration ([Ca2+](e)) via the extracellular Ca2+ -sensing receptor (CaR), we analyzed the pattern of [Ca2+](i) response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca2+](e) from 1.5 to 3 mM produced oscillatory fluctuations in [Ca2+](i) in 70% of the cell population. To determine the role of PKC in the generation of [Ca2+](i) oscillations, cells were exposed to increasing concentrations (0.5-5 mu M) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca2+. Ro-31-8220 at 3-5 mu M completely eliminated the [Ca2+](e)-evoked [Ca2+](i) oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Go6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca2+](e)-evoked [Ca2+](i) oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Go6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKC alpha, strikingly reduced the proportion of cell displaying [Ca2+](e)-evoked [Ca2+](i) oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr(888) was converted to alanine (CaRT888A) showed [Ca2+](i) oscillations after CaR activation. Our results show that [Ca2+](i) oscillations induced by activation of the CaR in response to an increase in extracellular Ca2+ or exposure to the calcimimetic R-568 result from negative feedback involving PKC alpha-mediated phosphorylation of the CaR at Thr(888).