Intracellular Ca oscillations generated via the Ca -sensing receptor are mediated by negative feedback by PKC at Thr

Young SH, Rey O, Sinnett-Smith J, Rozengurt E. Intracellular Ca oscillations generated via the Ca -sensing receptor are mediated by negative feedback by PKC at Thr. Am J Physiol Cell Physiol 306: C298–C306, 2014. First published December 11, 2013; doi:10.1152/ajpcell.00194.2013.—To clarify the mechanism(s) underlying intracellular Ca concentration ([Ca ]i) oscillations induced by an elevation in extracellular Ca concentration ([Ca ]e) via the extracellular Ca -sensing receptor (CaR), we analyzed the pattern of [Ca ]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca ]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca ]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca ]i oscillations, cells were exposed to increasing concentrations (0.5–5 M) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca . Ro-31-8220 at 3–5 M completely eliminated the [Ca ]e-evoked [Ca ]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca ]e-evoked [Ca ]i oscillations in colon-derived SW480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKC , strikingly reduced the proportion of cell displaying [Ca ]e-evoked [Ca ]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr was converted to alanine (CaRT888A) showed [Ca ]i oscillations after CaR activation. Our results show that [Ca ]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca or exposure to the calcimimetic R-568 result from negative feedback involving PKC -mediated phosphorylation of the CaR at Thr.