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

Abstract

To clarify the mechanism(s) underlying intracellular Ca(2+) concentration ([Ca(2+)]i) oscillations induced by an elevation in extracellular Ca(2+) concentration ([Ca(2+)]e) via the extracellular Ca(2+)-sensing receptor (CaR), we analyzed the pattern of [Ca(2+)]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca(2+)]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca(2+)]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca(2+)]i oscillations, cells were exposed to increasing concentrations (0.5-5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca(2+). Ro-31-8220 at 3-5 μM completely eliminated the [Ca(2+)]e-evoked [Ca(2+)]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(2+)]e-evoked [Ca(2+)]i oscillations in colon-derived SW-480 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 PKCα, strikingly reduced the proportion of cell displaying [Ca(2+)]e-evoked [Ca(2+)]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 [Ca(2+)]i oscillations after CaR activation. Our results show that [Ca(2+)]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca(2+) or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr(888).