Intracellular Ca 2 + oscillations generated via the Ca 2 - sensing receptor are 1 mediated by negative feedback by PKC α at Thr 888 2 3 4

23 To clarify the mechanism(s) underlying intracellular Ca concentration ([Ca]i) 24 oscillations induced by an elevation in extracellular Ca concentration ([Ca]e) via the 25 extracellular Ca-sensing receptor (CaR), we analyzed the pattern of [Ca]i response in 26 multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in 27 the [Ca]e from 1.5 mM to 3 mM produced oscillatory fluctuations in [Ca]i in 70% of the 28 cell population. To determine the role of PKC in the generation of [Ca]i oscillations, cells 29 were exposed to increasing concentrations (0.5-5 μM) of the preferential PKC inhibitor Ro30 31-8220 prior to stimulation by extracellular Ca. Ro-31-8220 at 3-5 μM completely 31 eliminated the [Ca]e-evoked [Ca]i oscillations and transformed the pattern to a peak and 32 sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or 33 Gö6983 produced an identical response. Similarly, treatment with Ro-31-8220 or GFI 34 eliminated [Ca]e-evoked [Ca]i oscillations in colon-derived SW-480 cells expressing the 35 CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 36 as well as siRNA-mediated knockdown of PKCα strikingly reduced the proportion of cell 37 displaying [Ca]e-evoked [Ca]i oscillations. Furthermore, none of the cells analyzed 38 expressing a CaR mutant in which the major PKC phosphorylation site, Thr, was 39 converted to alanine (CaRT888A) showed [Ca]i oscillations after CaR activation. Our results 40 show that [Ca]i oscillations induced by activation of the CaR in response to an increase in 41 extracellular Ca or exposure to the calcimimetic R-568 result from negative feedback 42 involving PKCα-mediated phosphorylation of the CaR at Thr. 43