Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

Abstract

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca2+ concentration ([Ca2+]i) oscillations elicited by an increase in the extracellular concentration of Ca2+ ([Ca2+]e) in human embryonic kidney 293 cells expressing the Ca2+sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca2+]i oscillations but without decreasing the overall response to increase in [Ca2+]e. Exposure to 100 nM phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca2+]i oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nM) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Coexpression of CaR with constitutively active mutants of PKC (either ε or β1 isoforms) also reduced [Ca2+]i oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca2+]i responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca2+]i oscillations mediated by this receptor.