Control of astrocyte Ca2+ oscillations and waves by oscillating translocation and activation of protein kinase C

Background: Glutamate-induced Ca2+ oscillations and waves coordinate astrocyte signaling responses, which in turn regulate neuronal excitability. Recent studies have suggested that the generation of these Ca2+ oscillations requires a negative feedback that involves the activation of conventional protein kinase C (cPKC). Here, we use total internal reflection fluorescence (TIRF) microscopy to investigate if and how periodic plasma membrane translocation of cPKC is used to generate Ca2+ oscillations and waves. Results: Glutamate stimulation of astrocytes triggered highly localized GFP-PKC gamma plasma membrane translocation events, induced rapid oscillations in GFP-PKC gamma translocation, and generated GFP-PKC gamma translocation waves that propagated across and between cells. These translocation responses were primarily mediated by the Ca2+-sensitive C2 domains of PKC gamma and were driven by localized Ca2+ spikes, by oscillations in Ca2+ concentration, and by propagating Ca2+ waves, respectively. Interestingly, GFP-conjugated C1 domains from PKC gamma or PKC delta that have been shown to bind diacylglycerol (DAG) also oscillated between the cytosol and the plasma membrane after glutamate stimulation, suggesting that PKC is repetitively activated by combined oscillating increases in Ca2+ and DAG concentrations. The expression of C1 domains, which increases the DAG buffering capacity and thereby delays changes in DAG concentrations, led to a marked prolongation of Ca2+ spikes, suggesting that PKC activation is involved in terminating individual Ca2+ spikes and waves and in defining the time period between Ca2+ spikes. Conclusions: Our study suggests that cPKCs have a negative feedback role on Ca2+ oscillations and waves that is mediated by their repetitive activation by oscillating DAG and Ca2+ concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca2+ spikes and waves and can define the Ca2+ spike and wave frequencies.