Background: Glutamate-induced Ca 2+ oscillations and waves coordinate astrocyte signaling responses, which in turn regulate neuronal excitability. Recent studies have suggested that the generation of these Ca 2+ 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 Ca 2+ oscillations and waves. Results: Glutamate stimulation of astrocytes triggered highly localized GFP-PKCγ plasma membrane translocation events, induced rapid oscillations in GFP-PKCγ translocation, and generated GFP-PKCγ translocation waves that propagated across and between cells. These translocation responses were primarily mediated by the Ca 2+-sensitive C2 domains of PKCγ and were driven by localized Ca 2+ spikes, by oscillations in Ca 2+ concentration, and by propagating Ca 2+ waves, respectively. Interestingly, GFP-conjugated C1 domains from PKCγ, or PKCδ 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 Ca 2+ 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 Ca 2+ spikes, suggesting that PKC activation is involved in terminating individual Ca 2+ spikes and waves and in defining the time period between Ca 2+ spikes. Conclusions: Our study suggests that cPKCs have a negative feedback role on Ca 2+ oscillations and waves that is mediated by their repetitive activation by oscillating DAG and Ca 2+ concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca 2+ spikes and waves and can define the Ca 2+ spike and wave frequencies.
ASJC Scopus subject areas
- Agricultural and Biological Sciences(all)