Real-time analysis of phospholipase C activity during different patterns of receptor-induced Ca 2+ responses in HEK293 cells

Abstract

[Ca 2+] i oscillations can either depend on oscillatory inositol-1,4,5-trisphosphate (InsP 3) formation by phospholipase C (PLC) or rely on local feedback mechanisms involving the InsP 3 receptor. To assess the PLC activity underlying carbachol-induced [Ca 2+] i oscillations in single HEK293 cells, we co-imaged [Ca 2+] i with fluorescent fusion proteins of protein kinase C (PKC) isotypes and the PH domain of PLC-δ1 (PLC-δ1(PH)). The translocation of PKCα-YFP in single cells followed two discrete patterns. Upon maximally effective agonist concentrations, a fast association and delayed dissociation ( k on> k off) was the predominant pattern. The delayed dissociation has been linked to diacylglycerol formation. Upon stimulation with submaximally effective agonist concentrations as well as during regenerative [Ca 2+] i waves, we mainly observed short translocations with k on≈ k off. Translocation time courses and efficiencies of the diacylglycerol-sensing PKCϵ-CFP and the InsP 3/phosphatidylinositol-4,5-bisphosphate-sensing YFP-PLC-δ1(PH) were closely correlated. Significant PLC activity was only detectable upon strong receptor stimulation, which typically failed to trigger [Ca 2+] i oscillations. During [Ca 2+] i oscillations induced by submaximal receptor stimulation, YFP-PLC-δ1(PH) did not translocate, whereas a fluorescent PKCϵ fusion protein has been reported to exhibit a slow, non-oscillatory accumulation at the plasma membrane. We conclude that carbachol-induced [Ca 2+] i oscillations in HEK293 cells develop at low levels of presumably non-oscillatory PLC activity.