Phospholipase C-η2 is activated by elevated intracellular Ca2+ levels

Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely uncharacterised. In this study the activity of PLCη2 was examined via [3H]inositol phosphate release in COS7 cells expressing the enzyme. PLCη2 activity increased approximately 5-fold in response to monensin, a Na+/H+ antiporter. This was significantly inhibited by CGP-37157 which implies that the effect of monensin was due, at least in part, to mitochondrial Na+/Ca2+-exchange. Direct activation of PLCη2 by < 1 μM Ca2+ was confirmed in permeabilised transfected cells. The roles of the PH and C2 domains in controlling PLCη2 activity via membrane association were also investigated. A PH domain-lacking mutant exhibited no detectable activity in response to monensin or Ca2+ due to an inability to associate with the cell membrane. Within the C2 domain, mutation of D920 to alanine at the predicted Ca2+-binding site dramatically reduced enzyme activity highlighting an important regulatory role for this domain. Mutation of D861 to asparagine also influenced activity, most likely due to altered lipid selectivity. Of the C2 mutations investigated, none altered sensitivity to Ca2+. This suggests that the C2 domain is not responsible for Ca2+ activation. Collectively, this work highlights an important new component of the Ca2+ signalling toolkit and given its sensitivity to Ca2+, this enzyme is likely to facilitate the amplification of intracellular Ca2+ transients and/or crosstalk between Ca2+-storing compartments in vivo.

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► The activation of PLCη2 in a cellular context was studied in transfected COS7 cells.
► Treatment with monensin activated the enzyme via mitochondrial Na+/Ca2+ exchange.
► Direct activation of the enzyme by < 1 μM Ca2+ was demonstrated in permeabilised cells.
► The PH domain of PLCη2 is essential for membrane binding and activity.
► The C2 domain of PLCη2 is important for activity but does not control Ca2+ sensitivity.