Detailed Analysis of Phospholipase C-β Activity in Living Cells

Abstract

Phospholipase C-β (PLC-β) catalyzes the hydrolysis of the plasma membrane (PM) lipid phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), thereby reducing the PM concentration of its precursor PI(4)P. A recent report proposed independent PI(4,5)P2 and PI(4)P PM pools with essential physiological functions, but it remains unknown whether PLC-β targets to these pools differentially.Here, we investigated the time course and specificity of PLC-β-dependent phospholipid depletion in living CHO cells using genetically-encoded phospholipid sensors, total internal reflection fluorescence microscopy and whole cell patch clamp. PLC-β mediated PI(4,5)P2 hydrolysis was detected reliably using KCNQ (Kv7)-mediated K+ currents, the PI(4,5)P2 reporters PLCδ1-PH and Epsin ENTH, but not with the C-terminus of Tubby protein. Analogous experiments showed that activation of PLC-β did not reduce the concentration of PI(4) detected with the PI(4)P-specific reporter Osbp-PH. When PLC-β3 was heterologously overexpressed, Tubby-Cterm and Osbp-PH reported on the PLC-β-induced changes of PI(4,5)P2 and PI(4)P, respectively.In summary, we present detailed real-time analysis of PLC-β activity in living cells. Our findings indicate that phospholipid sensors may detect different phospholipid pools that are accessible to PLC-β differentially. Hence, this work supports the presence of functional phospholipid pools in living cells.This work was supported by Deutsche Forschungsgemeinschaft through SFB 593 (TP A12) to D.O.