Exploring steps of the PI cycle by monitoring Ins(1,4,5)P3, DAG, and PtdIns(4,5)P2. (A) Schematic of Ins(1,4,5)P3 and DAG indicators. LIBRAvIII has CFP and YFP at opposite termini of an Ins(1,4,5)P3 binding domain. (B) Time course of intramolecular LIBRAvIII nFRETeff during a 10-min stimulation with 10 µM Oxo-M (gray bar) in cells with or without expression of M1R. Note inverted y axis for LIBRAvIII since FRET is reduced by binding of Ins(1,4,5)P3 to this receptor. (C) Time course of homologous nFRETeff between CFP- and YFP-tagged C1A domains with or without expression of M1R. CFP- and YFP-tagged C1A domains detect DAG at the PM and increase their FRET when DAG increases at the PM. (D) Comparison of pretreatment with 0.1 µM or 30 µM Wtn (shaded area) on PtdIns(4,5)P2 regeneration during M1R stimulation by 10 µM Oxo-M (gray, control; pink, 0.1 µM Wtn; and purple, 30 µM Wtn). (E) Effect of 2-h pretreatment with 10 mM Li+-enriched solution on regeneration of PtdIns(4,5)P2 (gray, control; green, Li+). (F) Mean PtdIns(4,5)P2 regeneration measured as homologous nFRETeff of CFP- and YFP-tagged PHPLCδ1 at 10 min of Oxo-M treatment. Control, n = 6 cells; 0.1 µM Wtn, n = 9; 30 µM Wtn, n = 9; Li+, n = 4. (G) Wtn (shaded area) limits Ins(1,4,5)P3 regeneration during M1R activation as measured with LIBRAvIII (pink, control; red: 30 µM Wtn). (H) Wtn depresses DAG production as measured with C1A probes (aqua, control; blue, 30 µM Wtn). (I) Mean Wtn effect on production of Ins(1,4,5)P3 (red bars) and DAG (blue bars) measured as nFRETeff at 10 min of Oxo-M treatment. Ins(1,4,5)P3: control, n = 4 cells and Wtn, n = 6; DAG: control, n = 9 and Wtn, n = 6 cells. *, P < 0.05; **, P < 0.005. n.s., not significant.