Increasing Sac1 at the PM decreases PM PI(4)P and PI(4,5)P₂. (A) Representative control cell. (top) Inverted TIRF micrographs of a tsA201 cells expressing CFP-FKBP-Cb₅ ER anchor (top row) and mCherry-P4M (bottom row), before (left) and after (right) the addition of rapamycin (5 µM). Cells also express LDR PM anchor. Bar, 5 µm. (bottom) Normalized intensity kinetics of overexpressed proteins from TIRF footprint. (B and C) Same experiment as A, except Sac1 is overexpressed (B) or cells are treated with siRNA against Sac1 (C). Representative cells displayed. (D, left) Inverted TIRF footprint from a single tsA201 cell expressing CFP-FKBP-Cb₅ and RFP-PH_PLCδ1 (bottom row), before (left) and after (right) the addition of rapamycin (5 µM). (righti) Analysis of changes in PH_PLCδ1 signal after the addition of rapamycin (means ± SEM, n = 12). (E) Same experiment as D, except GFP-Sac1 (top row) is overexpressed. Note that with GFP-Sac1 overexpression there is a faster decrease in PH_PLCδ1 signal (means ± SEM, n = 11). (F, left) Time course of KCNQ2/3 inhibition after the recruitment of endogenous Sac1 (eSac1) to the PM. (right) Summary of the percent decrease in KCNQ2/3 current (n = 7). (G) Time course of KCNQ2/3 inhibition after the recruitment of overexpressed GFP-Sac1 (oSac1) to the PM. (right) Summary of the percent decrease in KCNQ2/3 current (n = 6; *, P < 0.05). (H) Mass spectrometry analysis of total cellular PS (left), PIP (center), and PIP₂ (right) before and after the addition of rapamycin (5 µM) from tsA201 cells expressing CFP-FKBP-Cb₅, LDR, and GFP-Sac1. Means ± SEM, n = 4, t test; N.S., P > 0.05; *, P < 0.05). (I, left) steady-state distribution of GFP-Evt2-x2PH in control (top) or Sac1-overexpressing (bottom) tsA201 cells. PM/cytoplasmic (Cyto.) intensity of GFP-Evt2-x2PH (means ± SEM, n = 10, t test, N.S., not significant). (middle) Same experiment as A, except GFP-Evt2-x2PH is expressed (n = 10). (right) Same experiment as B, except GFP-Evt2-x2PH is expressed (n = 11). Bars, 5 µm.