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. 2011 May 3;589(Pt 13):3149–3162. doi: 10.1113/jphysiol.2011.208983

Figure 4. TASK-3 currents are insensitive to combined depletion of PI(4,5)P2 and PI(4)P by pseudojanin.

Figure 4

A, schematic representation of the pseudojanin membrane targeting construct (RF–PJ) and its recruitment to the plasma membrane by rapamycin. Confocal images of RF–PJ before (top) and after addition of 5 μm rapamycin (bottom) show efficient translocation of PJ. Scale bar, 10 μm. B, TIRF recordings were performed on cells co-transfected with PHPLCδ1-GFP, Lyn11-FRB, and either of the RF–PJ variants indicated. Dissociation of PHPLCδ1-GFP from the membrane upon application of rapamycin (shaded) was only observed with intact INPP5E 5-phosphatase domain. (n = 9 cells/5 independent experiments, 5/3, 10/4 and 21/8, for PJ, PJ-Sac, PJ-5ptase and PJ-Dead, respectively). C, TIRF recordings from cells co-transfected with PHOSH2×2-GFP, Lyn11-FRB and either of the RF–PJ variants indicated. Dissociation of PHOSH2×2-GFP from the membrane required functional Sac1 and INPP5E phosphatase domains (n = 14 cells /8 independent experiments, 11/5, 15/5 and 14/21, for PJ, PJ-Sac, PJ-5ptase and PJ-Dead, respectively). D, whole-cell voltage-clamp recordings from cells co-transfected with Lyn11-FRB, fully intact PJ and either TASK-3 or KCNQ4. While KCNQ currents were robustly inhibited by recruitment of PJ (residual currents, 30.6 ± 7.8%, n = 6), TASK currents were not affected (residual currents, 97.4 ± 3.9%, n = 7). Voltage protocols as shown in Fig. 1.