Phosphatidylinositol-3,4,5 trisphosphate (PIP₃) is a key second messenger in eukaryotic cells. Experimental tools to study its controlled synthesis and degradation are of widespread interest. Here we present a novel toolkit combining a farnesylated PIP₃-sensitive single molecule FRET-sensor derived from the PH-domain of the protein kinase Btk (InPBtk) and a G365A mutant of the voltage-activated PI-phosphatase Ci-VSP (CiV-G365A). Both proteins were expressed from a single ORF, using a self-cleaving viral 2A-peptide sequence. In voltage-clamped HEK293 cells transfected with this construct, robust FRET signals indicating depletion of PIP₃ could be induced by step depolarizations positive to -20 mV in a voltage- and time-dependent fashion. By replacing InPBtk by a pair of PIP₂-selective FRET sensors based on PH-domains of PLC¹ we studied selectivity of CiV-G365A for PIP₃. In contrast to a previous study using Xenopus oocytes², significant depletion of PIP₂ was induced by voltage activation of CiV-G365A, amounting to about 10% of the depletion by wild-type Ci-VSP. Kinetics of depletion and recovery were about one order of magnitude faster for PIP₂ as compared to PIP₃. By expressing this tool in atrial myocytes via adenoviral gene transfer, we could demonstrate that endogenous GIRK channels, which are highly sensitive to changes in the abundance of PIP₂, are not affected by depletion of PIP₃.

¹Hertel et al. (2011) Plos.One 6: e20855;

²Iwasaki et al. (2008), PNAS 105: 7970–75