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Acta Physiologica 2012; Volume 204, Supplement 689
91st Annual Meeting of The German Physiological Society
3/22/2012-3/25/2012
Dresden, Germany
A GENETICALLY ENCODED TOOLKIT FOR MANIPULATION AND MEASUREMENT OF PIP3 IN LIVING CELLS
Abstract number: O77
Kirmse1 *S., Kienitz1 M.-C., Hertel1 F., Pott1 L.
1Institut of Physiology, Dept. of Cellular Physiology, Bochum, Germany
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: 797075
To cite this abstract, please use the following information:
Acta Physiologica 2012; Volume 204, Supplement 689 :O77