Activation of Voltage-Sensitive Phosphatases with Pharmacological and Optogenetic Methods

Abstract

Voltage-sensitivephosphatases (VSPs) are proteins that dephosphorylate phosphoinositides (PIs) upon membrane voltage dependent activation. Particularly, Ci-VSP (Ciona intestinalis Voltage-Sensitive Phosphatase) is a 5-phosphatase of PI(4,5)P 2 and PI(3,4,5)P 3 and has so far been studied by electrophysiological means.Here we developed more broadly applicable methods of membrane depolarization and therefore phosphatase activation. First, depolarization was achieved by coexpression of the K þ channels TASK and KCNQ and elevation of extracellular potassium concentration. Second, we utilized the capsaicinactivated TRPV1 channel, and last the photosensitive Channel Rhodopsin, ChR2, in order to activate Ci-VSP. Cultured cell lines were transfected with the different channels, Ci-VSP and sensors for PIs, e.g. the tubby and the PLCd1-PH domain, each tagged with a fluorescent protein. Confocal microscopy was used to visualize and quantify the translocation of the PI-sensors from the membrane to the cytoplasm after the dephosphorylation of PIs. All three methods resulted in successful activation of Ci-VSP, with TRPV1 and ChR2 mediated depolarization to give the most robust results. Specifically, the ChR2 activation provides a very rapid response without the need of solution exchanges, simplifying the experimental procedures. To conclude, we suggest a series of methods that allow the manipulation of PI levels as well as the study of VSPs in living cells without electrophysiological instrumentation. This work was supported by a grant of the University Medical Center Giessen and Marburg (UKGM 32/2011 MR) to CRH and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to DO. 2256-Pos Board B275 Ion Induced Changes in Phosphoinositide Monolayers at Phisiological Concentrations