Ci-VSP and Ci-VSPTEN are phosphoinositide phosphatases regulated by a voltage sensing domain (VSD). Specifically, Ci-VSP is a 5'-phosphatase of PI(4,5)P₂ and PI(3,4,5)P₃ while Ci-VSPTEN, a chimera consisting of the VSD of Ci-VSP and the catalytic domain of PTEN, is a 3'-phosphatase of PI(3,4)P₂ and PI(3,4,5)P₃. The activation of these phosphatases has been mainly studied on the single-cell level using patch clamping. Our goal was to develop a more broadly applicable method for their activation.

Membrane depolarization and therefore phosphatase activation in a population of cultured cells was achieved through ion influx via heterologously expressed ion channels. The phosphatase activity was monitored by measuring the changes in phosphoinositide levels using fluorescent phosphoinositide binding probes and live cell confocal microscopy. More specifically, we examined different ion channels permeable to either K⁺ or Na⁺, different cell lines, and a range of phosphoinositide probes. Additionally, we tested two ways of measuring the phosphatase activity, one based on the direct observation of the probe's translocation from the membrane to the cytoplasm and a second based on Förster resonance energy transfer (FRET) measurements.

The activation of Na⁺ conductive transient receptor potential vanilloid 1 (TRPV1) channels proved to be the most reliable method to control the enzymatic activity of Ci-VSP and Ci-VSPTEN, using either probe translocation or FRET based measurements to detect the phosphatase activity.

This work was supported by a research grant of the University Medical Center Giessen and Marburg (UKGM 32/2011 MR) to C.R.H and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to D.O.