Phosphatidylinositol 4,5-bisphosphate (PIP₂) is not only a precursor of the second messengers IP₃ and DAG but is considered a bona fide second messenger itself. There is accumulating evidence that most ion channels and membrane transporters are regulated by the concentration of PIP₂ in the inner leaflet of the cell membrane. Thus far, no experimental data are available regarding the roles of individual kinases and phosphatases in homeostasis and dynamic regulation of PIP₂ and the impact on ion channels in cardiac myocytes. Ci-VSP is a protein cloned from the marine tunicate Ciona intestinalis consisting of a voltage sensor domain and an intracellular domain, which acts as a voltage-activated phosphinositide phosphatase (Murata,Y. et al. 2005. Nature 435, 1239-1243) and represents an emerging tool for manipulating PIP₂ levels without interfering with other pathways. Here we demonstrate that adenoviral expression of Ci-VSP can be used in mammalian cells, including adult atrial myocytes, as a powerful tool to manipulate PIP₂ levels in the cell membrane using depolarizing voltage steps of <=1 s in duration. In combination with co-expressed PH-PLCd₁-derived FRET-sensors for PIP₂, changes in PIP₂ and channel activity were measured in parallel. The onset of inhibition and recovery of endogenous Kir3.1/Kir3.4 current was slower than the corresponding FRET signal, in line with a lower affinity of the Kir3 channel subunits as compared to PH-PLCd₁. We suggest that this approach can be used for assessing relative affinities of channel proteins to PIP₂ and for analysing cell-specific pathways of PIP₂ homeostasis