Plasma membranes contain the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), which is a precursor of signaling molecules (IP₃, PIP₃, DAG) but is also considered a bona fide second messenger itself. There is accumulating evidence that most ion channels and membrane transporters are regulated by PIP₂. Investigation of PIP₂ dependent regulation of membrane proteins requires tools to manipulate the PIP₂ concentration and to simultaneously monitor dynamic changes. A voltage-activated phosphatase (Ci-VSP) cloned from the tunicate Ciona intestinalis represents a powerful tool for depleting PIP₂ in the plasma membrane in a graded and reversible manner without generating other signaling molecules (Iwasaki, H. et al. 2008. PNAS 23, 7970-7975 begin_of_the_skype_highlightingend_of_the_skype_highlighting). FRET sensors for PIP₂ (e.g. PH-PLCd1-CFP and PH-PLC1-YFP) permit high resolution monitoring of PIP₂ plasma membrane levels.

To allow for adequate expression of these three proteins in mammalian cells, a vector for a self- processing polyprotein was constructed by linking the individual cDNAs with viral 2A oligopeptide sequences to form a single open reading frame. 2A oligopeptides mediate contranslational cleavage at their C-terminus resulting in the production of separate proteins (De Felipe, P. et al. 2006. Trends Biotechnol. 24, 68-75). Using combined patch clamp and FRET measurements, functional expression of the three singular proteins in HEK293 transfected with the 2A-peptide vector could be verified. By comparing the rates of recovery of FRET and current we can demonstrate that co-expressed Kir3.1/Kir3.4 channels are highly sensitive to Ci-VSP-induced depletion of PIP₂, whereas Kir2.1 channels are much more resistant.