Dynamic changes in the phosphoinositide concentration ([PI]) in the cell membrane play an important role in the regulation of a plethora of cellular processes, e.g. regulation of ion channel function, of the cytoskeleton, and of gene expression. Tools that allow the monitoring of spatio-temporal changes in [PI] in living cells are essential to form an understanding of such [PI] regulated processes as well as processes that regulate [PI]. Of particular interest is the PI phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), which apart from its well known role as a precursor of the second messengers IP₃ and DAG was shown to be a signaling molecule of its own right that can e.g. modulate the function of certain ion channels.

Convenient tools to monitor PI levels are genetically encoded fluorescence labeled PI-binding probes such as the PI(4,5)P₂ specific PLCd₁-PH-GFP and tubby-GFP probes, since membrane association of these probes is directly related to PI content of the membrane. Of the tubby probe several mutants have been reported (Quinn et al. 2008, J. Physiol. 586, 2855–2871) that have different PI(4,5)P₂ affinities which may allow the monitoring of [PI(4,5)P₂] over a wide range (low affinity probes for high concentrations and high affinity probes for low concentrations). A comparison of PI(4,5)P₂ binding affinities in living cells has not been performed so far, but knowledge of the rank order of these affinities is essential to select the most appropriate probes and to interpret experimental results obtained with these probes.

To tackle this problem we employed the voltage dependent PI(4,5)P₂-5'-phosphatase Ci-VSP which alters cell membrane [PI(4,5)P₂] in a graded and reversible manner in response to changes in membrane voltage (Halaszovich et al., 2008, J. Biol. Chem., in press). We co-expressed Ci-VSP with different GFP tagged PI(4,5)P₂ binding probes in CHO cells and used total internal reflection (TIRF) microscopy to measure membrane association of the PI(4,5)P₂ probe under investigation at different membrane voltages. We observed a decrease in the TIRF signal at depolarized potentials, indicating the translocation of the probe from the cell membrane into the cytosol in response to the decrease in membrane [PI(4,5)P₂]. From the change in the TIRF signal we constructed fluorescence-voltage relations for each probe; comparing these we obtained the rank order of the affinities of the probes.

We conclude that Ci-VSP is a valuable tool in differentiating PI(4,5)P₂ affinities in living cells, which we demonstrated by analyzing the PI(4,5)P₂ affinities of fluorescence labeled probes. We want to point out that a similar approach can be used to examine and compare the [PI(4,5)P₂]-dependence of other cellular processes, like the regulation of ion channels, in a semi-quantitative way.

Supported by DFG grant OL 240/2–1