Quantitative Analysis of Binding Affinities of PI(4,5)P2 Sensor Domains in Living Cells by Using the Voltage-Controlled PI(4,5)P2-5’-Phosphatase, Ci-VSP

Abstract

Dynamic changes in the phosphoinositide (PI) concentration in the cell membrane play an important role in the regulation of many cellular processes. Of particular interest is the PI phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a genuine signaling molecule that, e.g., controls the function of some ion channels. Plasma membrane PI concentration in living cells is often monitored with genetically encoded fluorescence labeled PI-binding probes such as the PI(4,5)P2 specific PLCδ1-PH-GFP and tubby-GFP probes. Knowledge of their PI(4,5)P2 affinities is essential in selecting the most suitable probes and for appropriate interpretation of experimental results. These affinities of PI(4,5)P2 probes apparently differ and can be modified by mutagenesis; however, a quantitative analysis of affinity in vivo is lacking. To address this issue we employed the voltage dependent PI(4,5)P2-5′-phosphatase Ci-VSP which alters plasma membrane [PI(4,5)P2] as a function of membrane voltage in a graded and reversible manner (Halaszovich et al., 2009, JBC 284:2106-13). We co-expressed Ci-VSP with various GFP-tagged PI(4,5)P2 probes and used total internal reflection microscopy (TIRF-M) to measure membrane association of the probe at different membrane voltages. Generally, the TIRF signal decreased upon depolarization, indicating the translocation of the probe from the cell membrane into the cytosol in response to the decrease in [PI(4,5)P2]. Voltage-dependent changes of TIRF signals allowed construction of fluorescence-voltage relations for each probe that can be used as surrogate PI(4,5)P2 binding curves. Comparison of these curves defined the rank order of PI(4,5)P2 affinities of the various probes. We conclude that Ci-VSP is a valuable tool for analyzing PI(4,5)P2 affinities in living cells, which we demonstrated by determining the affinities of fluorescence labeled PI(4,5)P2-probes. Supported by DFG grant OL 240/2 and SFB 593 to D.O.