Where does all the PIP2 come from?

Richard D. Berlin Center for Cell Analysis and Modelling, University of Connecticut Health Center, Farmington, CT 06030, USA.
The Journal of Physiology (Impact Factor: 4.54). 08/2007; 582(Pt 3):945-51. DOI: 10.1113/jphysiol.2007.132860
Source: PubMed

ABSTRACT Despite its very low concentration in the plasma membrane, PIP(2) is the precursor for the important second messenger InsP(3) and, independently, is a key modulator of membrane signalling molecules such as ion channels. However, it has been difficult to determine the spatial and temporal characteristics of PIP(2) and InsP(3) during a cell signalling event. Our laboratory used bradykinin stimulation of N1E-115 neuroblastoma cells to infer the InsP(3) dynamics from calcium imaging studies, biochemical analysis and InsP(3) uncaging. We have used computational modelling with Virtual Cell to help analyse and interpret experimental data on the details of the calcium release process as well as to build a comprehensive image-based model of agonist-induced calcium release in a neuronal cell. These data provided a constraint for the further investigation of how low levels of cellular PIP(2) could provide sufficient InsP(3) for calcium release. Using biochemical assays, quantitative imaging of GFP-based probe translocation and computational analysis, it was shown that PIP(2) synthesis is stimulated concomitant with its hydrolysis. This mechanism should be important not just for consideration of PIP(2) as a precursor of InsP(3), but for any pathway that can be directly or indirectly modulated by PIP(2).

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    ABSTRACT: Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C-insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.
    The Journal of General Physiology 02/2014; 143(2):183-201. DOI:10.1085/jgp.201311033 · 4.57 Impact Factor
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    ABSTRACT: The Kv7/M current is one of the major mechanisms controlling neuronal excitability, which can be modulated by activation of the G protein coupled receptor (GPCR) via distinct signaling pathways. Membrane microdomains known as lipid rafts have been implicated in the specificity of various cell signaling pathways. The aim of this study was to understand the role of lipid rafts in the specificity of Kv7/M current modulation by activation of GPCR. Methyl-β-cyclodextrin (MβCD), often used to disrupt the integrity of lipid rafts, significantly reduced the bradykinin receptor (B2R)-induced but not muscarinic receptor (M1R)-induced inhibition of the Kv7/M current. B2R and related signaling molecules but not M1R were found in caveolin-containing raft fractions of the rat superior cervical ganglia (SCG). Furthermore, activation of B2R resulted in translocation of additional B2R into the lipid rafts, which was not observed for activation of M1R. The increase of B2R-induced intracellular Ca(2+) was also greatly reduced after MβCD treatment. Finally, B2R but not M1R was found to interact with the IP3 receptor. In conclusion, the present study implicates an important role for lipid rafts in mediating specificity for GPCR-mediated inhibition of the Kv7/M current.
    Neuroscience 09/2013; DOI:10.1016/j.neuroscience.2013.08.064 · 3.33 Impact Factor
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