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: 5.04). 08/2007; 582(Pt 3):945-51. DOI: 10.1113/jphysiol.2007.132860
Source: PubMed


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).

21 Reads
  • Source
    • "It has been suggested that phosphatidylinositol 4­phos­ phate is critical for replenishing PI(4,5)P 2 via its phos­ phorylation by PIP5K (Suh and Hille, 2002; Loew, 2007). "
    [Show abstract] [Hide abstract]
    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 · 4.79 Impact Factor
  • Source
    • "Thus, HIV-1 Gag membrane association is mediated by a specific bipartite determinant in the MA domain comprised of myristate and basic amino acid clusters [1] with Gag-PI(4,5)P2 binding serving as the basis for targeted membrane association. Gag's preferential association with the plasma membrane is due to two inherent features of PI(4,5)P2: (i) the PM is where most of cellular PI(4,5)P2 is located [60] and (ii) PI(4,5)P2 molecules are products of in situ synthesis (i.e., PM-localized molecules are produced at the PM; [69]). Thus, PI(4,5)P2 targeting provides a mechanism to direct Gag from its site of synthesis in the cell interior to the plasma membrane. "
    [Show abstract] [Hide abstract]
    ABSTRACT: More than a decade has elapsed since the link between the endosomal sorting complex required for transport (ESCRT) machinery and HIV-1 protein trafficking and budding was first identified. L domains in HIV-1 Gag mediate recruitment of ESCRT which function in bud abscission releasing the viral particle from the host cell. Beyond virus budding, the ESCRT machinery is also involved in the endocytic pathway, cytokinesis, and autophagy. In the past few years, the number of non-ESCRT host proteins shown to be required in the assembly process has also grown. In this paper, we highlight the role of recently identified cellular factors that link ESCRT machinery to calcium signaling machinery and we suggest that this liaison contributes to setting the stage for productive ESCRT recruitment and mediation of abscission. Parallel paradigms for non-ESCRT roles in virus budding and cytokinesis will be discussed.
    06/2012; 2012(4):851670. DOI:10.1155/2012/851670
  • Source
    • "8-pCPT suppressed K-ATP channels that additionally enhanced bursting. Activities of TRPM4 and K-ATP increased after brief exposure to bradykinin and decreased after incubation with wortmannin, the treatments often used to manipulate phosphatidylinositol 4,5-bisphosphate (PIP 2 ) levels (Loew 2007; Pian et al. 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: J. Neurochem. (2011) 117, 295–308. The exchange factor directly activated by cAMP (Epac) can couple cAMP production to the activation of particular membrane and cytoplasmic targets. Using patch-clamp recordings and calcium imaging in organotypic brainstem slices, we examined the role of Epac in pre-Bötzinger complex, an essential part of the respiratory network. The selective agonist 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT) sensitized calcium mobilisation from inositol-1,4,5-trisphosphate-sensitive internal stores that stimulated TRPM4 (transient receptor potential cation channel, subfamily M, Melastatin) channels and potentiated the bursts of action potentials. 8-pCPT actions were abolished after inhibition of phospholipase C with U73122 and depletion of calcium stores with thapsigargin. Caffeine-sensitive release channels were not modulated by 8-pCPT. Epac inhibited ATP-sensitive K+ channels that also led to the enhancement of bursting by 8-pCPT. Bursting activity, spontaneous calcium transients and activity of TRPM4 and ATP-sensitive K+ channels were potentiated after brief exposures to bradykinin and incubation with wortmannin produced opposite effects that can be explained by changes in phosphatidylinositol 4,5-bisphosphate levels. 8-pCPT stimulated the respiratory motor output in functionally intact preparations and the effects of bradykinin and wortmannin were identical to those observed in organotypic slices. The data thus indicate a novel pathway of controlling bursting activity in pre-Bötzinger complex neurons through Epac that can involved in reinforcement of the respiratory activity by cAMP.
    Journal of Neurochemistry 04/2011; 117(2):295-308. DOI:10.1111/j.1471-4159.2011.07202.x · 4.28 Impact Factor
Show more

Preview (2 Sources)

21 Reads
Available from