Multivalent Cation-Bridged PI(4,5)P2 Clusters Form at Very Low Concentrations

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂ or PIP2), is a key component of the inner leaflet of the plasma membrane in eukaryotic cells. In model membranes, PIP2 has been reported to form clusters, but whether these locally different conditions could give rise to distinct pools of unclustered and clustered PIP2 is unclear. By use of both fluorescence self-quenching and Förster resonance energy transfer assays, we have discovered that PIP2 self-associates at remarkably low concentrations starting below 0.05 mol% of total lipids. Formation of these clusters was dependent on physiological divalent metal ions, such as Ca²⁺, Mg²⁺, Zn²⁺, or trivalent ions Fe³⁺ and Al³⁺. Formation of PIP2 clusters was also headgroup-specific, being largely independent of the type of acyl chain. The similarly labeled phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol exhibited no such clustering. However, six phosphoinositide species coclustered with PIP2. The degree of PIP2 cation clustering was significantly influenced by the composition of the surrounding lipids, with cholesterol and phosphatidylinositol enhancing this behavior. We propose that PIP2 cation-bridged cluster formation, which might be similar to micelle formation, can be used as a physical model for what could be distinct pools of PIP2 in biological membranes. To our knowledge, this study provides the first evidence of PIP2 forming clusters at such low concentrations. The property of PIP2 to form such clusters at such extremely low concentrations in model membranes reveals, to our knowledge, a new behavior of PIP2 proposed to occur in cells, in which local multivalent metal ions, lipid compositions, and various binding proteins could greatly influence PIP2 properties. In turn, these different pools of PIP2 could further regulate cellular events.     

PI(4,5)P(2) regulation of surface membrane traffic.

Phosphatidylinositol 4,5-biphosphate (PI[4,5]P(2)) has emerged as an important signaling molecule in the membrane for regulating vesicle exo- and endocytosis and the accompanying actin cytoskeletal rearrangements. Localization studies with GFP-tagged binding domains and antibodies provide new views of the non-uniform, dynamic distribution of PI(4,5)P(2) in membranes and its organization in raft-like domains. The targeting of phosphoinositide kinases by GTPases can coordinate the reactions of membrane fusion and fission with cytoskeletal assembly, providing a basis for membrane movement.     

Regulation of voltage-gated potassium channels by PI(4,5)P2

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) regulates activities of numerous ion channels including inwardly rectifying potassium (K_ir) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K_V) channels might be regulated by PI(4,5)P₂. Wide expression of K_V channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K_V channels by PI(4,5)P₂, we have coexpressed several of them in tsA-201 cells with a G protein–coupled receptor (M₁R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P₂ with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P₂ at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K_V7.1, K_V7.2/7.3, and K_ir2.1 channel current by 90–95%. Activation of M₁R inhibited K_V7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P₂ regulation of activity of K_V1.1/K_Vβ1.1, K_V1.3, K_V1.4, and K_V1.5/K_Vβ1.3, K_V2.1, K_V3.4, K_V4.2, K_V4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K_V1.1/K_Vβ1.1 and K_V3.4, resulting in up-regulation of current density upon activation of M₁R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P₂ at the plasma membrane by enzymes does not seem to influence activity of most tested K_V channels, whereas it does strongly inhibit members of the K_V7 and K_ir families.     

Polyamines, PI(4,5)P2, and actin polymerization

Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol-4-phosphate 5-kinases (PI(4)P5K), enzymes that convert phosphatidylinositol-4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 +/- 3.5% and 81.5 +/- 4.0% of control, respectively. Accurately reversing DFMO-evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD-sensitive PI(4)P5K. PUT and SPM were not involved in DFMO-evoked changes in cellular PI(4,5)P2 contents. In DFMO-treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 +/- 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO-evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO-differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD-sensitive PI(4,5)P2 pools, probably formed via SPD-sensitive PI(4)P5K, that likely control actin polymerization.     

Molecular determinants of PI(4,5)P2 and PI(3,4,5)P3 regulation of the epithelial Na+ channel

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na(+) channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P(3) and PI(4,5)P(2) to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in beta- and gamma- but not alpha-ENaC as necessary for PI(3,4,5)P(2) but not PI(4,5)P(2) modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in beta- and gamma-ENaC are critical to PI(3,4,5)P(3) augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of beta- and gamma-ENaC were identified as being critical to down-regulation of ENaC activity and P(o) in response to depletion of membrane PI(4,5)P(2). These regions of the channel played no identifiable role in a PI(3,4,5)P(3) response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P(2) to increase open probability. We conclude that beta and gamma subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P(3) and PI(4,5)P(2). This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.     

Type Igamma PIP kinase is a novel uropod component that regulates rear retraction during neutrophil chemotaxis

Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility. Here, we demonstrate that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma661), which generates PtdIns(4,5)P(2), is enriched in the uropod during chemotaxis of primary neutrophils and differentiated HL-60 cells (dHL-60). Using time-lapse microscopy, we show that enrichment of PIPKIgamma661 at the cell rear occurs early upon chemoattractant stimulation and is persistent during chemotaxis. Accordingly, we were able to detect enrichment of PtdIns(4,5)P(2) at the uropod during chemotaxis. Overexpression of kinase-dead PIPKIgamma661 compromised uropod formation and rear retraction similar to inhibition of ROCK signaling, suggesting that PtdIns(4,5)P(2) synthesis is important to elicit the backness response during chemotaxis. Together, our findings identify a previously unknown function for PIPKIgamma661 as a novel component of the backness signal that regulates rear retraction during chemotaxis.     

Determinants of molecular specificity in phosphoinositide regulation. Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is the endogenous lipid regulating TRPV1

Once thought of as simply an oily barrier that maintains cellular integrity, lipids are now known to play an active role in a large variety of cellular processes. Phosphoinositides are of particular interest because of their remarkable ability to affect many signaling pathways. Ion channels and transporters are an important target of phosphoinositide signaling, but identification of the specific phosphoinositides involved has proven elusive. TRPV1 is a good example; although phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) can potently regulate its activation, we show that phosphatidylinositol (4)-phosphate (PI(4)P) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)) can as well. To determine the identity of the endogenous phosphoinositide regulating TRPV1, we applied recombinant pleckstrin homology domains to inside-out excised patches. Although a PI(4,5)P(2)-specific pleckstrin homology domain inhibited TRPV1, a PI(3,4,5)P(3)-specific pleckstrin homology domain had no effect. Simultaneous confocal imaging and electrophysiological recording of whole cells expressing a rapamycin-inducible lipid phosphatase also demonstrates that depletion of PI(4,5)P(2) inhibits capsaicin-activated TRPV1 current; the PI(4)P generated by the phosphatases was not sufficient to support TRPV1 function. We conclude that PI(4,5)P(2), and not other phosphoinositides or other lipids, is the endogenous phosphoinositide regulating TRPV1 channels.     

TXNIP interaction with GLUT1 depends on PI(4,5)P2

GLUT1 is a major glucose facilitator expressed ubiquitously among tissues. Upregulation of its expression plays an important role in the development of many types of cancer and metabolic diseases. Thioredoxin-interacting protein (TXNIP) is an α-arrestin that acts as an adaptor for GLUT1 in clathrin-mediated endocytosis. It regulates cellular glucose uptake in response to both intracellular and extracellular signals via its control on GLUT1‐4. In order to understand the interaction between GLUT1 and TXNIP, we generated GLUT1 lipid nanodiscs and carried out isothermal titration calorimetry and single-particle electron microscopy experiments. We found that GLUT1 lipid nanodiscs and TXNIP interact in a 1:1 ratio and that this interaction requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂ or PIP₂).     

Conversion of phosphatidylinositol (PI) to PI4‐phosphate (PI4P) and then to PI(4,5)P2 is essential for the cytosolic Ca2+ concentration under heat stress in Ganoderma lucidum

How cells drive the phospholipid signal response to heat stress (HS) to maintain cellular homeostasis is a fundamental issue in biology, but the regulatory mechanism of this fundamental process is unclear. Previous quantitative analyses of lipids showed that phosphatidylinositol (PI) accumulates after HS in Ganoderma lucidum, implying the inositol phospholipid signal may be associated with HS signal transduction. Here, we found that the PI‐4‐kinase and PI‐4‐phosphate‐5‐kinase activities are activated and that their lipid products PI‐4‐phosphate and PI‐4,5‐bisphosphate are increased under HS. Further experimental results showed that the cytosolic Ca²⁺ ([Ca²⁺]_c) and ganoderic acid (GA) contents induced by HS were decreased when cells were pretreated with Li⁺, an inhibitor of inositol monophosphatase, and this decrease could be rescued by PI and PI‐4‐phosphate. Furthermore, inhibition of PI‐4‐kinases resulted in a decrease in the Ca²⁺ and GA contents under HS that could be rescued by PI‐4‐phosphate but not PI. However, the decrease in the Ca²⁺ and GA contents by silencing of PI‐4‐phosphate‐5‐kinase could not be rescued by PI‐4‐phosphate. Taken together, our study reveals the essential role of the step converting PI to PI‐4‐phosphate and then to PI‐4,5‐bisphosphate in [Ca²⁺]_c signalling and GA biosynthesis under HS.     

The inositol 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis and is necessary for cytokinesis

During cytokinesis, constriction of an equatorial actomyosin ring physically separates the two daughter cells. At the cleavage furrow, the phosphoinositide PI(4,5)P2 plays an?important role by recruiting and regulating essential proteins of the cytokinesis machinery [1]. Accordingly, perturbation of PI(4,5)P2 regulation leads to abortive furrowing and binucleation [2-4]. To determine how PI(4,5)P2 is regulated during cytokinesis, we individually knocked down each of the enzymes controlling the phosphoinositide (PIP) cycle in Drosophila. We show that depletion of the Drosophila ortholog of human oculocerebrorenal syndrome of Lowe 1 (OCRL1), an inositol 5-phosphatase mutated in the X-linked disorder oculocerebrorenal Lowe syndrome, triggers a high rate of cytokinesis failure. In absence of dOCRL, several essential components of the cleavage furrow were found to be incorrectly localized on giant cytoplasmic vacuoles rich in PI(4,5)P2 and in endocytic markers. We demonstrate that dOCRL is associated with endosomes and that it dephosphorylates PI(4,5)P2 on internal membranes to restrict this phosphoinositide at the plasma membrane and thereby regulates cleavage furrow formation and ingression. Identification of dOCRL as essential for cell division may be important to understand the molecular basis of the phenotypic manifestations of Lowe syndrome.     

Local PI(4,5)P2 signaling inhibits fusion pore expansion during exocytosis

1. Download : Download high-res image (107KB)
2. Download : Download full-size image

     

Smooth muscle length-dependent PI(4,5)P2 synthesis and paxillin tyrosine phosphorylation

We studiedeffects of increasing the length of porcine trachealis muscle on 5.5 µM carbachol (CCh)-evoked phosphatidylinositol 4,5-bisphosphate[PI(4,5)P₂] synthesis and other parametersof phosphatidylinositol (PI) turnover.PI(4,5)P₂ resynthesis rates in muscle held at1.0 optimal length (L_o), measured over the first 6 min of CCh stimulation, were 140 ± 12 and 227 ± 14% ofvalues found in muscle held at 0.5 L_o and infree-floating muscle, respectively. Time-dependent changes in cellularmasses of PI(4,5)P₂, PI, and phosphatidicacid, and PI resynthesis rates, were also altered by the muscle lengthat which contraction occurred. In free-floating muscle, CCh did notevoke increases in tyrosine-phosphorylated paxillin (PTyr-paxillin), anindex of ₁-integrin signaling; however, there wereprogressive increases in PTyr-paxillin in muscle held at 0.5 and 1.0 L_o during contraction, which correlated withincreases in PI(4,5)P₂ synthesis rates. Thesedata indicate that PI(4,5)P₂ synthesis ratesand other parameters of CCh-stimulated inositol phospholipid turnoverare muscle length-dependent and provide evidence that supports thehypothesis that length-dependent ₁-integrin signals mayexert control on CCh-activated PI(4,5)P₂ synthesis.

     

A role for PtdIns(4,5)P2 and PIP5Kalpha in regulating stress-induced apoptosis

The phosphoinositide phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) is essential for many cellular processes and is linked to the etiology of numerous human diseases . PtdIns(4,5)P(2) has been indirectly implicated as a negative regulator of apoptosis ; however, it is unclear if apoptotic stimuli negatively regulate PtdIns(4,5)P(2) levels in vivo. Here, we show that two apoptotic-stress stimuli, hydrogen peroxide (H(2)O(2)) and UV irradiation, cause PtdIns(4,5)P(2) depletion during programmed cell death independently of and prior to caspase activation. Depletion of PtdIns(4,5)P(2) is essential for apoptosis because maintenance of PtdIns(4,5)P(2) levels by overexpression of PIP5Kalpha rescues cells from H(2)O(2)-induced apoptosis. PIP5Kalpha expression promotes both basal and sustained ERK1/2 activation after H(2)O(2) treatment, and importantly, pharmacological inhibition of ERK1/2 signaling blocks PIP5Kalpha-mediated cell survival. H(2)O(2) induces tyrosine phosphorylation and translocation of PIP5Kalpha away from its substrate at the plasma membrane, and both are dependent upon the activity of c-src family kinases. Furthermore, constitutively active c-src enhances tyrosine phosphorylation of PIP5Kalpha in vivo and is sufficient for the translocation of PIP5Kalpha away from the plasma membrane. These observations demonstrate that certain apoptotic stimuli initiate an essential signaling pathway during cell death, and this pathway leads to caspase-independent downregulation of PIP5Kalpha and its product PtdIns(4,5)P(2).     

Synaptojanin 1-mediated PI(4,5)P2 hydrolysis is modulated by membrane curvature and facilitates membrane fission

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P?] plays a fundamental role in clathrin-mediated endocytosis. However, precisely how PI(4,5)P? metabolism is spatially and temporally regulated during membrane internalization and the functional consequences of endocytosis-coupled PI(4,5)P? dephosphorylation remain to be explored. Using cell-free assays with liposomes of varying diameters, we show that the major synaptic phosphoinositide phosphatase, synaptojanin 1 (Synj1), acts with membrane curvature generators/sensors, such as the BAR protein endophilin, to preferentially remove PI(4,5)P? from curved membranes as opposed to relatively flat ones. Moreover, in vivo recruitment of Synj1's inositol 5-phosphatase domain to endophilin-induced membrane tubules results in fragmentation and condensation of these structures largely in a dynamin-dependent fashion. Our study raises the possibility that geometry-based mechanisms may contribute to spatially restricting PI(4,5)P? elimination during membrane internalization and suggests that the PI(4,5)P?-to-PI4P conversion achieved by Synj1 at sites of high curvature may cooperate with dynamin to achieve membrane fission.     

Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P2 hydrolysis

TRPV3 is a thermosensitive channel that is robustly expressed in skin keratinocytes and activated by innocuous thermal heating, membrane depolarization, and chemical agonists such as 2-aminoethyoxy diphenylborinate, carvacrol, and camphor. TRPV3 modulates sensory thermotransduction, hair growth, and susceptibility to dermatitis in rodents, but the molecular mechanisms responsible for controlling TRPV3 channel activity in keratinocytes remain elusive. We show here that receptor-mediated breakdown of the membrane lipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P(2)) regulates the activity of both native TRPV3 channels in primary human skin keratinocytes and expressed TRPV3 in a HEK-293-derived cell line stably expressing muscarinic M(1)-type acetylcholine receptors. Stimulation of PI(4,5)P(2) hydrolysis or pharmacological inhibition of PI 4 kinase to block PI(4,5)P(2) synthesis potentiates TRPV3 currents by causing a negative shift in the voltage dependence of channel opening, increasing the proportion of voltage-independent current and causing thermal activation to occur at cooler temperatures. The activity of single TRPV3 channels in excised patches is potentiated by PI(4,5)P(2) depletion and selectively decreased by PI(4,5)P(2) compared with related phosphatidylinositol phosphates. Neutralizing mutations of basic residues in the TRP domain abrogate the effect of PI(4,5)P(2) on channel function, suggesting that PI(4,5)P(2) directly interacts with a specific protein motif to reduce TRPV3 channel open probability. PI(4,5)P(2)-dependent modulation of TRPV3 activity represents an attractive mechanism for acute regulation of keratinocyte signaling cascades that control cell proliferation and the release of autocrine and paracrine factors.