Phosphatidylinositol Bisphosphate Research Articles

The spatial distribution of GPCR and Gβγ activity across a cell dictates PIP3 dynamics

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a plasma membrane-bound signaling phospholipid involved in many cellular signaling pathways that control crucial cellular processes and behaviors, including cytoskeleton remodeling, metabolism, chemotaxis, and apoptosis. Therefore, defective PIP3 signaling is implicated in various diseases, including cancer, diabetes, obesity, and cardiovascular diseases. Upon activation by G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2), generating PIP3. Though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show that subcellular redistributions of G proteins govern this PIP3 attenuation when GPCRs are activated globally, while localized GPCR activation induces sustained subcellular PIP3. Interestingly the observed PIP3 attenuation was Gγ subtype-dependent. Considering distinct cell-tissue-specific Gγ expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is regulated depending on the GPCR activity gradient across a cell, but also show how diversely cells respond to spatial and temporal variability of external stimuli.

Phosphoinositide acyl chain saturation drives CD8+ effector T cell signaling and function.

How lipidome changes support CD8+ effector T (Teff) cell differentiation is not well understood. Here we show that, although naive T cells are rich in polyunsaturated phosphoinositides (PIPn with 3-4 double bonds), Teff cells have unique PIPn marked by saturated fatty acyl chains (0-2 double bonds). PIPn are precursors for second messengers. Polyunsaturated phosphatidylinositol bisphosphate (PIP2) exclusively supported signaling immediately upon T cell antigen receptor activation. In late Teff cells, activity of phospholipase C-γ1, the enzyme that cleaves PIP2 into downstream mediators, waned, and saturated PIPn became essential for sustained signaling. Saturated PIP was more rapidly converted to PIP2 with subsequent recruitment of phospholipase C-γ1, and loss of saturated PIPn impaired Teff cell fitness and function, even in cells with abundant polyunsaturated PIPn. Glucose was the substrate for de novo PIPn synthesis, and was rapidly utilized for saturated PIP2 generation. Thus, separate PIPn pools with distinct acyl chain compositions and metabolic dependencies drive important signaling events to initiate and then sustain effector function during CD8+ T cell differentiation.

Conformational rearrangements in the second voltage sensor domain switch PIP2- and voltage-gating modes in two-pore channels

Two-pore channels (TPCs) are activated by phosphatidylinositol bisphosphate (PIP2) binding to domain I and/or by voltage sensing in domain II (DII). Little is known about how these two stimuli are integrated, and how each TPC subtype achieves its unique preference. Here, we show that distinct conformations of DII-S4 in the voltage-sensor domain determine the two gating modes. DII-S4 adopts an intermediate conformation, and forced stabilization in this conformation was found to result in a high PIP2-dependence in primarily voltage-dependent TPC3. In TPC2, which is PIP2-gated and nonvoltage-dependent, a stabilized intermediate conformation does not affect the PIP2-gated currents. These results indicate that the intermediate state represents the PIP2-gating mode, which is distinct from the voltage-gating mode in TPCs. We also found in TPC2 that the tricyclic antidepressant desipramine induces DII-S4-based voltage dependence and that naringenin, a flavonoid, biases the mode preference from PIP2-gating to desipramine-induced voltage gating. Taken together, our study on TPCs revealed an unprecedented mode-switching mechanism involving conformational changes in DII-S4, and its active role in integrating voltage and PIP2 stimuli.

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

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) is an important signaling phospholipid that is required for regulated exocytosis and some forms of endocytosis. The two processes share a topologically similar pore structure that connects the vesicle lumen with the outside. Widening of the fusion pore during exocytosis leads to cargo release, while its closure initiates kiss&run or cavicapture endocytosis. We show here, using live-cell total internal reflection fluorescence (TIRF) microscopy of insulin granule exocytosis, that transient accumulation of PI(4,5)P2 at the release site recruits components of the endocytic fission machinery and stalls the late fusion pore expansion that is required for peptide release. The absence of clathrin differentiates this mechanism from clathrin-mediated endocytosis. Knockdown of phosphatidylinositol-phosphate-5-kinase-1c or optogenetic recruitment of 5-phosphatase reduces PI(4,5)P2 transients and accelerates fusion pore expansion, suggesting that acute PI(4,5)P2 synthesis is involved. Thus, local phospholipid signaling inhibits fusion pore expansion and peptide release through an unconventional endocytic mechanism.

Critical regulation of the voltage-gated sodium channel, Nav1.4, by PIP2.

Voltage-gated sodium (NaV) channels are vital to the electrical signalling of cells. The voltage-dependent activation of NaV channels is followed almost instantaneously by fast inactivation, setting a timer on the excitability of tissues. NaV channels are protein complexes that consist of a large, pore forming alpha subunit and are often associated with auxiliary beta subunits. The alpha subunit is made up of four tandem repeat domains (DI-IV) that consist of six transmembrane segments (S1-S6) with S4 in each domain serving as its putative voltage sensor. The gating cycle of NaV channels is subject to tight regulation, with dysfunction leading to several disease states. Most ion channels are regulated by the membrane phospholipid, phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]; however, it is not known whether PI(4,5)P2 modulates the activity of NaV channels. Of the nine (NaV1.1 - NaV1.9) family members that have been identified and classified based on tissue distribution, we studied NaV1.4 which is the isoform primarily expressed in skeletal muscle. We used the optogenetically activated inositide phosphatase, pseudojanin, an enzymatic chimera of inositol polyphosphate 5 phosphatase E (INPP5E), which converts PI(4,5)P2 to PI4P and the S. cerevisiae sac1 phosphatase, which dephosphorylates PI(4)P, to evaluate the role of PI(4,5)P2 in NaV1.4 channel gating and regulation. Through a combination of patch-clamp recording and optogenetics we show that dephosphorylating PI(4,5)P2 shifts the voltage-dependent gating of NaV1.4 to more hyperpolarized membrane potentials. PI(4,5)P2 depletion also augments the late current that persists after fast inactivation, a phenotype that is associated with several mutations that lead to myotonia. Through this work, we provide evidence that PI(4,5)P2 is a necessary co-factor in the gating of NaV1.4 channels.

Altered voltage-dependence revealed in an inactivation-deficient mutant of Nav1.4 via optogenetic PI(4,5)P2 dephosphorylation.

Voltage-gated sodium (NaV) channels are a key membrane protein of most excitable cells that activate upon depolarization, which initiates an action potential by rapid influx of Na+ ions. Voltage-dependent activation is followed by fast inactivation, which confers the refractory period for excitable tissues. Because the gating processes of NaV channels are mechanically coupled, changes in the fast inactivation process also alter their voltage-dependent activation properties. Most ion channels are regulated by membrane phospholipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2), however the PI(4,5)P2 modulation of NaV channels has not been determined. The mutant channel NaV1.4-L435W-L437C-A438W (WCW) presents significantly attenuated fast-inactivation and expresses robustly in Xenopus oocytes. To study the effects of PI(4,5)P2 dephosphorylation on NaV1.4 activation in channels with attenuated fast inactivation, we expressed NaV1.4-WCW in Xenopus oocytes and recorded currents using two-electrode voltage-clamp (TEVC). To dephosphorylate PI(4,5)P2, we co-expressed 4’ or 5’ phospholipid phosphatases that are recruited to the cell membrane upon exposure to blue light (λ=470 nm). 4’ dephosphorylation of PI(4,5)P2 produced an 8 ± 2% increase in peak NaV1.4 WCW channel currents at a depolarizing pulse to −20 mV. Significantly greater channel currents at −15 mV and −25 mV were also observed. Similarly, 5’ dephosphorylation of PI(4,5)P2 resulted in a 15 ± 2% increase in Nav1.4 WCW peak channel currents. Significantly greater channel currents were observed in the voltage range from −20 mV to +20 mV. Although the peak currents were augmented, the half-maximal activation voltage (V1/2) and slope factor (Kact) of the WCW mutant were both unaffected by either dephosphorylation, suggesting that the altered voltage-dependence of this channel mutant is at least partially due to the effect of PI(4,5)P2 dephosphorylation on fast inactivation.

Elucidating the mechanism of PI3K when interacting with TRPV1.

The interaction between Transient Receptor Potential Cation Channel Subfamily V member 1 (TRPV1), Phosphoinositide 3-Kinase (PI3K), and Receptor Tyrosine Kinase (RTK) provides us with a positive feedback loop that is responsible for recruiting Pi3K, a protein responsible for phosphorylating Phosphatidylinositol Bisphosphate (PIP2) into Phosphatidylinositol Trisphosphate (PIP3), and recruit TRPV1, an ion channel responsible for regulating heat and pain sensation, to specific areas in the plasma membrane (PM). In this study, we use ELISA immunoassay, microscopy, and other techniques to elucidate PI3K's functionality under the influence of TRPV1. Ultimately, we want to investigate this positive feedback loop and determine if we can interfere with excess TRPV1 activation and potentially stop excess pain and inflammation.

Regulation of Kir2.1 channels by ubiquitin-like modifier proteins.

The strong inward-rectifying subclass of potassium channel Kir2.1 produces IK1 current in the human myocardium which stabilizes the resting membrane potential of the heart and shapes the cardiac action potential. Perturbation or disruption of IK1 is associated with various arrhythmic phenotypes. Multiple studies have provided extensive evidence for the modulation of potassium channels by endogenous regulatory proteins. This group has studied extensively the relationship between the small ubiquitin-like modifier SUMO and Kir2.1 biophysical properties. Using patch clamp and Förster resonance energy transfer (FRET) studies in heterologous cells and rat ventricular cardiomyocytes (RVCM), we have demonstrated that SUMO interacts with Kir2.1 at a key lysine residue K49; the proximate physical association of which SUMO associates with the channel interferes with the binding pocket for phosphatidylinositol bisphosphate 2 (PIP2) required for channel gating, resulting in decreased IK1 current. Recent studies have posited an important role for another ubiquitin-like modifier, FAT10 in the regulation of ion channels that are expressed in the myocardium. While our group has characterized that acute hypoxia inhibits IK1 current in RVCM by an increased SUMOylation mechanism, another group has shown that FAT10 expression is markedly increased in hypoxia RVCMs. Together with studies showing evidence of competition for common target proteins between SUMO and FAT10, we seek to elucidate a mechanism of FAT10ylation of Kir2.1. Using FRET, we show disrupted SUMOylation in the presence of FAT10 in the heterologous cell system. Using electrophysiological techniques, we also demonstrate a change in Kir2.1 channel dynamics when FAT10 is co-expressed in both Xenopus laevis oocytes and HEK293T cells. Lastly, this work establishes a foundation for the study of Kir2.1 channel FAT10ylation in the native cardiomyocyte system in which FAT10 is posited to have a cardioprotective effect.

Frizzled does not get bent out of shape by Wnt

Ligands of the Wnt family activate the Frizzled-LRP5/6 receptor complex to initiate intracellular signaling. In this issue of Science Signaling, Mahoney et al. reveal a Wnt-stimulated positive feedback loop that involves local production of the lipid phosphatidylinositol(4,5)bisphosphate [PI(4,5)P2], which promotes Dishevelled recruitment and additional PI(4,5)P2 production, to facilitate LRP5/6 phosphorylation.

PIKfyve-Dependent Phosphoinositide Dynamics in Megakaryocyte/Platelet Granule Integrity and Platelet Functions.

Secretory granules are key elements for platelet functions. Their biogenesis and integrity are regulated by fine-tuned mechanisms that need to be fully characterized. Here, we investigated the role of the phosphoinositide 5-kinase PIKfyve and its lipid products, PtdIns5P (phosphatidylinositol 5 monophosphate) and PtdIns(3,5)P2 (phosphatidylinositol (3,5) bisphosphate) in granule homeostasis in megakaryocytes and platelets. For that, we invalidated PIKfyve by pharmacological inhibition or gene silencing in megakaryocytic cell models (human MEG-01 cell line, human imMKCLs, mouse primary megakaryocytes) and in human platelets. We unveiled that PIKfyve expression and its lipid product levels increased with megakaryocytic maturation. In megakaryocytes, PtdIns5P and PtdIns(3,5)P2 were found in alpha and dense granule membranes with higher levels in dense granules. Pharmacological inhibition or knock-down of PIKfyve in megakaryocytes decreased PtdIns5P and PtdIns(3,5)P2 synthesis and induced a vacuolar phenotype with a loss of alpha and dense granule identity. Permeant PtdIns5P and PtdIns(3,5)P2 and the cation channel TRPML (transient receptor potential mucolipin) 1 and TPC (two pore segment channel) 2 activation were able to accelerate alpha and dense granule integrity recovery following release of PIKfyve pharmacological inhibition. In platelets, PIKfyve inhibition specifically impaired the integrity of dense granules culminating in defects in their secretion, platelet aggregation, and thrombus formation. These data demonstrated that PIKfyve and its lipid products PtdIns5P and PtdIns(3,5)P2 control granule integrity both in megakaryocytes and platelets.

Membrane insertion mechanism of the caveola coat protein Cavin1

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical–basal polarity. Here, we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

Phosphorylation of cPLA2α at Ser505 Is Necessary for Its Translocation to PtdInsP2-Enriched Membranes.

Group IVA cytosolic phospholipase A2α (cPLA2α) is a key enzyme in physiology and pathophysiology because it constitutes a rate-limiting step in the pathway for the generation of pro- and anti-inflammatory eicosanoid lipid mediators. cPLA2α activity is tightly regulated by multiple factors, including the intracellular Ca2+ concentration, phosphorylation reactions, and cellular phosphatidylinositol (4,5) bisphosphate levels (PtdInsP2). In the present work, we demonstrate that phosphorylation of the enzyme at Ser505 is an important step for the translocation of the enzyme to PtdInsP2–enriched membranes in human cells. Constructs of eGFP-cPLA2 mutated in Ser505 to Ala (S505A) exhibit a delayed translocation in response to elevated intracellular Ca2+, and also in response to increases in intracellular PtdInsP2 levels. Conversely, translocation of a phosphorylation mimic mutant (S505E) is fully observed in response to cellular increases in PtdInsP2 levels. Collectively, these results suggest that phosphorylation of cPLA2α at Ser505 is necessary for the enzyme to translocate to internal membranes and mobilize arachidonic acid for eicosanoid synthesis.

Relationship between HIV-1 Gag Multimerization and Membrane Binding.

HIV-1 viral particle assembly occurs specifically at the plasma membrane and is driven primarily by the viral polyprotein Gag. Selective association of Gag with the plasma membrane is a key step in the viral assembly pathway, which is traditionally attributed to the MA domain. MA regulates specific plasma membrane binding through two primary mechanisms including: (1) specific interaction of the MA highly basic region (HBR) with the plasma membrane phospholipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2], and (2) tRNA binding to the MA HBR, which prevents Gag association with non-PI(4,5)P2 containing membranes. Gag multimerization, driven by both CA–CA inter-protein interactions and NC-RNA binding, also plays an essential role in viral particle assembly, mediating the establishment and growth of the immature Gag lattice on the plasma membrane. In addition to these functions, the multimerization of HIV-1 Gag has also been demonstrated to enhance its membrane binding activity through the MA domain. This review provides an overview of the mechanisms regulating Gag membrane binding through the MA domain and multimerization through the CA and NC domains, and examines how these two functions are intertwined, allowing for multimerization mediated enhancement of Gag membrane binding.

Nitric oxide alleviates chilling injury by regulating the metabolism of lipid and cell wall in cold-storage peach fruit

Peach fruit are prone to development of chilling injury during cold storage at around 0–7 °C. Nitric oxide (NO) has been proven to alleviate chilling injury, but the mechanism was still unclear. In this study, peach fruit were immersed in a NO donor (sodium nitroprusside, SNP) solution for 10 min, then stored at 0 °C. The SNP alleviated chilling injury, including decreasing the internal browning index, malondialdehyde content, electrolyte leakage, and lipoxygenase activity, and maintaining firmness. Furthermore, SNP maintenance of fruit firmness was associated with reduction of xyloglucan endotransglycosylase/hydrolase family member gene expression and decrease of cell wall hydrolase activity, especially the activities of polygalacturonase, xyloglucan endoglycosyl transferase, cellulase, and β-galactosidase. Meanwhile, SNP regulated the lipid metabolism by up-regulating the expression of genes encoding glycerol-3-phosphate acyltransferase, ketoacy-ACP synthase, phosphatidylinositol bisphosphate and long-chain acyl-CoA. Thus, the results of this study indicate that SNP alleviates chilling injury of post-harvest peach fruit by regulating cell wall and lipid metabolism.

Gain-of-Function Properties of a Dynamin 2 Mutant Implicated in Charcot-Marie-Tooth Disease.

Mutations in the gene encoding dynamin 2 (DNM2), a GTPase that catalyzes membrane constriction and fission, are associated with two autosomal-dominant motor disorders, Charcot-Marie-Tooth disease (CMT) and centronuclear myopathy (CNM), which affect nerve and muscle, respectively. Many of these mutations affect the pleckstrin homology domain of DNM2, yet there is almost no overlap between the sets of mutations that cause CMT or CNM. A subset of CMT-linked mutations inhibit the interaction of DNM2 with phosphatidylinositol (4,5) bisphosphate, which is essential for DNM2 function in endocytosis. In contrast, CNM-linked mutations inhibit intramolecular interactions that normally suppress dynamin self-assembly and GTPase activation. Hence, CNM-linked DNM2 mutants form abnormally stable polymers and express enhanced assembly-dependent GTPase activation. These distinct effects of CMT and CNM mutations are consistent with current findings that DNM2-dependent CMT and CNM are loss-of-function and gain-of-function diseases, respectively. In this study, we present evidence that at least one CMT-causing DNM2 mutant (ΔDEE; lacking residues 555DEE557) forms polymers that, like the CNM mutants, are resistant to disassembly and display enhanced GTPase activation. We further show that the ΔDEE mutant undergoes 2-3-fold higher levels of tyrosine phosphorylation than wild-type DNM2. These results suggest that molecular mechanisms underlying the absence of pathogenic overlap between DNM2-dependent CMT and CNM should be re-examined.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α confined to the surface of lipid droplets and adjacent narrow cytoplasm in progesterone-producing cells of in situ ovaries of adult mice

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) produced by phosphatidylinositol phosphate 5 kinase (PIP5K) plays not only as a precursor of second messengers in the phosphoinositide signal transduction, but also multiple roles influencing a variety of cellular activities. From this viewpoint, the present study attempted to localize PIP5Kα in the ovaries in situ of adult mice. PIP5Kα-immunoreactivity was confined to the surfaces of lipid droplets (LDs) and their adjacent cytoplasm in progesterone-producing cells of the interstitial glands, corpora lutea and theca interna. The LDs often contained membranous tubules/lamellae along their surfaces and within their interior whose membranes were continuous with those delineating LDs composed of a monolayer of phospholipids and were partially PIP5Kα-immunoreactive. Although granulosa cells of healthy-looking follicles were immunonegative, as the atresia progressed, PIP5Kα-immunoreactivity first appeared in sparsely dispersed dot forms in mural cells of the follicular epithelia, and then were dominant in almost all mural cells that remained after desquamation of the antral cells. The present study provides evidence suggesting that PI(4,5)P2 locally synthesized by PIP5K in LDs is involved in the lipid transfer between lipid droplets (LDs) and the endoplasmic reticulum, which eventually regulates ovarian progesterone production through control of multiple dynamic activities of LDs. It is also suggested that PIP5Kα and PI(4,5)P2 are implicated in the modulation of programmed cell death and/or acquiring the ability of progesterone production in some follicular cells surviving atresia.

LDL receptor-related protein 1 and its interacting partners in tissue homeostasis.

LDL receptor-related protein 1 (LRP1) is a multifunctional protein with endocytic and signal transduction properties due to its interaction with numerous extracellular ligands and intracellular proteins. This brief review highlights key developments in identifying novel functions of LRP1 in liver, lung, and the central nervous system in disease pathogenesis. In hepatocytes, LRP1 complexes with phosphatidylinositol 4-phosphate 5-kinase-1 and its related protein to maintain intracellular levels of phosphatidylinositol (4,5) bisphosphate and preserve lysosome and mitochondria integrity. In contrast, in smooth muscle cells, macrophages, and endothelial cells, LRP1 interacts with various different extracellular ligands and intracellular proteins in a tissue-dependent and microenvironment-dependent manner to either enhance or suppress inflammation, disease progression or resolution. Similarly, LRP1 expression in astrocytes and oligodendrocyte progenitor cells regulates cell differentiation and maturation in a developmental-dependent manner to modulate neurogenesis, gliogenesis, and white matter repair after injury. LRP1 modulates metabolic disease manifestation, inflammation, and differentiation in a cell-dependent, time-dependent, and tissue-dependent manner. Whether LRP1 expression is protective or pathogenic is dependent on its interaction with specific ligands and intracellular proteins, which in turn is dependent on the cell type and the microenvironment where these cells reside.

Mechanical properties of anionic asymmetric bilayers from atomistic simulations.

Mechanotransduction, the biological response to mechanical stress, is often initiated by activation of mechanosensitive (MS) proteins upon mechanically induced deformations of the cell membrane. A current challenge in fully understanding this process is in predicting how lipid bilayers deform upon the application of mechanical stress. In this context, it is now well established that anionic lipids influence the function of many proteins. Here, we test the hypothesis that anionic lipids could indirectly modulate MS proteins by alteration of the lipid bilayer mechanical properties. Using all-atom molecular dynamics simulations, we computed the bilayer bending rigidity (KC), the area compressibility (KA), and the surface shear viscosity (ηm) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) lipid bilayers with and without phosphatidylserine (PS) or phosphatidylinositol bisphosphate (PIP2) at physiological concentrations in the lower leaflet. Tensionless leaflets were first checked for each asymmetric bilayer model, and a formula for embedding an asymmetric channel in an asymmetric bilayer is proposed. Results from two different sized bilayers show consistently that the addition of 20% surface charge in the lower leaflet of the PC bilayer with PIP2 has minimal impact on its mechanical properties, while PS reduced the bilayer bending rigidity by 22%. As a comparison, supplementing the PIP2-enriched PC membrane with 30% cholesterol, a known rigidifying steroid lipid, produces a significant increase in all three mechanical constants. Analysis of pairwise splay moduli suggests that the effect of anionic lipids on bilayer bending rigidity largely depends on the number of anionic lipid pairs formed during simulations. The potential implication of bilayer bending rigidity is discussed in the framework of MS piezo channels.

Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study

SummaryC2 domains facilitate protein interactions with lipid bilayers in either a Ca2+-dependent or -independent manner. We used molecular dynamics (MD) simulations to explore six Ca2+-independent C2 domains, from KIBRA, PI3KC2α, RIM2, PTEN, SHIP2, and Smurf2. In coarse-grained MD simulations these C2 domains formed transient interactions with zwitterionic bilayers, compared with longer-lived interactions with anionic bilayers containing phosphatidylinositol bisphosphate (PIP2). Type I C2 domains bound non-canonically via the front, back, or side of the β sandwich, whereas type II C2 domains bound canonically, via the top loops. C2 domains interacted strongly with membranes containing PIP2, causing bound anionic lipids to cluster around the protein. Binding modes were refined via atomistic simulations. For PTEN and SHIP2, CG simulations of their phosphatase plus C2 domains with PIP2-containing bilayers were also performed, and the roles of the two domains in membrane localization compared. These studies establish a simulation protocol for membrane-recognition proteins.