PIP2 Molecules Research Articles

Regulation of anionic lipids in binary membrane upon the adsorption of polyelectrolyte: A Monte Carlo simulation

We employ Monte Carlo simulations to investigate the interaction between an adsorbing linear flexible cationic polyelectrolyte and a binary fluid membrane. The membrane contains neutral phosphatidyl–choline, PC) and multivalent anionic (phosphatidylinositol, PIP2) lipids. We systematically study the influences of the solution ionic strength, the chain length and the bead charge density of the polyelectrolyte on the lateral rearrangement and the restricted mobility of the multivalent anionic lipids in the membrane. Our findings show that, the cooperativity effect and the electrostatic interaction of the polyelectrolyte beads can significantly affect the segregation extent and the concentration gradients of the PIP2 molecules, and further cooperate to induce the complicated hierarchical mobility behaviors of PIP2 molecules. In addition, when the polyelectrolyte brings a large amount of charges, it can form a robust electrostatic well to trap all PIP2 and results in local overcharge of the membrane. This work presents a mechanism to explain the membrane heterogeneity formation induced by the adsorption of charged macromolecule.

Physiological Calcium Concentrations Induce PI(4,5)P2 Clustering: PI(4,5)P2 as a Lipidic Calcium Sensor

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the plasma membrane of eukaryotic cells that is essential for several cellular mechanisms, including organization and remodeling of the actin cytoskeleton, membrane trafficking, endocytosis and exocytosis. The regulation of these processes is thought to involve localized enrichment of PI(4,5)P2 in the plasma membrane at particular timings. Particularly in calcium-triggered SNARE-dependent exocytosis, PI(4,5)P2 is known to interact and co-segregate with certain synaptic proteins. However, the mechanisms responsible for PI(4,5)P2 clustering in particular sites of the plasma membrane are still not completely understood.Using membrane model systems and fluorescently labeled PI(4,5)P2, we were able to demonstrate that PI(4,5)P2 lateral distribution is sensitive to physiologic calcium concentrations, in the low micromolar range. Calcium concentrations of 0-100 μM were used to mimic neuron intracellular calcium concentrations before and after a stimulus. Fluorescence self-quenching and energy migration data obtained from steady-state and time-resolved fluorescence methods clearly showed that Ca2+ ions promoted the clustering of PI(4,5)P2 molecules in both liquid disordered and liquid ordered membranes. Sensitivity of PI(4,5)P2 clustering to [Ca2+] is shown to be highly dependent on both PI(4,5)P2 density and membrane lipid phase.In this way, the lateral distribution of PI(4,5)P2 presents significant sensitivity to changes in calcium concentration of only a few micromolar, allowing this lipid to function as a lipidic calcium sensor. In the particular case of SNARE-dependent exocytosis, it is expected that localized increases in calcium concentration will dramatically change the pattern of PI(4,5)P2 interaction with synaptic proteins, possibly enhancing fusion rates and promoting exocytosis.Acknowledgements: FCT Portugal is acknowledged for financial support (PTDC/QUI-BIQ/112067/2009; PTDC/QUI-BIQ/099947/2008; SFRH/BPD/64320/2009; SFRH/BD/80575/2011).

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Langevin Dynamics Simulations of Protein Fencing of PIP2

Anionic phosphotidylinositol 4,5-bisphosphate (PIP2) occupies only 2∼3% of the phospholipids in the inner leaflet of the plasma membrane, but participates in numerous cellular processes. Concentrated pools of PIP2 on the membrane surface appear to be the source for such PIP2-involved cellular processes, and recent experimental observations indicate that a “protein fence” prevents PIP2 from diffusing out of the pool. Here the necessary properties of the protein fence are examined using Langevin dynamics simulation with effective potential maps. PIP2 molecules are modeled as explicitly diffusing spheres, and the rest of the system represented by steric and electrostatic potentials. Simple models of charged or porous rods, as well as rigid proteins are considered. Retardation by electrostatic forces from a rod is only significant when the charges are highly concentrated and close to the diffusion plane; charge densities and geometries of proteins are insufficient to effectively block diffusion. Hence, fencing appears to be dominated by steric effects, though even a small (> 1%) opening in a porous rod in contact with the surface results in substantial leakage. The three protein fence candidates, actin, human septin, and yeast septin, are known to self-assemble into filaments on the cell surface and to interact with PIP2. Simulations reveal that actin is a poor fence for all depths and orientations simulated because its arch-type shape leaves passageways for diffusion. In contrast, the septins effectively block PIP2 diffusion when buried in the membrane to 10∼15 A from the lipid surface. Free energy calculations using an implicit membrane model support the possibility of burial of septins at these depths. It is also possible that the septins are not buried as deeply, but associate with other proteins or peptides to make a fence.

PIP2-Binding to an Open State Model of Kir1.1 Probed by Multiscale Biomolecular Simulations

Phosphatidylinositol bisphosphate (PIP2) is an activator of mammalian inwardly rectifying potassium (Kir) channels. We have used multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics to explore the interactions of PIP2 molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on both open and closed state models of the Kir1.1 (ROMK) channel. Coarse-grained simulations of the channels in PIP2-containing lipid bilayers identified the PIP2-binding site on each channel. These models of the PIP2-channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. The binding site in the closed state agrees with previous mutagenesis data of Kir1.1 as well as with previous modeling studies of related Kir channels. Intriguingly, analysis of the open state model reveals a differential interaction of PIP2 with key residues thought to be involved in PIP2 activation of the channel. These models will serve as a framework for the functional validation of PIP2 interactions with Kir1.1 and provide a fresh insight into how PIP2 stabilizes the open state of the Kir channel.

Investigating the PIP2 Binding Site in Kir Channels Via Multi-Scale Biomolecular Simulations

Mammalian inwardly rectifying potassium (Kir) channels are activated by the anionic lipid, Phosphatidylinositol bisphosphate (PIP2). In this study, coarse-grained simulations followed by atomistic molecular dynamics have revealed the interactions made by PIP2 molecules with Kir channels. The three channels investigated are X-ray structures of KirBac1.1 and the Kir3.1-KirBac1.3 chimera, and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP2-containing POPC lipid bilayers identified the PIP2 binding site on each channel. These models of the PIP2/channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with known functional data and is in close proximity to the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations agree well with H-bonding interactions between the channels and PIP2 in the atomistic simulations, enabling identification of key sidechains, which are primarily basic in nature. Notable differences within the KirBac1.1 and Kir6.2 binding sites are apparent; providing hypotheses for why PIP2 activates Kir6.2 channels whilst inhibiting the opening of KirBac1.1 channels.

Hard to Fence You in: Computational Approaches to Explore the Hypothesis that Actin Filaments Impede PIP2 Diffusion in Membranes

Experiments described by Golebiewska et al. at this meeting suggest the existence of a ‘fence’that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate (PIP2) into and out of nascent phagosomes in macrophages. Although the nature of the fence remains an enigma, actin filaments are plausible components. They are highly negatively charged (as is PIP2), are swept away from the central region and are concentrated at the perimeter of the forming phagosome. To explore the actin fence hypothesis, we have used (1) Poisson-Boltzmann continuum electrostatics and a grid-based repulsive potential to describe a fence model made of a single layer of actin filaments, and (2) Brownian dynamics to describe the diffusion of PIP2 molecules modeled as single spheres. The simulations with actin filaments positioned parallel to the membrane indicate that a single filament without attached proteins does not significantly impede the diffusion of PIP2. A helical stripe of basic residues on the acidic actin filament provides a hole in the putative fence through which PIP2 can diffuse, no matter how close the filament is positioned to the membrane. Results from simulations of PIP2 diffusion out of corrals formed of multiple layers of actin filaments, and mazes of non-electrostatic barriers will also be presented.

PIP2-Binding Site in Kir Channels: Definition by Multiscale Biomolecular Simulations

Phosphatidylinositol bisphosphate (PIP2) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP2 molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1−KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP2-containing lipid bilayers identified the PIP2-binding site on each channel. These models of the PIP2−channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP2 in the atomistic simulations, enabling identification of key side chains.

Molecular Dynamics simulations of mixture of POPC and PIP2 bilayer

PI(4,5)P2 is a phospholipid that plays a role in a wide variety of cellular signaling processes. We have developed force field parameters for the molecular dynamics simulations of PI(4,5)P2 that are consistent with the modified GROMACS parameters 43A1-S3. Furthermore, we performed a long time simulation of hydrated bilayers of mixtures of POPC and 5 mol% PIP2 lipids. The simulated system consists of 800 POPC molecules. This is system is large enough to demonstrate the effect of small quantities of PIP2 and yet small enough to be trackable for molecular dynamics techniques. In this simulation we study the structural properties of poly-unsaturated chains of PIP2 molecules and electrostatic interactions at the bilayer-water interface that enable PIP2 to play significant roles in singling processes.

Quantitative Analysis of the Binding of Ezrin to Large Unilamellar Vesicles Containing Phosphatidylinositol 4,5 Bisphosphate

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP2) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP2. For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be ∼5μM for PIP2-LUVs and 20-to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP2-LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of ∼30nm2) binding to a single PIP2 can block access to neighboring PIP2 molecules and thus contributes to lower the accessible PIP2 concentration. In addition, no evidence exists for a clustering of PIP2 induced by ezrin addition.

Hydrogen Bonding and Binding of Polybasic Residues with Negatively Charged Mixed Lipid Monolayers

Phosphoinositides, phosphorylated products of phosphatidylinositol, are a family of phospholipids present in tiny amounts (1% or less) in the cytosolic surface of cell membranes, yet they play an astonishingly rich regulatory role, particularly in signaling processes. In this letter, we use molecular dynamics simulations on a model system of mixed lipid monolayers to investigate the interaction of phosphatidylinositol 4,5-bisphosphate (PIP2), the most common of the phosphoinositides, with a polybasic peptide consisting of 13 lysines. Our results show that the polybasic peptide sequesters three PIP2 molecules, forming a complex stabilized by the formation of multiple hydrogen bonds between PIP2 and the Lys residues. We also show that the polybasic peptide does not sequester other charged phospholipids such as phosphatidylserine because of the inability to form long-lived stable hydrogen bonds.

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Phosphatidylinositol bisphosphate (PIP2) is the most abundant phosphoinositide in the plasma membranes of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP2 for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP2. We first showed that PIP2 regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP2 levels. Moreover, by controlling the number of PIP2-sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP2, where each of the four open states corresponds to the channel activated by one to four PIP2 molecules.

Metabolic regulation of sodium–calcium exchange by intracellular acyl CoAs

The sodium-calcium exchanger (NCX) is a critical mediator of calcium homeostasis. In the heart, NCX1 predominantly operates in forward mode to extrude Ca(2+); however, reverse-mode NCX1 activity during ischemia/reperfusion (IR) contributes to Ca(2+) loading and electrical and contractile dysfunction. IR injury has also been associated with altered fat metabolism and accumulation of long-chain acyl CoA esters. Here, we show that acyl CoAs are novel, endogenous activators of reverse-mode NCX1 activity, exhibiting chain length and saturation dependence, with longer chain saturated acyl moieties being the most effective NCX1 activators. These results implicate dietary fat composition as a plausible determinant of IR injury. We further show that acyl CoAs may interact directly with the XIP (exchanger inhibitory peptide) sequence, a known region of anionic lipid modulation, to dynamically regulate NCX1 activity and Ca(2+) homeostasis. Additionally, our findings have broad implications for the coupling of Ca(2+) homeostasis to fat metabolism in a variety of tissues.

Molecular dynamics study of a gelsolin‐derived peptide binding to a lipid bilayer containing phosphatidylinositol 4,5‐bisphosphate

Gelsolin is an actin-severing protein whose action is initiated by Ca(2+) and inhibited by binding to phosphorylated inositol lipid or phosphoinositides. The regions of gelsolin responsible for phosphoinositide binding are comprised of residues 150-169 (G150-169) and 135-142 (G135-142). The corresponding peptides possess similar binding potency as native gelsolin. Their common feature is the presence of arginine and lysine residues that can bind to negatively charged phosphate groups of phosphoinositides. In this work the binding of the G150-169 peptide to a phosphatidylinositol 4,5-bisphosphate (PIP2) cluster in a lipid membrane model was investigated by molecular dynamics calculations (MD) with the AMBER 4.1 force field, taking into account explicit solvent molecules. Initially the structure of G150-169 was simulated by using the electrostatically driven Monte Carlo (EDMC) and MD methods, and the resulting structure agreed within 3.7 A backbone-atom root mean square deviation with the corresponding experimentally derived structure (PDB code: 1SOL). Using this model for the peptide, a subsequent MD simulation of G150-169 in a periodic box containing a model of dimyristoyl-phosphatidylcholine (DMPC) lipids with a cluster of four PIP2 molecules was carried out. During the simulation G150-169 interacted strongly with PIP2 molecules, initially by formation of salt bridges between its N-terminal basic groups and the phosphate groups of PIP2, followed by formation of hydrophobic bonds between the hydrophobic side chains of the peptide and the fatty acid tail of the lipid. As a result of the formation of hydrophobic bonds, the PIP2 molecules were pulled out from the lipid bilayer. This mode of binding differs from those of other PIP2-binding protein motifs such as PH domains that interact solely with the hydrophilic head group of PIP2. These results suggest that dissociation of gelsolin from actin by PIP2 lipids may involve entering of the PIP2 molecules to the gelsolin-actin interface, thereby weakening the interactions between these proteins.

Ultracentrifugation technique for measuring the binding of peptides and proteins to sucrose-loaded phospholipid vesicles.

AbstractThe binding of extrinsic proteins to membranes is important for their biological activity in many different systems. Consider a few examples from the calcium/phospholipid second-messenger system (reviewed in refs. 1 and 2). First, membrane binding facilitates interaction of the Gq protein with both its activated receptor and its membrane-bound effector, phosphoinositide-specific phospholipase C (PLC-β). Second, it enhances the ability of PLC to hydrolyze its membrane-bound substrate phosphatidylmositol 4,5bis-phosphate (PIP2) in a precessive manner (i.e., the enzyme can “scoot” over the surface, hydrolyzing many PIP2 molecules before desorbing from the membrane). Third, membrane binding increases the probability that protein kinase C (PKC) will phosphorylate its membrane-bound substrates, which include pp60src (Src) and MARCKS (mynstoylated alanine-rich C kinase substrate). Fourth, membrane binding is required for the biological activity of proteins such as Src and Ras. All these proteins bind to the membrane by a combination of electrostatic and hydrophobic interactions (see refs. 3 and 4 and references therem). This chapter describes a simple ultracentrifugation technique for measuring the binding of extrinsic proteins and peptides to membranes.KeywordsLipid ConcentrationScintillation VialSalt BufferMembrane BindingExtrinsic ProteinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Dependence of the activity of phospholipase C beta on surface pressure and surface composition in phospholipid monolayers and its implications for their regulation.

We have examined the influence of surface pressure and phospholipid composition on hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C beta 1 (PLC beta 1) and PLC beta 2 in mixed composition phospholipid monolayers. Increasing the monolayer surface pressure from 15 to 36 mN/m reduced the rate at which PIP2 was hydrolyzed by PLC beta 1 and PLC beta 2 by 4-6-fold, although PLC beta 1 was more active than PLC beta 2, even at high surface pressure. Reduced enzyme activity was accompanied by an increase in reaction induction times, suggesting that increasing surface pressure reduced the penetration rate of the enzymes into the monolayer. Quantitation of interfacial enzyme concentration using 35S-labeled PLC beta 1 confirmed that less enzyme was associated with the monolayer at higher pressures. The relationship between PLC activity and substrate concentration was examined at a single surface pressure of 30 mN/m. This relationship was not hyperbolic, and increases in the mole percentage (mol %) of PIP2 in the monolayer resulted in an upwardly-curving increase in PLC activity. Thus, PLC beta 1 activity increased 7-fold and PLC beta 2 activity increased 4-fold when the mol % of PIP2 in the monolayer increased from 17.9% to 29%, increasing further thereafter. Paradoxically, increasing the mol % of PIP2 from 0 to 60% was accompanied by a 3-fold decrease in interfacial enzyme concentrations. Taken together, these data show that the catalytic activity of PLC beta involves some element of penetration of lipid interfaces, and suggest that the organization of the substrate facilitates PLC activity, giving credence to the substrate theory of interfacial activation of phospholipases. We present a hypothesis suggesting that PIP2 molecules coalesce into enriched lateral domains which favor PLC beta activity.

Drug-induced transmembrane lipid scrambling in erythrocytes and in liposomes requires the presence of polyanionic phospholipids.

The asymmetric transmembrane distribution of phospholipids between the two bilayer halves of erythrocyte can be modified upon addition of cationic amphiphilic drugs, such as chlorpromazine or verapamil. We studied this phenomenon in erythrocytes and in lipid vesicles using spin-labelled analogues of the endogenous phospholipids. The extent of the rapid disappearance of the analogues from the erythrocyte outer leaflet depended on the concentration of the drug. Up to 40% of spin-labelled sphingomyelin moved to the inner erythrocyte leaflet in 10 min in the presence of 1.5 mm chlorpromazine. Verapamil or vinblastine gave similar results. On the other hand, the inside-outside movement of the aminophospholipid analogues was less evident, and did not exceed 10%. This apparent discrepancy between inward and outward movements could result from the formation of an endovesicle which is known to occur upon drug addition at high concentration. A fraction of lipids would be trapped in the intravesicular leaflet, corresponding to the cell outer leaflet, and be inaccessible both from the cytoplasm and the extracellular medium. In cells submitted to a metabolic depletion of cellular ATP the intensity of the scrambling induced by the amphipaths was drastically lowered. We attribute this effect to the important reduction of the membrane content in phosphatidylinositol-4,5-bisphosphate (PIP2). The involvement of the latter lipid in triggering scrambling was partly confirmed by experiments carried out with artificial membranes. Indeed, in large unilamellar vesicles PIP2 is required in order to obtain a rapid redistribution of phospholipids between the two leaflets upon addition of drugs. However, the extent of phospholipid redistribution was limited to 15-20%. This redistribution was also induced when the vesicle membrane contained di-anionic phospholipids (phosphatidylinositol-4-monophosphate or diphosphatidylglycerol), but did not occur when it contained mono-anionic phospholipids (phosphatidylserine or phosphatidylinositol). Some drugs such as methochlorpromazine, active in artificial membranes, were ineffective in erythrocyte membranes, probably because they could not cross the membrane and reach PIP2 molecules at the cytoplasmic leaflet.

A short sequence responsible for both phosphoinositide binding and actin binding activities of cofilin.

Cofilin is a widely distributed actin-modulating protein that has abilities to bind along the side of F-actin and to depolymerize F-actin. Both abilities of cofilin can be inhibited by phosphoinositides such as phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate (PIP2). We have previously shown that the synthetic dodecapeptide corresponding to Trp104-Met115 of cofilin is a potent inhibitor of actin polymerization (Yonezawa, N., Nishida, E., Iida, K., Kumagai, H., Yahara, I., and Sakai, H. (1991) J. Biol. Chem. 266, 10485-10489). In this study, we have found that the inhibitory effect of the synthetic dodecapeptide on actin polymerization is canceled specifically by phosphatidylinositol, phosphatidylinositol 4-monophosphate and PIP2. We further show that the dodecapeptide as well as cofilin binds to PIP2 molecules and inhibits PIP2 hydrolysis by phospholipase C. Thus, the actin-binding dodecapeptide sequence of cofilin may constitute a multifunctional domain in cofilin.

Gelsolin-Polyphosphoinositide Interaction: Full Expression of Gelsolin-inhibiting Function by Polyphosphoinositides in Vesicular form and Inactivation by Dilution, Aggregation, or Masking of the Inositol Head Group

Abstract Calcium activates, and the polyphosphoinositides phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) inhibit the mechanical severing of actin filaments by gelsolin. Previous work indicated that the physical state of the two phospholipids is important for their effects in this system. This study correlates tests of gelsolin's severing function with quasielastic light scattering measurements of the size of mixed lipid particles and shows that the previously demonstrated diminution of the maximal effect of PIP2 in micellar form by aggregation of the micelles or mixing with other phospholipids is not the result of an absolute requirement for small lipid particles, but rather the masking of critical sites by aggregation, by sequestration in multilamellar vesicles, or by dilution of the polyphosphoinositides below a critical concentration. Large unilamellar vesicles of PIP and, importantly, PIP2 at low molar ratios (less than 3%) in mixed lipid vesicles of composition similar to plasma membranes are as active as PIP2 micelles. Aggregation or masking of polyphosphoinositide head groups by neomycin or profilin, respectively, blocked inhibition of gelsolin. Experiments with bilayer-forming phospholipids or with Triton X-100 indicate that a critical number of PIP2 molecules may be required for incipient effects on a gelsolin molecule. The actin and polyphosphoinositide binding protein profilin competed with gelsolin for binding PIP2 with a stoichiometry also suggesting binding to multiple PIP2 molecules. The membrane constituents sphingosine and cholesterol blocked the effect of PIP2 on gelsolin when added alone, but did not affect PIP2 when incorporated into mixed lipid bilayers containing phosphatidylinositol. The results suggest that profilin, small changes in membrane lipid composition, and, especially, membrane PIP2 concentration could have large effects on the modulation of gelsolin function in vivo.