Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP₂), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP₂, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.     

Mechanism of PLC-Mediated Kir3 Current Inhibition

A large number of ion channels maintain their activity through direct interactions with phosphatidylinositol bisphosphate (PIP₂). For such channels, hydrolysis of PIP₂ causes current inhibition. It has become controversial whether the inhibitory effects on channel activity represent direct effects of PIP₂ hydrolysis or of downstream PKC action. We studied Phospholipase C (PLC)-dependent inhibition of G protein-activated inwardly rectifying K⁺ (Kir3) channels. By monitoring simultaneously channel activity and PIP2 hydrolysis, we determined that both direct PIP₂ depletion and PKC actions contribute to Kir3 current inhibition. We show that the PKC-induced effects strongly depend on the PIP₂ levels in the membrane. At the same time, we show that PKC destabilizes Kir3/PIP₂ interactions and enhances the effects of PIP₂ depletion on channel activity. These results demonstrate that PIP₂ depletion and PKC-mediated effects reinforce each other and suggest that both of these interdependent mechanisms contribute to Kir3 current inhibition. This mechanistic insight may explain how even minor changes in PIP₂ levels can have profound effects on Kir3 activity. We also show that stabilization of Kir3PIP₂ interactions with Gβγ attenuates both PKC and Gq-mediated current inhibition, suggesting that diverse pathways regulate Kir3 activity through modulation of channel interactions with PIP₂.     

Interactions of the Auxilin-1 PTEN-like Domain with Model Membranes Result in Nanoclustering of Phosphatidyl Inositol Phosphates

Auxilin-1 is a neuron-specific membrane-binding protein involved in a late stage of clathrin-mediated endocytosis. It recruits Hsc70, thus initiating uncoating of the clathrin-coated vesicles. Interactions of auxilin-1 with the vesicle membrane are crucial for this function and are mediated via an N-terminal PTEN-like domain. We have used multiscale molecular dynamics simulations to probe the interactions of the auxilin-1 PTEN-like domain with lipid bilayers containing differing phospholipid composition, including bilayers containing phosphatidyl inositol phosphates. Our results suggest a novel, to our knowledge, model for the auxilin/membrane encounter and subsequent interactions. Negatively charged lipids (especially PIP₂) enhance binding of auxilin to lipid bilayers and facilitate its correct orientation relative to the membrane. Mutations in three basic residues (R301E/R307E/K311E) of the C2 subdomain of the PTEN-like domain perturbed its interaction with the bilayer, changing its orientation. The interaction of membrane-bound auxilin-1 PTEN-like domain with negatively charged lipid headgroups results in nanoclustering of PIP₂ molecules in the adjacent bilayer leaflet.     

HIV-1 Gag specificity for PIP2 is regulated by macromolecular electric properties of both protein and membrane local environments

HIV-1 assembly occurs at the plasma membrane, with the Gag polyprotein playing a crucial role. Gag association with the membrane is directed by the matrix domain (MA), which is myristoylated and has a highly basic region that interacts with anionic lipids. Several pieces of evidence suggest that the presence of phosphatidylinositol-(4,5)-bisphosphate (PIP₂) highly influences this binding. Furthermore, MA also interacts with nucleic acids, which is proposed to be important for the specificity of GAG for PIP₂-containing membranes. It is hypothesized that RNA has a chaperone function by interacting with the MA domain, preventing Gag from associating with unspecific lipid interfaces. Here, we study the interaction of MA with monolayer and bilayer membrane systems, focusing on the specificity for PIP₂ and on the possible effects of a Gag N-terminal peptide on impairing the binding for either RNA or membrane. We found that RNA decreases the kinetics of the protein association with lipid monolayers but has no effect on the selectivity for PIP₂. Interestingly, for bilayer systems, this selectivity increases in presence of both the peptide and RNA, even for highly negatively charged compositions, where MA alone does not discriminate between membranes with or without PIP₂. Therefore, we propose that the specificity of MA for PIP₂-containing membranes might be related to the electrostatic properties of both membrane and protein local environments, rather than a simple difference in molecular affinities. This scenario provides a new understanding of the regulation mechanism, with a macromolecular view, rather than considering molecular interactions within a ligand-receptor model.     

Phosphoinositides alter lipid bilayer properties

Phosphatidylinositol-4,5-bisphosphate (PIP₂), which constitutes ∼1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP₂ regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid–protein interactions, but the mechanisms by which PIP₂ regulates many of its various targets remain to be fully elucidated. Because many PIP₂ targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states—and thereby protein function. Taking advantage of the gramicidin A (gA) channels’ sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP₂s (brain PIP₂, which is predominantly 1-stearyl-2-arachidonyl-PIP₂, and dioleoyl-PIP₂) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP₂s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP₂ was a more potent modifier than dioleoyl-PIP₂. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC₈-phosphoinositol (PI), PI(4,5)P₂, PI(3,5)P₂, PI(3,4)P₂, and PI(3,4,5)P₃. The diC₈ phosphoinositides, except for PI(3,5)P₂, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC₈ phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP₂ levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP₂, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments.     

Membrane targeting of the yeast exocyst complex

The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits — Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP₂) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP₂. We found that PIP₂ is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP₂ in the adjacent leaflet. We further revealed that PIP₂ is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP₂ for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation.     

Molecular dynamics simulations on the interaction of the transmembrane NavAb channel with cholesterol and lipids in the membrane

Increased cholesterol levels are associated with multiple pathological conditions. In this work, molecular dynamics simulations were applied to observe the influence of membrane cholesterol levels on a voltage-gated sodium channel. Different lipid compositions are modeled around the channel to obtain information about the possible effects by which cholesterol influences Na_vAb channels. Cholesterol was normally not directly interacting with either the closed or inactivated conformation. Cholesterol increased lipid packing implying that it plays a crucial role in restricting lipid movement in the region around 1 nm of the channel in a 1-palmitoyl-2-oeleoyl phosphatidylcholine matrix. Our results provide the first computational indication of an indirect modulation of Na_vAb channels by membrane cholesterol.     

Dynamic phospholipid interaction of β2e subunit regulates the gating of voltage-gated Ca2+ channels

High voltage-activated Ca²⁺ (Ca_V) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating Ca_V channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on Ca_V2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP₂) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed Ca_V2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP₂ were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of Ca_V2.2(β2e) currents was enhanced. Activation of the M₁ muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of Ca_V2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in Ca_V channel gating. The phospholipid–protein interaction observed here provides structural insight into mechanisms of membrane–protein association and the role of phospholipids in ion channel regulation.     

Two structural components in CNGA3 support regulation of cone CNG channels by phosphoinositides

Cyclic nucleotide-gated (CNG) channels in retinal photoreceptors play a crucial role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric cone CNGA3 (A3) + CNGB3 (B3) channels are inhibited by membrane phosphoinositides (PIP_n), including phosphatidylinositol 3,4,5-triphosphate (PIP₃) and phosphatidylinositol 4,5-bisphosphate (PIP₂), demonstrating a decrease in apparent affinity for cyclic guanosine monophosphate (cGMP).Unlike homomeric A1 or A2 channels, A3-only channels paradoxically did not show a decrease in apparent affinity for cGMP after PIP_n application. However, PIP_n induced an ∼2.5-fold increase in cAMP efficacy for A3 channels. The PIP_n-dependent change in cAMP efficacy was abolished by mutations in the C-terminal region (R643Q/R646Q) or by truncation distal to the cyclic nucleotide-binding domain (613X). In addition, A3-613X unmasked a threefold decrease in apparent cGMP affinity with PIP_n application to homomeric channels, and this effect was dependent on conserved arginines within the N-terminal region of A3. Together, these results indicate that regulation of A3 subunits by phosphoinositides exhibits two separable components, which depend on structural elements within the N- and C-terminal regions, respectively. Furthermore, both N and C regulatory modules in A3 supported PIP_n regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIP_n sensitivity to heteromeric channels formed with PIP_n-insensitive A subunits. Finally, channels formed by mixtures of PIP_n-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIP_n regulation, implying that intersubunit N–C interactions help control the phosphoinositide sensitivity of cone CNG channels.     

IQGAP1 is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration

Phosphatidylinositol 4,5 bisphosphate (PIP₂) is a key lipid messenger for regulation of cell migration. PIP₂ modulates many effectors, but the specificity of PIP₂ signalling can be defined by interactions of PIP₂‐generating enzymes with PIP₂ effectors. Here, we show that type Iγ phosphatidylinositol 4‐phosphate 5‐kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP₂ effector that directly binds PIP₂ through a polybasic motif and PIP₂ binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP₂ binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration.     

Functional mechanisms of neurotransmitter transporters regulated by lipid–protein interactions of their terminal loops

The physiological functions of neurotransmitter:sodium symporters (NSS) in reuptake of neurotransmitters from the synapse into the presynaptic nerve have been shown to be complemented by their involvement, together with non-plasma membrane neurotransmitter transporters, in the reverse transport of substrate (efflux) in response to psychostimulants. Recent experimental evidence implicates highly anionic phosphatidylinositol 4,5-biphosphate (PIP₂) lipids in such functions of the serotonin (SERT) and dopamine (DAT) transporters. Thus, for both SERT and DAT, neurotransmitter efflux has been shown to be strongly regulated by the presence of PIP₂ lipids in the plasma membrane, and the electrostatic interaction of the N-terminal region of DAT with the negatively charged PIP₂ lipids. We examine the experimentally established phenotypes in a structural context obtained from computational modeling based on recent crystallographic data. The results are shown to set the stage for a mechanistic understanding of physiological actions of neurotransmitter transporters in the NSS family of membrane proteins. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Dual-mode phospholipid regulation of human inward rectifying potassium channels

The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using ⁸⁶Rb⁺ flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP₂) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP₂ increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP₂, and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP₂ and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity.     

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates Shaker K⁺ channels and voltage-gated Ca²⁺ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP₂ regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP₂ regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K⁺ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP₂ regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K⁺ channel family. Open-state stabilization by PIP₂ has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP₂ strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP₂ has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4–S5 linker) and R479 near the S6 activation gate are required for PIP₂ to inhibit voltage activation. The ability of PIP₂ to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP₂ on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP₂-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP₂ can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP₂ regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.     

The Cytosolic GH Loop Regulates the Phosphatidylinositol 4,5-Bisphosphate-induced Gating Kinetics of Kir2 Channels

Inwardly rectifying K⁺ (Kir) channels set the resting membrane potential and regulate cellular excitability. The activity of Kir channels depends critically on the phospholipid PIP₂. The molecular mechanism by which PIP₂ regulates Kir channel gating is poorly understood. Here, we utilized a combination of computational and electrophysiological approaches to discern structural elements involved in regulating the PIP₂-induced gating kinetics of Kir2 channels. We identify a novel role for the cytosolic GH loop. Mutations that directly or indirectly affect GH loop flexibility (e.g. V223L, E272G, D292G) increase both the on- and especially the off-gating kinetics. These effects are consistent with a model in which competing interactions between the CD and GH loops for the N terminus regulate the gating of the intracellular G loop gate.     

Giant unilamellar vesicles containing phosphatidylinositol(4,5)bisphosphate: characterization and functionality

Biomimetic systems such as giant unilamellar vesicles (GUVs) are increasingly used for studying protein/lipid interactions due to their size (similar to that of cells) and to their ease of observation by light microscopy techniques. Biophysicists have begun to complexify GUVs to investigate lipid/protein interactions. In particular, composite GUVs have been designed that incorporate lipids that play important physiological roles in cellulo, such as phosphoinositides and among those the most abundant one, phosphatidylinositol(4,5)bisphosphate (PIP₂). Fluorescent lipids are often used as tracers to observe GUV membranes by microscopy but they can not bring quantitative information about the insertion of unlabeled lipids. In this study, we carried out ζ-potential measurements to prove the effective incorporation of PIP₂ as well as that of phosphatidylserine in the membrane of GUVs prepared by electroformation and to follow the stability of PIP₂-containing GUVs. Using confocal microscopy, we found that long-chain (C16) fluorescent PIP₂ analogs used as tracers (0.1% of total lipids) show a uniform distribution in the membrane whereas PIP₂ antibodies show PIP₂ clustering. However, the clustering effect, which is emphasized when tertiary antibodies are used in addition to secondary ones to enhance the size of the detection complex, is artifactual. We showed that divalent ions (Ca²⁺ and Mg²⁺) can induce aggregation of PIP₂ in the membrane depending on their concentration. Finally, the interaction of ezrin with PIP₂-containing GUVs was investigated. Using either labeled ezrin and unlabeled GUVs or both labeled ezrin and GUVs, we showed that clusters of PIP₂ and proteins are formed.     

Alternative Splicing Governs Cone Cyclic Nucleotide-gated (CNG) Channel Sensitivity to Regulation by Phosphoinositides

Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ∼2-fold greater shift in K_1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIP_n) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIP_n sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIP_n regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIP_n sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIP_n. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides.     

Membrane Docking Geometry and Target Lipid Stoichiometry of Membrane-Bound PKCα C2 Domain: A Combined Molecular Dynamics and Experimental Study

Protein kinase Cα (PKCα) possesses a conserved C2 domain (PKCα C2 domain) that acts as a Ca²⁺-regulated membrane targeting element. Upon activation by Ca²⁺, the PKCα C2 domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca²⁺ concentrations, binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca²⁺ concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP₂) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCα C2 domain docked to (i) PS alone and (ii) both PS and PIP₂ simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP₂ headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP₂-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP₂:C2 stoichiometry approaching 2:1 at a high PIP₂ mole density. Direct binding measurements titrating the C2 domain with PIP₂ in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP₂ mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP₂ stoichiometries are discussed.     

Role of GAP-43 in sequestering phosphatidylinositol 4,5-bisphosphate to Raft bilayers

The lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) is critical for a number of physiological functions, and its presence in membrane microdomains (rafts) appears to be important for several of these spatially localized events. However, lipids like PIP₂ that contain polyunsaturated hydrocarbon chains are usually excluded from rafts, which are enriched in phospholipids (such as sphingomyelin) containing saturated or monounsaturated chains. Here we tested a mechanism by which multivalent PIP₂ molecules could be transferred into rafts through electrostatic interactions with polybasic cytoplasmic proteins, such as GAP-43, which bind to rafts via their acylated N-termini. We analyzed the interactions between lipid membranes containing raft microdomains and a peptide (GAP-43P) containing the linked N-terminus and the basic effector domain of GAP-43. In the absence or presence of nonacylated GAP-43P, PIP₂ was found primarily in detergent-soluble membranes thought to correspond to nonraft microdomains. However, when GAP-43P was acylated by palmitoyl coenzyme A, both the peptide and PIP₂ were greatly enriched in detergent-resistant membranes that correspond to rafts; acylation of GAP-43P changed the free energy of transfer of PIP₂ from detergent-soluble membranes to detergent-resistant membranes by −1.3 kcal/mol. Confocal microscopy of intact giant unilamellar vesicles verified that in the absence of GAP-43P PIP₂ was in nonraft microdomains, whereas acylated GAP-43P laterally sequestered PIP₂ into rafts. These data indicate that sequestration of PIP₂ to raft microdomains could involve interactions with acylated basic proteins such as GAP-43.     

Novel Kv7.1-Phosphatidylinositol 4,5-Bisphosphate Interaction Sites Uncovered by Charge Neutralization Scanning

K_v7.1 to K_v7.5 α-subunits belong to the family of voltage-gated potassium channels (K_v). Assembled with the β-subunit KCNE1, K_v7.1 conducts the slowly activating potassium current I_Ks, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of K_v7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP₂). PIP₂ increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between K_v7.1 and PIP₂. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC₈-PIP₂. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP₂ regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP₂ binding, molecular dynamics simulations of K_v7.1/KCNE1 complexes containing two PIP₂ molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP₂ and K_v7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP₂ binding.