Lipid Binding Assays Research Articles

The minor house dust mite allergen Der p 13 is a fatty acid-binding protein and an activator of a TLR2-mediated innate immune response.

The house dust mite (HDM) allergen Der p 13 could be a lipid-binding protein able to activate key innate signaling pathways in the initiation of the allergic response. We investigated the IgE reactivity of recombinant Der p 13 (rDer p 13), its lipid-binding activities, and its capacity to stimulate airway epithelium cells. Purified rDer p 13 was characterized by mass spectrometry, circular dichroism, fluorescence-based lipid-binding assays, and in silico structural prediction. IgE-binding activity and allergenic potential of Der p 13 were examined by ELISA, basophil degranulation assays, and in vitro airway epithelial cell activation assays. Protein modeling and biophysical analysis indicated that Der p 13 adopts a β-barrel structure with a predominately apolar pocket representing a potential binding site for hydrophobic ligands. Fluorescent lipid-binding assays confirmed that the protein is highly selective for ligands and that it binds a fatty acid with a dissociation constant typical of lipid transporter proteins. The low IgE-binding frequency (7%, n = 224) in Thai HDM-allergic patients as well as the limited propensity to activate basophil degranulation classifies Der p 13 as a minor HDM allergen. Nevertheless, the protein with its presumptively associated lipid(s) triggered the production of IL-8 and GM-CSF in respiratory epithelial cells through a TLR2-, MyD88-, NF-kB-, and MAPK-dependent signaling pathway. Although a minor allergen, Der p 13 may, through its lipid-binding capacity, play a role in the initiation of the HDM-allergic response through TLR2 activation.

Mirror Images of Antimicrobial Peptides Provide Reflections on Their Functions and Amyloidogenic Properties.

Enantiomeric forms of BTD-2, PG-1, and PM-1 were synthesized to delineate the structure and function of these β-sheet antimicrobial peptides. Activity and lipid-binding assays confirm that these peptides act via a receptor-independent mechanism involving membrane interaction. The racemic crystal structure of BTD-2 solved at 1.45 Å revealed a novel oligomeric form of β-sheet antimicrobial peptides within the unit cell: an antiparallel trimer, which we suggest might be related to its membrane-active form. The BTD-2 oligomer extends into a larger supramolecular state that spans the crystal lattice, featuring a steric-zipper motif that is common in structures of amyloid-forming peptides. The supramolecular structure of BTD-2 thus represents a new mode of fibril-like assembly not previously observed for antimicrobial peptides, providing structural evidence linking antimicrobial and amyloid peptides.

Factors Modulating the Interaction of Huntingtin with Lipid Membranes: Implications for Huntington's Disease

Huntington disease (HD) is a genetic neurodegenerative disease caused by an expanded polyglutamine (polyQ) domain in the first exon of the huntingtin (Htt) protein. This polyQ expansions leads directly to the formation of proteinaceous aggregates including oligomers and fibrils. Htt interacts with a variety of membraneous structures within the cell, and the first seventeen amino acids (Nt17) of Htt, which directly flanks the polyQ domain, is an amphiphathic α-helix (AH) lipid-binding domain. Here, the role that lipid/htt interactions play in aggregation was investigated with a specific emphasis on the role of Nt17 acetylation and membrane cholesterol content. N-terminal acetylation of htt has been implicated in the etiology of HD, and cholesterol homeostasis is altered in HD. A variety of aggregation assays, lipid binding assays, mass spectrometry, and atomic force microscopy were used. The model membrane systems were vesicles and supported bilayers comprised of total brain lipid extract (TBLE), and these were exposed to a variety of purified or synthetic peptides modeled after htt exon1. Acetylation of htt was achieved using a selective covalent label sulfo-N-hydroxysuccinimide (NHSA). Acetylation retarded fibril formation, promoted the formation of larger globular aggregates, and altered htt's ability to bind lipid membranes. Exogenous cholesterol was incorporated into our model TBLE membranes. As cholesterol content increased, morphological and mechanical changes in the bilayer associated with exposure to Htt were drastically altered. Collectively, these observations suggest that the acetylation and cholesterol content of bilayers play a critical role in htt binding to and aggregation on lipid membranes.

Computational Prediction of Lipid Binding Profiles for Sec14-Like Domains

Sec14 is a phosphatidylinositol (PI) transfer protein that functions in transport from the Golgi apparatus and regulation of lipid metabolism in Saccharomyces cerevisiae. This protein is the namesake of the Sec14 family of proteins that includes relatives in a wide variety of eukaryotic organisms. Structural models of the Sec14 lipid binding domain predicted it would have increased selectivity for particular phosphorylated PI derivatives, such as PI5P. Lipid binding assays for Sec14 confirmed this previously unreported preference, which has also been observed for the homologous Sec14 protein Patellin from Arabidopsis thaliana. We also have extended our computational modeling to Sec14 proteins from the moss Physcomitrella patens in order to predict their lipid binding profiles. Ongoing work is aimed at experimentally confirming these computational predictions. A robust computational model for Sec14 lipid binding would simplify functional predictions, providing insight into the physiological function for these proteins.

PII: S0891-5849(15)00979-X

Ischemic injury to the heart induces mitochondrial dysfunction due to increasing oxidative stress. MG53, also known as TRIM72, is highly expressed in striated muscle, is secreted as a myokine after exercise, and is essential for repairing damaged plasma membrane of many tissues by interacting with the membrane lipid phosphatidylserine (PS). We hypothesized MG53 could preserve mitochondrial integrity after an ischemic event by binding to the mitochondrial-specific lipid, cardiolipin (CL), for mitochondria protection to prevent mitophagy. Fluorescent imaging and Western blotting experiments showed recombinant human MG53 (rhMG53) translocated to the mitochondria after ischemic injury in vivo and in vitro. Fluorescent imaging indicated rhMG53 treatment reduced superoxide generation in ex vivo and in vitro models. Lipid-binding assay indicated MG53 binds to CL. Transfecting cardiomyocytes with the mitochondria-targeted mt-mKeima showed inhibition of mitophagy after MG53 treatment. Overall, we show that rhMG53 treatment may preserve cardiac function by preserving mitochondria in cardiomyocytes. These findings suggest MG53's interactions with mitochondria could be an attractive avenue for developing MG53 as a targeted protein therapy for cardioprotection.

Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-γ that regulates neoangiogenesis.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that can function both extracellularly and intracellularly to mediate a variety of cellular processes. Using lipid affinity matrices and a radiolabeled lipid binding assay, we reveal that S1P directly interacts with the transcription factor peroxisome proliferator-activated receptor (PPAR)γ. Herein, we show that S1P treatment of human endothelial cells (ECs) activated a luciferase-tagged PPARγ-specific gene reporter by ∼12-fold, independent of the S1P receptors. More specifically, in silico docking, gene reporter, and binding assays revealed that His323 of the PPARγ ligand binding domain is important for binding to S1P. PPARγ functions when associated with coregulatory proteins, and herein we identify that peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1)β binds to PPARγ in ECs and their progenitors (nonadherent endothelial forming cells) and that the formation of this PPARγ:PGC1β complex is increased in response to S1P. ECs treated with S1P selectively regulated known PPARγ target genes with PGC1β and plasminogen-activated inhibitor-1 being increased, no change to adipocyte fatty acid binding protein 2 and suppression of CD36. S1P-induced in vitro tube formation was significantly attenuated in the presence of the PPARγ antagonist GW9662, and in vivo application of GW9662 also reduced vascular development in Matrigel plugs. Interestingly, activation of PPARγ by the synthetic ligand troglitazone also reduced tube formation in vitro and in vivo. To support this, Sphk1(-/-)Sphk2(+/-) mice, with low circulating S1P levels, demonstrated a similar reduction in vascular development. Taken together, our data reveal that the transcription factor, PPARγ, is a bona fide intracellular target for S1P and thus suggest that the S1P:PPARγ:PGC1β complex may be a useful target to manipulate neovascularization.

Tetrahymena Expresses More than a Hundred Proteins with Lipid‐binding MORN Motifs that can Differ in their Subcellular Localisations

Proteins with membrane occupation and recognition nexus (MORN) motifs are associated with cell fission in apicomplexan parasites, chloroplast division in Arabidopsis and the motility of sperm cells. We found that ciliates are among those that encode the largest variety of MORN proteins. Tetrahymena thermophila expresses 129 MORN protein-encoding genes, some of which are specifically up-regulated during conjugation. A lipid-binding assay underpins the assumption that the predominant function of MORN motifs themselves is to confer the ability of lipid binding. The localisation of four MORN candidate proteins with similar characteristics highlights the functional diversity of this group especially in ciliates.

CD1d serves as a surface receptor for oxidized cholesterol induction of peroxisome proliferator-activated receptor-γ

ObjectiveThe cluster of differentiation-1d (CD1d) recognizes and presents the lipid antigens to NK-T lymphocytes. Atherosclerotic lesions contain atherogenic lipids, mainly cholesterol and its oxides. Peroxisome proliferator-activated receptor-γ (PPARγ) is also known to exist in atherosclerotic lesions, participating in regulation of lipid metabolism. The current study tested whether CD1d acts as a surface receptor that mediates induction and activation of PPARγ by oxysterols commonly found in atherosclerotic lesions. Methods and resultsCD1d overexpression in HEK 293 cells transfected with CD1d cDNA was confirmed by fluorescence, flow cytometry, Western blotting and mRNA expression. Tritiated (3H) 7-ketocholesterol (7K) was used for lipid binding assays. Radioactive assessment demonstrated an increased 7K-binding activity HEK 293 cells with CD1d overexpression. The 7K binding could be blocked by another oxysterol, 25-hydroxycholesterol, but not by native free cholesterol. Addition of CD1d:IgG dimer protein or an anti-CD1d antibody, but not control IgG, significantly diminished 7K binding to CD1d-expressing HEK 293 cells. CD1d deficiency markedly diminished the 7K-binding in macrophages and smooth muscle cells. Western blot and gel shift assays demonstrated that CD1d-mediated 7K binding induced expression and activation of PPARγ. The PPARγ agonist PGJ2 enhances the 7K stimulatory effect on PPARγ expression and activity but the antagonist GW9662 inhibits the 7K effect on the CD1d-expressing cells. ConclusionsCD1d acts as a cell surface receptor that recognizes and binds oxysterols and initializes a pathway connecting oxysterol binding to PPARγ activation.

Cholesterol Modulates the Binding and Subsequent Aggregation of Huntingtin on Lipid Bilayers

Huntington disease (HD) is an autosomal dominant inherited neurodegenerative disease caused by abnormally long CAG-repeats in the huntingtin gene. This mutation encodes an expanded polyglutamine (polyQ) domain in the N-terminus of the huntingtin (htt) protein which directly leads to its disease-related aggregation. Htt is found highly associated with a variety of cellular and subcellular membranes, which are predominately comprised of lipids. The interaction of htt with lipid membranes is facilitated by its first 17 amino acids, whose secondary structure is an amphipathic α- helix. There are alterations in the relative amounts of specific membrane components in the brains of HD patients, and in particular, cholesterol homeostasis is altered. Here, we investigate how cholesterol modifies the interaction of htt with lipid bilayers. Using atomic force microscopy (AFM), we track aggregation of htt on supported lipid bilayer containing varying amounts of exogenously added cholesterol. As the amount of cholesterol in the bilayer increased, htt binding to the membrane, and subsequent aggregation, was reduced. This reduced lipid-membrane interaction was further validated using a colorimetric polydiacetylene (PDA) lipid binding assay. More interestingly, morphological changes on the bilayer induced by exposure to htt are significantly altered upon addition of cholesterol.

Yeast Pmp3p has an important role in plasma membrane organization.

Pmp3p-related proteins are highly conserved proteins that exist in bacteria, yeast, nematodes and plants, and its transcript is regulated in response to abiotic stresses, such as low temperature or high salinity. Pmp3p was originally identified in Saccharomyces cerevisiae, and it belongs to the sensitive to Na(+) (SNA)-protein family, which comprises four members--Pmp3p/Sna1p, Sna2p, Sna3p and Sna4p. Deletion of the PMP3 gene conferred sensitivity to cytotoxic cations, whereas removal of the other SNA genes did not lead to clear phenotypic effects. It has long been believed that Pmp3p-related proteins have a common and important role in the modulation of plasma membrane potential and in the regulation of intracellular ion homeostasis. Here, we show that several growth phenotypes linked to PMP3 deletion can be modulated by the removal of specific genes involved in sphingolipid synthesis. These genetic interactions, together with lipid binding assays and epifluorescence microscopy, as well as other biochemical experiments, suggest that Pmp3p could be part of a phosphoinositide-regulated stress sensor.

Acetylation Regulates the Interaction of Huntingtin with Lipid Membranes: Implications for Huntington Disease

Huntington disease (HD) is a genetic neurodegenerative disorder caused by an expanded polyglutamine (polyQ) domain in the N-terminus of the huntingtin (htt) protein which facilitates its aggregation. The first 17 amino acids (Nt17) in htt is an amphiphathic α-helix lipid-binding domain that promotes the formation of a diverse population of nanoscale aggregates. This domain undergoes numerous posttranslational modifications that modulate htt's toxicity, subcellular localization, and trafficking of vesicles. More specifically, N-terminal acetylation of htt has been implicated in the etiology of HD. Given the importance of acetylation in HD, we employed mass spectrometry (MS), both in situ and ex situ atomic force microscopy (AFM), and spectroscopic techniques to evaluate the impact of lysine acetylation on htt's aggregation kinetics in solution and on model lipid bilayers. Acetylation of htt exon 1 (51Q), and synthetic truncated htt exon 1 peptide (Nt17Q35P10KK) was achieved using a selective covalent label sulfo-N-hydroxysuccinimide (NHSA) in molar ratios of 1x, 2x, and 3x NHSA per peptide. With these molar ratios, all three lysine residues (K6, K9, and K15) in Nt17 were significantly labeled, as verified by MS. N-terminal htt acetylation retarded fibril formation in solution; however, the resulting fibril morphology was unaltered. Htt acetylation strongly impacted the protein's ability to bind lipid membranes, as demonstrated by a combination of lipid binding assays and AFM. Acetylated htt was found to bind to lipid vesicles, and disrupt lipid bilayer morphology less aggressively compared to the unlabeled htt. Our results highlight that N-terminal acetylation influences the aggregation of htt and its interaction with lipid bilayers.

Neuronal porosome lipidome.

Cup-shaped lipoprotein structures called porosomes are the universal secretory portals at the cell plasma membrane, where secretory vesicles transiently dock and fuse to release intravesicular contents. In neurons, porosomes measure ∼15 nm and are comprised of nearly 40 proteins, among them SNAREs, ion channels, the Gαo G-protein and several structural proteins. Earlier studies report the interaction of specific lipids and their influence on SNAREs, ion channels and G-protein function. Our own studies demonstrate the requirement of cholesterol for the maintenance of neuronal porosome integrity, and the influence of lipids on SNARE complex assembly. In this study, to further understand the role of lipids on porosome structure-function, the lipid composition of isolated neuronal porosome was determined using mass spectrometry. Using lipid-binding assays, the affinity of porosome-associated syntaxin-1A to various lipids was determined. Our mass spectrometry results demonstrate the presence of phosphatidylinositol phosphates (PIP's) and phosphatidic acid (PA) among other lipids, and the enriched presence of ceramide (Cer), lysophosphatidylinositol phosphates (LPIP) and diacylglycerol (DAG). Lipid binding assays demonstrate the binding of neuronal porosome to cardiolipin, and confirm its association with PIP's and PA. The ability of exogenous PA to alter protein–protein interaction and neurotransmitter release is further demonstrated from the study.

Human junctophilin-2 undergoes a structural rearrangement upon binding PtdIns(3,4,5)P3 and the S101R mutation identified in hypertrophic cardiomyopathy obviates this response

JP2 (junctophilin-2) is believed to hold the transverse tubular and jSR (junctional sarcoplasmic reticulum) membranes in a precise geometry that facilitates excitation–contraction coupling in cardiomyocytes. We have expressed and purified human JP2 and shown using electron microscopy that the protein forms elongated structures ~15 nm long and 2 nm wide. Employing lipid-binding assays and quartz crystal microbalance with dissipation we have determined that JP2 is selective for PS (phosphatidylserine), with a Kd value of ~0.5 μM, with the N-terminal domain mediating this interaction. JP2 also binds PtdIns(3,4,5)P3 at a different site than PS, resulting in the protein adopting a more flexible conformation; this interaction is modulated by both Ca2+ and Mg2+ ions. We show that the S101R mutation identified in patients with hypertrophic cardiomyopathy leads to modification of the protein secondary structure, forming a more flexible molecule with an increased affinity for PS, but does not undergo a structural transition in response to binding PtdIns(3,4,5)P3. In conclusion, the present study provides new insights into the structural and lipid-binding properties of JP2 and how the S101R mutation may have an effect upon the stability of the dyad organization with the potential to alter JP2–protein interactions regulating Ca2+ cycling.

Secreted Progranulin Is a Homodimer and Is Not a Component of High Density Lipoproteins (HDL)

Progranulin is a secreted glycoprotein, and the GRN gene is mutated in some cases of frontotemporal dementia. Progranulin has also been implicated in cell growth, wound healing, inflammation, and cancer. We investigated the molecular nature of secreted progranulin and provide evidence that progranulin exists as a homodimer. Although recombinant progranulin has a molecular mass of ∼85 kDa by SDS-PAGE, it elutes in fractions corresponding to ∼170-180 kDa by gel-filtration chromatography. Additionally, recombinant progranulin can be intermolecularly cross-linked, yielding a complex corresponding to a dimer (∼180 kDa), and progranulins containing different epitope tags physically interact. In plasma, progranulin similarly forms complexes of ∼180-190 kDa. Although progranulin partially co-fractionated with high density lipoproteins (HDL) by gel-filtration chromatography, we found no evidence that progranulin in mouse or human plasma is a component of HDL either by ultracentrifugation or by lipid binding assays. We conclude that circulating progranulin exists as a dimer and is not likely a component of HDL.

Elucidating the Mechanism of Ebola Virus Assembly and Budding

Ebola virus from the filoviridae family is characterized by internal and external bleeding in primates due to coagulation abnormalities induced by the virus at the onset of the infection. With no vaccines or treatment, Ebola is classified as bio-safety level IV agent with the potential to be used as a biological weapon. Details of virus assembly are poorly understood. Evidence suggests that matrix protein VP40 is the main driving force for assembly and budding. Generation of new virus involves a cascade of cellular events that recruit the viral genome, the matrix proteins and subsequent acquisition of the viral envelope from the host cell. The new virus like particle (VLP) forms at a bud site at the inner leaflet of the plasma membrane and can serve as a primary therapeutic target for inhibiting Ebola virus replication. Preliminary results demonstrate that VP40 alone assembles in mammalian cells into VLPs independent of other viral proteins. It binds to the plasma membrane with nanomolar affinity and possesses the ability to modify membrane structure. VP40 induces membrane curvature changes, an important step for bud formation and egress of the newly formed virus. This project is aimed at elucidating the mechanistic details of VP40 assembly on the plasma membrane using an interdisciplinary approach. Specifically, we have employed in vitro lipid binding and curvature assays with cellular scanning and single molecule microscopy to investigate the basis of VP40 lipid binding, membrane bending and viral egress. Our results further demonstrate that VP40 oligomerizes on the plasma membrane in a PS-dependent manner and also remodels actin network for assembly and maturation. Our results represent a key step to understanding the general principles governing the remodeling of membrane by matrix proteins from lipid enveloped viruses such as Ebola and HIV.

The Role of Phosphatidylinositol Phosphates (PIPs) in Poliovirus Replication Complexes Trafficking to Viral Replication Organelles

Viruses are intracellular parasites which utilize their host cell's subcellular machinery for their own replication by remodeling of the host's intracellular membranes. Lipids with phosphatidylinositol-4-phosphate (PI4P) head groups have been found to be crucial for the formation of these replication organelles in enteroviruses and flaviviruses. Lipid binding assays show that poliovirus protease 3Cpro specifically binds to PI4P. Using NMR spectroscopy, binding with other phosphatidylinositol phosphates (PIPs) has been detected and specific residues has been identified which exhibits chemical shift changes upon binding with 3Cpro. PI3P, PI4P, and PI5P have been found to exhibit similar chemical shift perturbation patterns upon binding with 3Cpro. Our results indicate that a novel PIP binding site has been identified, with some residues exhibiting long range interactions (or a secondary binding site).

PTEN-lipid Binding Assay

The lipid and protein interactions are an integral and important part of many cellular signaling pathways. The understanding of the selective and specific interaction of the given lipid molecule with the target protein is required for studying cellular signaling. In this assay, different lipids are spotted onto a nitrocellulose membrane to which they attach. Then the membrane is incubated with a lipid binding protein possessing an epitope tag. The protein binds to the lipid which is detected by immunoblotting with an antibody recognizing the epitope tag (see Figure 1). PTEN is an important tumor suppressor which functions as both protein and lipid phosphatase. The primary physiological substrate of PTEN is signaling lipid PtdIns (3, 4, 5) P3, by dephosphrylating PtdIns (3, 4, 5) P3 to PtdIns (4, 5) P2 PTEN negatively regulates PI3K signaling and mediates its tumor-suppressor function by inactivating downstream oncogenic AKT-mediated signaling. The PTEN lipid binding assay is conducted to study the specific binding of PTEN to different lipid molecules.

Structural insights into the inhibition of actin-capping protein by interactions with phosphatidic acid and phosphatidylinositol (4,5)-bisphosphate.

The actin cytoskeleton is a dynamic structure that coordinates numerous fundamental processes in eukaryotic cells. Dozens of actin-binding proteins are known to be involved in the regulation of actin filament organization or turnover and many of these are stimulus-response regulators of phospholipid signaling. One of these proteins is the heterodimeric actin-capping protein (CP) which binds the barbed end of actin filaments with high affinity and inhibits both addition and loss of actin monomers at this end. The ability of CP to bind filaments is regulated by signaling phospholipids, which inhibit the activity of CP; however, the exact mechanism of this regulation and the residues on CP responsible for lipid interactions is not fully resolved. Here, we focus on the interaction of CP with two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP2). Using different methods of computational biology such as homology modeling, molecular docking and coarse-grained molecular dynamics, we uncovered specific modes of high affinity interaction between membranes containing PA/phosphatidylcholine (PC) and plant CP, as well as between PIP2/PC and animal CP. In particular, we identified differences in the binding of membrane lipids by animal and plant CP, explaining previously published experimental results. Furthermore, we pinpoint the critical importance of the C-terminal part of plant CPα subunit for CP–membrane interactions. We prepared a GST-fusion protein for the C-terminal domain of plant α subunit and verified this hypothesis with lipid-binding assays in vitro.

The lysine-rich motif of intrinsically disordered stress protein CDeT11-24 from Craterostigma plantagineum is responsible for phosphatidic acid binding and protection of enzymes from damaging effects caused by desiccation

The late embryogenesis abundant (LEA)-like protein CDeT11-24 is one of the major desiccation-related phosphoproteins of the resurrection plant Craterostigma plantagineum. In this study, it was shown that CDeT11-24 is mostly intrinsically disordered and protects two different enzymes, citrate synthase and lactate dehydrogenase, against damaging effects caused by desiccation. Lipid-binding assays revealed that CDeT11-24 is able to interact with phosphatidic acid, although electrostatic repulsion was expected due to the overall negative net charge of the protein under the tested physiological conditions. CDeT11-24 carries an N-terminal lysine-rich sequence, which is predicted to form an amphipathic α-helix. Analysis of the truncated CDeT11-24 protein identified this region to be responsible for both activities: enzyme protection and phosphatidic acid interaction. Possible functions of the CDeT11-24 protein are discussed in the context of desiccation tolerance. Abbreviations:ABAabscisic acid;CDcircular dichroism;CScitrate synthase;IDPintrinsically disordered protein;LDHlactate dehydrogenase;LEAlate embryogenesis abundant;PAphosphatidic acid;PCphosphatidylcholine;TFEtrifluoroethanol.

Phosphatidylinositol phosphate-dependent regulation ofXenopusENaC by MARCKS protein

Phosphatidylinositol phosphates (PIPs) are known to regulate epithelial sodium channels (ENaC). Lipid binding assays and coimmunoprecipitation showed that the amino-terminal domain of the β- and γ-subunits of Xenopus ENaC can directly bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidic acid (PA). Similar assays demonstrated various PIPs can bind strongly to a native myristoylated alanine-rich C-kinase substrate (MARCKS), but weakly or not at all to a mutant form of MARCKS. Confocal microscopy demonstrated colocalization between MARCKS and PIP(2). Confocal microscopy also showed that MARCKS redistributes from the apical membrane to the cytoplasm after PMA-induced MARCKS phosphorylation or ionomycin-induced intracellular calcium increases. Fluorescence resonance energy transfer studies revealed ENaC and MARCKS in close proximity in 2F3 cells when PKC activity and intracellular calcium concentrations are low. Transepithelial current measurements from Xenopus 2F3 cells treated with PMA and single-channel patch-clamp studies of Xenopus 2F3 cells treated with a PKC inhibitor altered Xenopus ENaC activity, which suggest an essential role for MARCKS in the regulation of Xenopus ENaC activity.