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BBA - Biomembranes (v.1798, #9)

Editorial Board (pp. i).

Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles by Koki Kamiya; Jun Kobayashi; Tetsuro Yoshimura; Kanta Tsumoto (pp. 1625-1631).

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus ( Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0–6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0–6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic β₂ receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.

Keywords: Giant unilamellar vesicle; Liposome; Membrane fusion; Baculovirus; Enveloped virus; Confocal laser scanning microscopy

Folding of lipid monolayers containing lung surfactant proteins SP-B₁_–₂₅ and SP-C studied via coarse-grained molecular dynamics simulations by Susan L. Duncan; Ronald G. Larson (pp. 1632-1650).

To explore the role of lung surfactant proteins SP-B and SP-C in storing and redelivering lipid from lipid monolayers during the compression and re-expansion occurring in lungs during breathing, we simulate the folding of lipid monolayers with and without these proteins. We utilize the MARTINI coarse-grained force field to simulate monolayers containing pure dipalmitoylphosphatidylcholine (DPPC) and DPPC mixed with palmitoyloleoylphosphatidylglycerol (POPG), palmitic acid (PA), and/or peptides. The peptides considered include the 25-residue N-terminal fragment of SP-B (SP-B₁_–₂₅), SP-C, and several SP-B₁_–₂₅ mutants in which charged and hydrophilic residues are replaced by hydrophobic ones, or vice-versa. We observe two folding mechanisms: folding by the amplification of undulations and folding by nucleation about a defect. The first mechanism is observed in monolayers containing either POPG or peptides, while the second mechanism is observed only with peptides present, and involves the lipid-mediated aggregation of the peptides into a defect, from which the fold can nucleate. Fold nucleation from a defect displays a dependence on the hydrophobic character of the peptides; if the number of hydrophobic residues is decreased significantly, monolayer folding does not occur. The addition of POPG or peptides to the DPPC monolayer has a fluidizing effect, which assists monolayer folding. In contrast, the addition of PA has a charge-dependent condensing affect on DPPC monolayers containing SP-C. The peptides appear to play a significant role in the folding process, and provide a larger driving force for folding than POPG. In addition to promoting fold formation, the peptides also display fusogenic behavior, which can lead to surface refining.

Keywords: Pulmonary surfactant; Surfactant reservoir; Monolayer collapse; Surface aggregate; Order parameter; Fluidization

Potential role of the membrane in hERG channel functioning and drug-induced long QT syndrome by Étienne Chartrand; Alexandre A. Arnold; Andrée Gravel; Sarah Jenna; Isabelle Marcotte (pp. 1651-1662).

The human ether-à-go-go related gene (hERG) potassium channels are located in the myocardium cell membrane where they ensure normal cardiac activity. The binding of drugs to this channel, a side effect known as drug-induced (acquired) long QT syndrome (ALQTS), can lead to arrhythmia or sudden cardiac death. The hERG channel is a unique member of the family of voltage-gated K⁺ channels because of the long extracellular loop connecting its transmembrane S5 helix to the pore helix in the pore domain. Considering the proximal position of the S5-P linker to the membrane surface, we have investigated the interaction of its central segment I⁵⁸³–Y⁵⁹⁷ with bicelles. Liquid and solid-state NMR experiments as well as circular dichroism results show a strong affinity of the I⁵⁸³–Y⁵⁹⁷ segment for the membrane where it would sit on the surface with no defined secondary structure. A structural dependence of this segment on model membrane composition was observed. A helical conformation is favoured in detergent micelles and in the presence of negative charges. Our results suggest that the interaction of the S5-P linker with the membrane could participate in the stabilization of transient channel conformations, but helix formation would be triggered by interactions with other hERG domains. Because potential drug binding sites on the S5-P linker have been identified, we have explored the role of this segment in ALQTS. Four LQTS-liable drugs were studied which showed more affinity for the membrane than this hERG segment. Our results, therefore, identify two possible roles for the membrane in channel functioning and ALQTS.

Keywords: NMR spectroscopy; Bicelles; Membrane-protein interaction; Drug–protein interaction; Cardiotoxic drugs; Voltage-gated potassium channel

Mimicking SP-C palmitoylation on a peptoid-based SP-B analogue markedly improves surface activity by Michelle T. Dohm; Nathan J. Brown; Shannon L. Seurynck-Servoss; Jorge Bernardino de la Serna; Annelise E. Barron (pp. 1663-1678).

Hydrophobic lung surfactant proteins B and C (SP-B and SP-C) are critical for normal respiration in vertebrates, and each comprises specific structural attributes that enable the surface-tension-reducing ability of the lipid–protein mixture in lung surfactant. The difficulty in obtaining pure SP-B and SP-C on a large scale has hindered efforts to develop a non-animal-derived surfactant replacement therapy for respiratory distress. Although peptide-based SP-C mimics exhibit similar activity to the natural protein, helical peptide-based mimics of SP-B benefit from dimeric structures. To determine if in vitro surface activity improvements in a mixed lipid film could be garnered without creating a dimerized structural motif, a helical and cationic peptoid-based SP-B mimic was modified by SP-C-like N-terminus alkylation with octadecylamine. “Hybridized” mono- and dialkylated peptoids significantly decreased the maximum surface tension of the lipid film during cycling on the pulsating bubble surfactometer relative to the unalkylated variant. Peptoids were localized in the fluid phase of giant unilamellar vesicle lipid bilayers, as has been described for SP-B and SP-C. Using Langmuir–Wilhelmy surface balance epifluorescence imaging (FM) and atomic force microscopy (AFM), only lipid-alkylated peptoid films revealed micro- and nanostructures closely resembling films containing SP-B. AFM images of lipid-alkylated peptoid films showed gel condensed-phase domains surrounded by a distinct phase containing “nanosilo” structures believed to enhance re-spreading of submonolayer material. N-terminus alkylation may be a simple, effective method for increasing lipid affinity and surface activity of single-helix SP-B mimics.

Keywords: Abbreviations; LS; lung surfactant; γ; surface tension in mN m; −; ¹; SP-B; surfactant protein B; SP-C; surfactant protein C; a/l; air/liquid; SRT; surfactant replacement therapy; IRDS; infant respiratory distress syndrome; ARDS; acute respiratory distress syndrome; SP-B; _1–25; N; -terminus fragment of SP-B consisting of amino acids 1–25; dSP-B; _1–25; dimeric SP-B; _1–25; TL; Tanaka lipids, 68:22:9 [wt] DPPC:POPG:PA; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-; rac; -(1-glycerol); PA; palmitic acid; CD; circular dichroism; PBS; pulsating bubble surfactometer; GUVs; giant unilamellar vesicles; LWSB; Langmuir–Wilhelmy surface balance; FM; epifluorescence microscopy; AFM; atomic force microscopy; Boc; di-tert-butyl dicarbonate; N; Lys; N; -(4-aminobutyl)glycine; N; spe; (; N; )-(; S; )-(1-phenylethyl)glycine; N; ocd; (; N; )-(octadecyl)glycine; TFA; trifluoroacetic acid; TR-DHPE; Texas Red®, 1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine, triethylammonium salt; SPPS; solid phase peptide synthesis; RP-HPLC; reverse-phase high performance liquid chromatography; ESI/MS; electrospray ionization mass spectrometry; MALDI-TOF/MS; matrix-assisted laser desorption/ionization time of flight mass spectrometry; λ; wavelength in nm; UV/Vis; ultraviolet/visible; SA; surface area; γ; _eq/max/min; equilibrium/maximum/minimum surface tension in mN m; −; ¹; π; surface pressure in mN m; −; ¹; A; area in Å; ²; per molecule; LE; liquid-expanded; LC; liquid-condensedLung surfactant; SP-B; SP-C; Peptoid; Lipid monolayer; Lipid bilayer

Altered plasmodial surface anion channel activity and in vitro resistance to permeating antimalarial compounds by Godfrey Lisk; Margaret Pain; Morgan Sellers; Philip A. Gurnev; Ajay D. Pillai; Sergey M. Bezrukov; Sanjay A. Desai (pp. 1679-1688).

Erythrocytes infected with malaria parasites have increased permeability to various solutes. These changes may be mediated by an unusual small conductance ion channel known as the plasmodial surface anion channel (PSAC). While channel activity benefits the parasite by permitting nutrient acquisition, it can also be detrimental because water-soluble antimalarials may more readily access their parasite targets via this channel. Recently, two such toxins, blasticidin S and leupeptin, were used to select mutant parasites with altered PSAC activities, suggesting acquired resistance via reduced channel-mediated toxin uptake. Surprisingly, although these toxins have similar structures and charge, we now show that reduced permeability of one does not protect the intracellular parasite from the other. Leupeptin accumulation in the blasticidin S-resistant mutant was relatively preserved, consistent with retained in vitro susceptibility to leupeptin. Subsequent in vitro selection with both toxins generated a double mutant parasite having additional changes in PSAC, implicating an antimalarial resistance mechanism for water-soluble drugs requiring channel-mediated uptake at the erythrocyte membrane. Characterization of these mutants revealed a single conserved channel on each mutant, albeit with distinct gating properties. These findings are consistent with a shared channel that mediates uptake of ions, nutrients and toxins. This channel's gating and selectivity properties can be modified in response to in vitro selective pressure.

Keywords: Ion channel mutants; Gating; Selectivity; Noise analysis; Plasmodium falciparum; PSAC

Electrophoretic mobility of sarcoplasmic reticulum vesicles is determined by amino acids of A+P+N domains of Ca²⁺–ATPase by Pavel Smejtek; Laura E. Satterfield; Robert C. Word; Jonathan J. Abramson (pp. 1689-1697).

Establishing the origin of electrophoretic mobility of sarcoplasmic reticulum (SR) vesicles is the primary goal of this work. It was found that the electrophoretic mobility originates from ionizable amino acids of cytoplasmic domains of the Ca²⁺–ATPase, the calcium pump of SR. The mobility was measured at pH 4.0, 4.7, 5.0, 6.0, 7.5, and 9.0 in the region of ionic strength from 0.05 to 0.2M. Mobility measurements were supplemented by studies of SR vesicles by photoelectron microscopy. The median diameter of SR vesicles was 260nm. Ca²⁺–ATPases were not resolved. The mobility data were standardized by interpolation to a reference ionic strength of 0.1M. The mobility of the SR vesicles is determined by the charge of the Ca²⁺–ATPase. It is due to the ionizable amino acids selected from the amino acid sequence of SERCA1a Ca²⁺–ATPase. The pH dependence of charge residing in various domains of Ca²⁺–ATPase was computed using pKa values in free water. The charge correlated with measured mobility. It was shown that a linear relationship exists between the mobility of the SR vesicles, μ, and the total computed charge, Q, on three cytoplasmic domains of Ca²⁺–ATPase: A, P, and N. It is given by μ= α+ β Q where the fitted values β=(0.043±0.002)×10⁻⁸m²V⁻¹s⁻¹e⁻¹ and α=(0.16±0.02)×10⁻⁸m²V⁻¹s⁻¹. Since β and α values do not change from pH 4 to pH 9, one concludes that the hydrodynamic friction of the cytoplasmic domains of SR is independent of their charge.

Keywords: Electrophoretic mobility; Sarcoplasmic reticulum; Photoelectron microscopy; Size distribution; Ca2+–ATPase; Calcium pump; Zeta potential; pH dependence; Cytoplasmic domain; SR vesicle

Mitochondria-targeted penetrating cations as carriers of hydrophobic anions through lipid membranes by Tatyana I. Rokitskaya; Natalia V. Sumbatyan; Vadim N. Tashlitsky; Galina A. Korshunova; Yuri N. Antonenko; Vladimir P. Skulachev (pp. 1698-1706).

High negative electric potential inside mitochondria provides a driving force for mitochondria-targeted delivery of cargo molecules linked to hydrophobic penetrating cations. This principle is utilized in construction of mitochondria-targeted antioxidants (MTA) carrying quinone moieties which produce a number of health benefitting effects by protecting cells and organisms from oxidative stress. Here, a series of penetrating cations including MTA were shown to induce the release of the liposome-entrapped carboxyfluorescein anion (CF), but not of glucose or ATP. The ability to induce the leakage of CF from liposomes strongly depended on the number of carbon atoms in alkyl chain ( n) of alkyltriphenylphosphonium and alkylrhodamine derivatives. In particular, the leakage of CF was maximal at n about 10–12 and substantially decreased at n=16. Organic anions (palmitate, oleate, laurylsulfate) competed with CF for the penetrating cation-induced efflux. The reduced activity of alkylrhodamines with n=16 or n=18 as compared to that with n=12 was ascribed to a lower rate of partitioning of the former into liposomal membranes, because electrical current relaxation studies on planar bilayer lipid membranes showed rather close translocation rate constants for alkylrhodamines with n=18 and n=12. Changes in the alkylrhodamine absorption spectra upon anion addition confirmed direct interaction between alkylrhodamines and the anion. Thus, mitochondria-targeted penetrating cations can serve as carriers of hydrophobic anions across bilayer lipid membranes.

Keywords: Abbreviations; BLM; bilayer lipid membrane; CF; carboxyfluorescein; C; ₂; R1; ethylrhodamine 19 or rhodamine 6G; C; ₈; R1; octylrhodamine 19; C; ₁₀; R1; decylrhodamine 19; C; ₁₂; R1; dodecylrhodamine 19; C; ₁₆; R1; octadecylrhodamine 19; C; ₁₂; R4; dodecylrhodamine B; C; ₁₈; R4 or R18; octadecylrhodamine B; C; ₁₂; TPP; dodecyltriphenylphosphonium; DPhPC; diphytanoylphosphatidylcholine; EYPC; egg yolk phosphatidylcholine; MES; 2-(; N; -morpholino)ethanesulfonic acid; MTA; mitochondria-targeted antioxidant; MitoQ; compound of ubiquinone and decyl triphenylphosphonium; ROS; reactive oxygen species; SkQ1; compound of plastoquinone and decyl triphenylphosphonium; SkQR1; compounds of plastoquinone and decylrhodamine 19; SRB; sulforhodamine B; TPP; tetraphenylphosphoniumMitochondria; Liposome; Leakage; Hydrophobic cation; Flip-flop

Lactose permease lipid selectivity using Förster resonance energy transfer by Laura Picas; Suarez-Germa Carme Suárez-Germà; M. Teresa Montero; Vazquez-Ibar José L. Vázquez-Ibar; Hernandez-Borrell Jordi Hernández-Borrell; Manuel Prieto; Luís M.S. Loura (pp. 1707-1713).

The phospholipid composition that surrounds a membrane protein is critical to maintain its structural integrity and, consequently, its functional properties. To understand better this in the present work we have performed FRET measurements between the single tryptophan residue of a lactose permease Escherichia coli mutant (single-W151/C154G LacY) and pyrene-labeled phospholipids (Pyr-PE and Pyr-PG) at 37°C. We have reconstituted this LacY mutant in proteoliposomes formed with heteroacid phospholipids, POPE and POPG, and homoacid phospholipids DOPE and DPPE, resembling the same PE/PG proportion found in the E. coli inner membrane (3:1, mol/mol). A theoretical model has been fitted to the experimental data. In the POPE/POPG system, quantitative model calculations show accordance with the experimental values that requires an annular region composed of approximately ∼90mol% PE. The experimental FRET efficiencies for the gel/fluid phase-separated DOPE/POPG system indicate a higher presence of PG in the annular region, from which it can be concluded that LacY shows clear preference for the fluid phase. Similar conclusions are obtained from analysis of excimer-to-monomer ( E/M) pyrene ratios. To test the effects of this on cardiolipin (CL) on the annular region, myristoyl-CL and oleoyl-CL were incorporated in the biomimetic POPE/POPG matrix. The experimental FRET efficiency values, slightly larger for Pyr-PE than for Pyr-PG, suggest that CL displaces POPE and, more extensively, POPG from the annular region of LacY. Model fitting indicates that CL enrichment in the annular layer is, in fact, solely produced by replacing PG and that myristoyl-CL is not able to displace PE in the same way that oleoyl-CL does. One of the conclusions of this work is the fact that LacY inserts preferentially in fluid phases of membranes.

Keywords: Lipid selectivity; Lactose permease; FRET

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells by G.A. dos Santos; Thome C.H. Thomé; G.A. Ferreira; J.S. Yoneda; T.M. Nobre; K.R.P. Daghastanli; P.S. Scheucher; H.L. Gimenes-Teixeira; M.G. Constantino; K.T. de Oliveira; Faca V.M. Faça; Falcao R.P. Falcão; L.J. Greene; E.M. Rego; P. Ciancaglini (pp. 1714-1723).

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9μM for 72h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25μM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7μM, respectively. The critical micellar concentration (CMC) of ODPC was 200μM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature ( T_c) of 41.5°C and an enthalpy (∆H) variation of 7.3kcal mol⁻¹. The presence of 25μM ODPC decreased T_c and ∆H to 39.3°C and 4.7kcal mol⁻¹, respectively. ODPC at 250μM destabilized the liposomes (36.3°C, 0.46kcal mol⁻¹). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.

Keywords: 10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate; Lipids; Liposome; Alkylphospholipids; Leukemia

Numerical studies of the membrane fluorescent dyes dynamics in ground and excited states by Justyna Barucha-Kraszewska; Sebastian Kraszewski; Piotr Jurkiewicz; Christophe Ramseyer; Martin Hof (pp. 1724-1734).

Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.

Keywords: Molecular dynamics; Fluorescent probe; Membrane; Excited state; Solvent relaxation; Prodan; Laurdan

Quantitation of cholesterol incorporation into extruded lipid bilayers by Maitane Ibarguren; Alicia Alonso; Boris G. Tenchov; Goni Felix M. Goñi (pp. 1735-1738).

Cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. To this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures 33–75mol% cholesterol have been measured and compared with the original mixture before lipid hydration. There is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the extruded bilayers is much lower than predicted. A quantitative analysis of the vesicles is thus required before any experimental study is undertaken.

Keywords: Abbreviations; Chol; cholesterol; DAG; diacylglycerol; DAPC; diarachidonoyl phosphatidylcholine; DLPC; dilinoleoyl phosphatidylcholine; DOPC; dioleoyl phosphatidylcholine; DPPC; dipalmitoyl phosphatidylcholine; LUV; large unilamellar vesicles; MLV; multilamellar vesicles; PC; phosphatidylcholine; PE; phosphatidylethanolamine; POPC; 1-palmitoyl-2-oleoyl phosphatidylcholine; SM; sphingomyelinCholesterol; Lipid bilayers; Inverted hexagonal phases; Phospholipid; Sphingomyelin; Extrusion vesicles

The plasma membrane plays a central role in cells response to mechanical stress by Sandra V. Verstraeten; Gerardo G. Mackenzie; Patricia I. Oteiza (pp. 1739-1749).

The mechanisms by which lymphocytes recognize and interpret mechanical stimuli and translate these into the triggering of select signaling cascades that are critical for lymphocyte function are still not fully understood. In this work, we investigated the association of mechanical stress (MS)-induced changes in membrane physical properties with changes in cytoskeleton dynamics and cell signaling. In Jurkat T cells, MS was associated with the immediate and transient depolymerization of both β-tubulin and F-actin. The fluidity of the plasma membrane measured in the hydrophobic region of the bilayer, increased 0.5min post-MS, recovering the initial value in the following 2min. This effect was accompanied by the rearrangement of lipids in the lateral phase of the plasma membrane, transient lipid rafts' alteration, and membrane hyperpolarization. The consequent increase in cellular [Ca²⁺] triggered the activation of the transcription factors NFAT, AP-1, and NF-κB. Results indicate that the cytoplasmic membrane, through changes in membrane physical properties, senses MS, and transduces an initial physical stimulus into microtubules rearrangements, Ca²⁺ mobilization, and the subsequent changes in cell signaling.

Keywords: Abbreviations; 16-AP; 16-(9-anthroyloxy) palmitic acid; 6-AS; 6-(9-anthroyloxy)stearic acid; AP-1; activator protein-1; BODIPY-GM; ₁; BODIPY® FL C5-ganglioside GM; ₁; CRAC; Ca; ²⁺; release-activated Ca; ²⁺; channels; CsA; cyclosporin A; CTX–FITC; FITC conjugate of subunit B of cholera toxin; DiBAC; ₄; (3); bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DTT; dithiothreitol; FBS; fetal bovine serum; HRP; horseradish peroxidase; IKKβ; inhibitor of nuclear factor kappa B kinase beta subunit; IκBα; I-kappa-B kinase alpha; JNK; Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; MAPKK; kinase of mitogen-activated protein kinase; MS; mechanical stress; NFAT; nuclear factor of activated T cells; NF-kB; nuclear factor kappa B; PKC; protein kinase C; PLCγ; phospholipase C gamma; p-NFAT; phosphorylated nuclear factor of activated T cells; TNFα; tumor necrosis factor alphaMechanical stress; Membrane fluidity; Calcium; Signal transduction; Lipid lateral mobility

Identification and experimental verification of a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels by David B. Caldwell; Hannah R. Malcolm; Donald E. Elmore; Joshua A. Maurer (pp. 1750-1756).

Studies of bacterial ion channels have provided significant insights into the structure–function relationships of mechanosensitive and voltage-gated ion channels. However, to date, very few bacterial channels that respond to small molecules have been identified, cloned, and characterized. Here, we use bioinformatics to identify a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels containing a channel domain related by sequence homology to the mechanosensitive channel of small conductance (MscS). In this initial report, we clone selected members of this channel family, use electrophysiological measurements to verify their ability to directly gate in response to cyclic nucleotides, and use osmotic downshock to demonstrate their lack of mechanosensitivity. In addition to providing insight into bacterial physiology, these channels will provide researchers with a useful model system to investigate the role of ligand-gated ion channels (LGICs) in the signaling processes of higher organisms. The identification of these channels provides a foundation for structural and functional studies of LGICs that would be difficult to perform on mammalian channels. Moreover, the discovery of bCNG channels implies that bacteria have cyclic nucleotide-gated and cyclic nucleotide-modulated ion channels, which are analogous to the ion channels involved in eukaryotic secondary messenger signaling pathways.

Keywords: Ligand-gated ion channel; Cyclic nucleotide-gated (CNG) channel; Bacterial ion channel; Data mining; Membrane proteins

Divergent signature motifs of nucleotide binding domains of ABC multidrug transporter, CaCdr1p of pathogenic Candida albicans, are functionally asymmetric and noninterchangeable by Antresh Kumar; Suneet Shukla; Ajeet Mandal; Sudhanshu Shukla; Suresh V. Ambudkar; Rajendra Prasad (pp. 1757-1766).

Nucleotide binding domains (NBDs) of the multidrug transporter of Candida albicans, CaCdr1p, possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved signature motifs, while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site-directed mutagenesis, we made three categories of mutant variants. These included mutants where all the signature motif residues were replaced with either alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where signature motifs were swapped to have variants with either both the conserved or degenerated entire signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2 and swapped signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogation in ATPase and R6G efflux activities. However, some of the mutants displayed a selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show not only that certain conserved residues of NBD1 signature sequence (S304, G306, and E307) are important in ATP hydrolysis and R6G efflux but also that a few divergent residues (N1002 and E1004) of NBD2 signature motif have evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the signature motifs of CaCdr1p, whether it is divergent or conserved, are nonexchangeable and are functionally critical for ATP hydrolysis.

Keywords: Abbreviations; ABC; ATP binding cassette; PDR; pleiotropic drug resistance; NBD; nucleotide binding domain; TMD; transmembrane domain; TMS; transmembrane segment; PM; plasma membrane; FLC; fluconazole; CYH; cycloheximide; ANI; anisomycin; R6G; rhodamine 6G; [; ¹²⁵; I] IAAP; [; ¹²⁵; I] iodoarylazidoprazosinABC transporter; Nucleotide binding domain; Signature motif; Drug resistance; Drug transport; ATPase activity

Erythrocyte morphological states, phases, transitions and trajectories by Sergey V. Rudenko (pp. 1767-1778).

Morphological response (MR) of red blood cells represents a triphasic sequence of spontaneously occurring shape transformation between different shape states upon transfer the cells into isotonic sucrose solution in the order: S₀ (initial discoid shape in physiological saline)→S₁ (echinocytic shape at the beginning of MR, phase 1)→S₂ (intermediate discoid shape, phase 2)→S₃ (final stomatocytic shape, phase 3). In this paper, the dynamics of cell shape changes was investigated by non-invasive light fluctuation method and optical microscopy. Among 12 possible transitions between four main shape states, we experimentally demonstrate here an existence of nine transitions between neighbour or remote states in this sequence. Based on these findings and data from the literature, we may conclude that red blood cells are able to change their shape through direct transitions between four main states except transition S₁→S₀, which has not been identified yet. Some shape transitions and their temporal sequence are in accord with predictions of bilayer couple concept, whereas others for example transitions between remote states S₃→S₁, S₁→S₃ and S₃→S₀ are difficult to explain based solely on the difference in relative surface areas of both leaflets of membrane suggesting more complex mechanisms involved. Our data show that MR could represents a phenomenon in which the major role can play pH and chloride-sensitive sensor and switching mechanisms coupled with transmembrane signaling thus involving both cytoskeleton and membrane in coordinated shape response on changes in cell ionic environment.

Keywords: Abbreviations; HBS; HEPES-buffered saline; RBC; red blood cells; MR; morphological response; SI; shape index; Cpr; chlorpromazine; SSS; standard sucrose solution; NSVDC; non-selective voltage dependent channelRed blood cell; Morphological response; Shape; Low ionic strength; Anion; pH

Investigation of the interaction between modified ISCOMs and stratum corneum lipid model systems by Henriette Baun Madsen; Helle M. Arboe-Andersen; Noemi Rozlosnik; Flemming Madsen; Peter Ifversen; Marina R. Kasimova; Hanne Mørck Nielsen (pp. 1779-1789).

The modified ISCOMs, so-called Posintro™ nanoparticles, provide an opportunity for altering the surface charge of the particles, which influences their affinity for the negatively charged antigen sites, cell membranes and lipids in the skin. Hypothetically, this increases the passage of the ISCOMs (or their components) and their load through the stratum corneum. The subsequent increase in the uptake by the antigen-presenting cells results in enhanced transcutaneous immunization. To understand the nature of penetration of Posintro™ nanoparticles into the intercorneocyte space of the stratum corneum, the interaction between the nanoparticles and lipid model systems in form of liposomes and/or supported lipid bilayer was studied. As a lipid model we used Stratum Corneum Lipid (SCL), a mixture similar in composition to the lipids of the intercorneocyte space. By Förster Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Electrochemical Impedance Spectroscopy (EIS) and cryo-Transmission Electron Microscopy (cryo-TEM) it was shown that application of nanoparticles to the SCL bilayers results in lipid disturbance. Investigation of this interaction by means of Isothermal Titration Calorimetry (ITC) confirmed existence of an enthalpically unfavorable reaction. All these methods demonstrated that the strength of electrostatic repulsion between the negatively charged SCL and the nanoparticles affected their interaction, as decreasing the negative charge of the Posintro™ nanoparticles leads to enhanced disruption of lipid organization.

Keywords: ISCOMs; Skin; Interaction; ITC; FRET; AFM; Drug delivery

Different molecular arrangements of the tetrameric annexin 2 modulate the size and dynamics of membrane aggregation by Françoise Illien; Stefanie Finet; Olivier Lambert; Jesus Ayala-Sanmartin (pp. 1790-1796).

Annexin 2, a member of the annexin family of Ca²⁺-dependent membrane binding proteins is found in monomeric and heterotetrameric forms and has been involved in different membrane related functions. The heterotetrameric annexin 2 is composed of a dimer of S100A10, a member of the S100 family of Ca²⁺ binding proteins and two annexin 2 molecules ((Anx2-S100A10)₂). Different molecular models including tetramers and octamers in which S100A10 is localized in the centre of the complex with the annexin 2 molecules positioned around S100A10 had been proposed. Herein, the organization of the (Anx2-S100A10)₂ complex in conditions in which membranes are able to bridge was studied. We performed Cryo-electron microscopy observations of the tetrameric annexin 2 on the membrane surface, and study the S100A10 accessibility to antibodies by flow “cytometry”. We also studied the kinetics and size evolution of vesicle aggregates by dynamic light scattering. The results show that the protein is able to organize in three different arrangements depending on the presence of Ca²⁺ and pH and that the aggregation is faster in the presence of Ca²⁺ compared with the aggregation in its absence. In one arrangement the S100A10 molecule is exposed to the solvent allowing its interaction with other proteins. The presented results will serve as a molecular basis to explain some of the functions of the tetrameric annexin 2.

Keywords: Abbreviations; Anx2; annexin 2; (AnxA2-S100A10)2; heterotetrameric annexin 2; DLS; Dynamic light scattering; LUV; Large unilamellar vesicles; PG; Phosphatidylglycerol; PS; glycerophosphatidylserineAnnexin 2; Calcium; Conformational change; Membrane bridging; Mild acidic pH

P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells by Patrick Constantinescu; Bin Wang; Kati Kovacevic; Iman Jalilian; Giel J.C.G.M. Bosman; James S. Wiley; Ronald Sluyter (pp. 1797-1804).

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium⁺ uptake into MEL cells in a concentration-dependent fashion and with an EC₅₀ of ∼154μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ and YO-PRO-1²⁺ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.

Keywords: Abbreviations; BzATP; 2′- and 3′-0(4-benzoylbenzoyl) ATP; PS; phosphatidylserine; MP; microparticle; MEL; murine erythroleukemia; MTT; thiazolyl blue tetrazolium bromide; mAb; monoclonal antibody; 7AAD; 7-aminoactinomycin D; Ab; antibody; MFI; mean fluorescence intensityP2X receptor; Red blood cell; Macrophage; Apoptosis; Microparticle

The N-terminal fragment of human islet amyloid polypeptide is non-fibrillogenic in the presence of membranes and does not cause leakage of bilayers of physiologically relevant lipid composition by Khemtemourian Lucie Khemtémourian; Maarten F.M. Engel; Rob M.J. Liskamp; Hoppener Jo W.M. Höppener; J. Antoinette Killian (pp. 1805-1811).

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). The formation of hIAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet β-cells during the pathogenesis of DM2. Previous studies have shown that the N-terminal part of hIAPP, hIAPP_1–19, plays a major role in the initial interaction of hIAPP with lipid membranes. However, the exact role of this N-terminal part of hIAPP in causing membrane damage is unknown. Here we investigate the structure and aggregation properties of hIAPP₁_–₁₉ in relation to membrane damage in vitro by using membranes of the zwitterionic lipid phosphatidylcholine (PC), the anionic lipid phosphatidylserine (PS) and mixtures of these lipids to mimic membranes of islet cells. Our data reveal that hIAPP_1–19 is weakly fibrillogenic in solution and not fibrillogenic in the presence of membranes, where it adopts a secondary structure that is dependent on lipid composition and stable in time. Furthermore, hIAPP_1–19 is not able to induce leakage in membranes of PC/PS or PC bilayers, indicating that the membrane interaction of the N-terminal fragment by itself is not responsible for membrane leakage under physiologically relevant conditions. In bilayers of the anionic lipid PS, the peptide does induce membrane damage, but this leakage is not correlated to fibril formation, as it is for mature hIAPP. Hence, membrane permeabilization by the N-terminal fragment of hIAPP in anionic lipids is most likely an aspecific process, occurring via a mechanism that is not relevant for hIAPP-induced membrane damage in vivo.

Keywords: Abbreviations; CD; circular dichroism; DMSO; dimethyl sulfoxide; hIAPP; human islet amyloid polypeptide; HFIP; 1,1,1,3,3,3-hexafluoro-2-propanol; LUV; large unilamellar vesicle; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-; l; -serine; ThT; Thioflavin TAmyloid; Protein–membrane interaction; Model membrane; Amylin; Type II diabetes mellitus

Interaction of two intrinsically disordered plant stress proteins (COR15A and COR15B) with lipid membranes in the dry state by Anja Thalhammer; Michaela Hundertmark; Antoaneta V. Popova; Robert Seckler; Dirk K. Hincha (pp. 1812-1820).

COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly α-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic α-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids.

Keywords: Abbreviations; CD; circular dichroism; COR; cold regulated; DLnPC; 1, 2-dilinolenoyl-sn-glycero-3-phosphatidylcholine; DLnPE; 1, 2-dilinolenoyl-sn-glycero-3-phosphatidylethanolamine; EPE; egg phosphatidylethanolamine; FTIR; Fourier-transform infrared; H; _II; hexagonal II phase; IDP; intrinsically disordered protein; LDH; lactate dehydrogenase; LEA; late embryogenesis abundant; MGDG; monogalactosyldiacylglycerol; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; T; _Hex; bilayer to hexagonal II phase transition temperature; T; _m; gel to liquid-crystalline phase transition temperatureDesiccation; FTIR spectroscopy; Intrinsically disordered proteins; LEA proteins; Lipid phase transitions; Protein secondary structure

Interference between two modulators of N-type (CaV2.2) calcium channel gating demonstrates that ω-conotoxin GVIA disrupts open state gating by Viktor Yarotskyy; Keith S. Elmslie (pp. 1821-1828).

N-type calcium channels play an important role in synaptic transmission and a drug that blocks these channels has become an important tool in controlling chronic pain. The development of new N-channel-targeted drugs is dependent on a better understanding of the gating of these channels and how that gating can be modulated. We have previously concluded that ω-conotoxin GVIA (GVIA) is a gating modifier that acts by destabilizing the N-channel open state. However, this conclusion was largely based on our modeling results and requires experimental support. Roscovitine, a tri-substituted purine, has been shown to stabilize the N-channel open state to slow gating charge relaxation, which provides a direct test of our hypothesis for GVIA-induced gating modification. We found that roscovitine could modulate gating current in the presence of GVIA, which shows that roscovitine can still affect the gating of the GVIA-bound N-channel. However, the magnitude of the roscovitine-induced slowing of Off-gating current was significantly reduced. In addition to confirming our hypothesis, our evidence supports an additional effect of GVIA to alter gating transitions between N-channel closed states. By strongly limiting access to the N-channel open state, GVIA analogs that selectively induce this modulation could provide the basis for the next generation drugs that treat chronic pain.

Keywords: Abbreviations; GVIA; ω-conotoxin GVIA; LaMg; lanthanum and magnesium external solution; Q; gating charge; Q; _On; On-gating charge; Q; _Off; Off-gating charge; V; _0.5; Boltzmann half maximal voltage; ∆; V; _0.5; change in; V; _0.5; k; Boltzmann slope factor; ∆; k; change in Boltzmann slope factor; Rosc; R-roscovitine; CFP; Cyan Fluorescent Protein; NMG; N-methyl-; d; -glucamineGating current; Roscovitine; Modulation; Markov model; ω-conopeptide

Adhesive and conformational behaviour of mycolic acid monolayers by Zhenyu Zhang; Yu Pen; Robert G. Edyvean; Steven A. Banwart; Robert M. Dalgliesh; Mark Geoghegan (pp. 1829-1839).

We have studied the pH-dependent interaction between mycolic acid (MA) monolayers and hydrophobic and hydrophilic surfaces using molecular (colloidal probe) force spectroscopy. In both cases, hydrophobic and hydrophilic monolayers (prepared by Langmuir–Blodgett and Langmuir–Schaefer deposition on silicon or hydrophobized silicon substrates, respectively) were studied. The force spectroscopy data, fitted with classical DLVO (Derjaguin, Landau, Verwey, and Overbeek) theory to examine the contribution of electrostatic and van der Waals forces, revealed that electrostatic forces are the dominant contribution to the repulsive force between the approaching colloidal probe and MA monolayers. The good agreement between data and the DLVO model suggest that beyond a few nm away from the surface, hydrophobic, hydration, and specific chemical bonding are unlikely to contribute to any significant extent to the interaction energy between the probe and the surface. The pH-dependent conformation of MA molecules in the monolayer at the solid-liquid interface was studied by ellipsometry, neutron reflectometry, and with a quartz crystal microbalance. Monolayers prepared by the Langmuir–Blodgett method demonstrated a distinct pH-responsive behaviour, while monolayers prepared by the Langmuir–Schaefer method were less sensitive to pH variation. It was found that the attachment of water molecules plays a vital role in determining the conformation of the MA monolayers.

Keywords: Mycolic acid; Monolayer; Neutron reflectometry; Force spectroscopy; DLVO; Quartz crystal microbalance

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BBA - Biomembranes (v.1798, #9)