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BBA - Biomembranes (v.1808, #12)

Editorial Board (pp. i).

Distribution and shedding of the membrane phosphatidylserine during maturation and aging of erythroid cells by Inna Freikman; Eitan Fibach (pp. 2773-2780).

Maturation and aging of erythroid cells are accompanied by extensive remodeling of the membrane and a marked decrease in cell size, processes that are mediated by externalization and shedding of phosphatidylserine (PS). In the present study, we investigated the redistribution of PS in the plasma membrane of erythroid precursors during their maturation and of mature RBCs during senescence, and the involvement of changes in calcium (Ca)-flux in these processes. Maturation was studied by analyzing normal human bone marrow cells as well as cultured human normal erythroid precursors induced by erythropoietin and murine erythroleukemia cells induced by hexamethylene-bisacetamide. Senescence was studied in normal human peripheral RBCs following density fractionation. PS and Ca were determined by flow cytometry using annexin-V and Flu-3, respectively. The outer, inner and shed PS were quantified by a novel two-step binding inhibitory assay. The results indicate a bi-phasic modulation of intracellular Ca and PS externalization/shedding; both of which decreased during maturation and increased during aging. The role of intracellular Ca in PS externalization/shedding was demonstrated by modulating intracellular Ca: Ca was decreased by incubating the cells with an ion chelator (EDTA) or with decreasing concentrations of Ca, whereas treatment with the ionophore A23187 elevated intracellular Ca. The results showed that low Ca resulted in decreased outer and shed PS, whereas high Ca had the opposite effect. The results suggest that PS externalization and shedding are mediated by increased cellular Ca-flux, and that they play an important role in erythroid maturation and RBC senescence.► We studied the phosphatidylserine (PS) distribution during RBC maturation and aging.► Blood, bone marrow and cultured human and murine erythroid cells were studied. ► PS externalization/shedding decreased during maturation but increased during aging. ► PS externalization and shedding were regulated by changes in the cellular Ca flux. ► We suggest that this redistribution plays a role in RBC maturation and senescence.

Keywords: Abbreviations; Ca; calcium; HMBA; hexamethylene bisacetamide; HNE; human normal erythroid; GPA; glycophorin A; MFI; mean fluorescence intensity; MEL; murine erythroleukemia; PS; phosphatidylserine; PBS; phosphate-buffered-saline; RBC; red blood cell; SD; standard deviationErythroid cell; Phosphatidylserine; Calcium; Maturation; RBC senescence

New molecular rods — Characterization of their interaction with membranes by Jörg Nikolaus; Sylvia Czapla; Mollnitz Kristian Möllnitz; Hofer Chris T. Höfer; Andreas Herrmann; Pablo Wessig; Muller Peter Müller (pp. 2781-2788).

Molecular rods are synthetical molecules consisting of a hydrophobic backbone which are functionalized with varying terminal groups. Here, we report on the interaction of a recently described new class of molecular rods with lipid and biological membranes. In order to characterize this interaction, different fluorescently labeled rods were synthesized allowing for the application of fluorescence spectroscopy and microscopy based approaches. Our data show that the rods are incorporated into membranes with a perpendicular orientation to the membrane surface and enrich preferentially in liquid-disordered lipid domains. These characteristics underline that rods can be applied as stable membrane-associated anchors for functionalizing membrane surfaces.Display Omitted► Fluorescent hydrophobic rods incorporate into lipid and biological membranes. ► Rods accumulate in liquid disordered domains. ► Rods embed into the bilayer perpendicular to the membrane surface. ► Rods do not disturb membrane structure and dynamics.

Keywords: Abbreviations; Chol; Cholesterol; DOPC; 1,2-Dioleoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-Dipalmitoyl-; sn; -glycero-3-phosphocholine; GUVs; Giant unilamellar vesicle(s); HBS; HEPES buffered salt solution; LUVs; Large unilamellar vesicle(s); MLVs; Multilamellar vesicles; NBD; 7-nitro-2-1,3-benzoxadiazol-4-yl; NBD-PC; 1-Palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-caproyl]-; sn; -glycero-3-phosphocholine; NR; NBD labeled rod; N-Rh-DOPE; 1,2-Dioleoyl-; sn; -glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); OSK; Oligospiroketal; PC; Phosphocholine; POPC; 1-Palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PR; Pyrene labeled rod; RBC; Red blood cell(s); RR; Rhodamine labeled rod; SL-POPC; 1-Palmitoyl-2-oleoyl-(16-doxyl)-; sn; -glycero-3-phosphocholine; SSM; N-Stearoyl-; d; -sphingomyelin; TCSPC; Time correlated single photon countingMolecular rod; Phospholipid; Lipid domain; Spiro compound

Electrophysiological properties of heteromeric TRPV4–C1 channels by Xin Ma; Bernd Nilius; Judy Wei-Yan Wong; Yu Huang; Xiaoqiang Yao (pp. 2789-2797).

We previously reported that TRPV4 and TRPC1 can co-assemble to form heteromeric TRPV4–C1 channels [12]. In the present study, we characterized some basic electrophysiological properties of heteromeric TRPV4–C1 channels. 4α-Phorbol 12,13-didecanoate (4α-PDD, a TRPV4 agonist) activated a single channel current in HEK293 cells co-expressing TRPV4 and TRPC1. The activity of the channels was abrogated by a TRPC1-targeting blocking antibody T1E3. Conductance of the channels was ~95pS for outward currents and ~83pS for inward currents. The channels with similar conductance were also recorded in cells expressing TRPV4–C1 concatamers, in which assembled channels were expected to be mostly 2V4:2C1. Fluorescence Resonance Energy Transfer (FRET) experiments confirmed the formation of a protein complex with 2V4:2C1 stoichiometry while suggesting an unlikeliness of 3V4:1C1 or 1V4:3C1 stoichiometry. Monovalent cation permeability profiles were compared between heteromeric TRPV4–C1 and homomeric TRPV4 channels. For heteromeric TRPV4–C1 channels, their permeation profile was found to fit to Eisenman sequence VI, indicative of a strong field strength cation binding site, whereas for homomeric TRPV4 channels, their permeation profile corresponded to Eisenman sequence IV for a weak field strength binding site. Compared to homomeric TRPV4 channels, heteromeric TRPV4–C1 channels were slightly more permeable to Ca²⁺ and had a reduced sensitivity to extracellular Ca²⁺ inhibition. In summary, we found that, when TRPV4 and TRPC1 were co-expressed in HEK293 cells, the predominate assembly type was 2V4:2C1. The heteromeric TRPV4–C1 channels display distinct electrophysiological properties different from those of homomeric TRPV4 channels.► The predominate assembly type of heteromeric TRPV4–C1 channels is 2V4:2C1. ► Properties of TRPV4–C1 channels were examined at single-channel and whole-cell level. ► Monovalent cation permeability profiles of TRPV4–C1 were fit to Eisenman sequence VI. ► Heteromeric TRPV4–C1 channels were slightly more permeable to Ca²⁺. ► TRPV4–C1 channels had a reduced sensitivity to extracellular Ca²⁺ inhibition.

Keywords: Abbreviations; [Ca; ²⁺; ]i; intracellular Ca; ²⁺; concentration; CFP; cyan fluorescent protein; YFP; yellow fluorescent protein; FRET; Fluorescence Resonance Energy Transfer; 4α-PDD; 4α-phorbol 12,13-didecanoate; TRP; transient receptor potentialTRPV4–C1; Heteromeric channel; Cation permeability profile

Lipophilic penetration enhancers and their impact to the bilayer structure of stratum corneum lipid model membranes: Neutron diffraction studies based on the example Oleic Acid by Tanja N. Engelbrecht; Annett Schroeter; Hauss Thomas Hauß; Reinhard H.H. Neubert (pp. 2798-2806).

The present study analyzes the effect of the lipophilic penetration enhancer oleic acid on the bilayer structure of stratum corneum (SC) lipid model membranes based on Ceramide AP by using the neutron diffraction technique. Our results indicate the formation of a single lamellar phase in the presence of oleic acid under the chosen experimental conditions; a separated fluid-like oleic acid-rich phase was not detected in the present study. By comparing the internal membrane structure received from Fourier synthesis with the model system lacking oleic acid, considerable structural changes in terms of impairment of the lamellar order were found after incorporation of the penetration enhancer into the bilayers. In addition, by using specifically deuterated oleic acid we were able to prove the integration of the enhancer molecules into the model bilayers and moreover, to determine the exact position of oleic acid inside the SC lipid model membrane. From the present results we conclude a strong perturbation of lamellar SC lipid arrangement due to the intercalated penetration enhancer which can account for the promoting effects on drug penetration across the SC known for oleic acid.► We study the impact of oleic acid to the assembly of stratum corneum model membranes. ► Deuterium-labeling is used to localize oleic acid inside the membrane. ► The presence of oleic acid is associated with a significant loss of lamellar order. ► Perturbation of lipid assembly is crucial for penetration enhancer activity of oleic acid.

Keywords: Abbreviations; SC; stratum corneum; CER; ceramide; OA; oleic acid; OA-D2; oleic acid-9,10-D2; CER[AP]; N-(α-hydroxyoctadecanoyl)-phytosphingosine; FFA; free fatty acid; PA; palmitic acid; CHOL; cholesterol; ChS; cholesterol sulfate; SLD; scattering length density; RH; relative humidityStratum corneum; Ceramide; Penetration enhancer; Neutron diffraction; Lipid model membrane

The over-expression of TRPC6 channels in HEK-293 cells favours the intracellular accumulation of zinc by Julien Gibon; Peng Tu; Sylvain Bohic; Pierre Richaud; Josiane Arnaud; Mike Zhu; Guylain Boulay; Alexandre Bouron (pp. 2807-2818).

TRPC6 are plasma membrane cation channels. By means of live-cell imaging and spectroscopic methods, we found that HEK cells expressing TRPC6 channels (HEK-TRPC6) are enriched in zinc and sulphur and have a reduced copper content when compared to HEK cells and HEK cells expressing TRPC3 channels (HEK-TRPC3). Hence, HEK-TRPC6 cells have larger pools of mobilizable Zn²⁺ and are more sensitive to an oxidative stress. Synchrotron X-ray fluorescence experiments showed a higher zinc content in the nuclear region indicating that the intracellular distribution of this metal was influenced by the over-expression of TRPC6 channels. Their properties were investigated with the diacylglycerol analogue SAG and the plant extract hyperforin. Electrophysiological recordings and imaging experiments with the fluorescent Zn²⁺ probe FluoZin-3 demonstrated that TRPC6 channels form Zn²⁺-conducting channels. In cortical neurons, hyperforin-sensitive channels co-exist with voltage-gated channels, AMPA and NMDA receptors, which are known to transport Zn²⁺. The ability of these channels to regulate the size of the mobilizable pools of Zn²⁺ was compared. The data collected indicate that the entry of Zn²⁺ through TRPC6 channels can up-regulate the size of the DTDP-sensitive pool of Zn²⁺. By showing that TRPC6 channels constitute a Zn²⁺ entry pathway, our study suggests that they could play a role in zinc homeostasis.► TRPC6 proteins form cation channels found in many tissues and organs. ► The over-expression of TRPC6 channels causes an intracellular accumulation of Zinc. ► The entry of Zn²⁺ through TRPC6 regulates the size of internal pools of Zn²⁺. ► Our data unveil an unanticipated role of TRPC6 in cellular zinc homeostasis.

Keywords: Brain; Oxidative stress; TRPC channel; Zinc; TRPC6

Fluorescence spectroscopy studies of HEK293 cells expressing DOR-G_i1α fusion protein; the effect of cholesterol depletion by Brejchova Jana Brejchová; Sykora Jan Sýkora; Dlouha Kateřina Dlouhá; Roubalova Lenka Roubalová; Ostasov Pavel Ostašov; Vosahlikova Miroslava Vošahlíková; Martin Hof; Petr Svoboda (pp. 2819-2829).

Biophysical studies of fluorescence anisotropy of DPH and Laurdan generalized polarization were performed in plasma membranes (PM) isolated from control and cholesterol-depleted HEK293 cells stably expressing pertussis toxin (PTX)-insensitive DOR-G_i1α (Cys³⁵¹–Ile³⁵¹) fusion protein. PM isolated from control, PTX-untreated, cells were compared with PM isolated from PTX-treated cells. Results from both types of PM indicated that i) hydrophobic membrane interior was made more accessible to water molecules and more chaotically organized in cholesterol-depleted samples, ii) cholesterol depletion resulted in an overall increase in surface area of membrane, membrane fluidity, and mobility of its constituents.Analysis of DOR-G_i1α coupling in PTX-treated and PTX-untreated cells indicated that cholesterol depletion did not alter the agonist binding site of DOR (B_max and K_d) but the ability of DOR agonist DADLE to activate G proteins was markedly impaired. In PTX-untreated membranes, EC₅₀ for DADLE-stimulated [³⁵S]GTPγS binding was shifted by one order of magnitude to the right: from 4.3±1.2×10−⁹M to 2.2±1.3×10−⁸M in control and cholesterol-depleted membrane samples, respectively. In PTX-treated membranes, EC₅₀ was shifted from 4.5±1.1×10−⁹M to 2.8±1.4×10−⁸M.In summary, the perturbation of optimum PM organization by cholesterol depletion deteriorates functional coupling of DOR to covalently bound G_i1α as well as endogenously expressed PTX-sensitive G proteins of G_i/G_o family while receptor ligand binding site is unchanged. The biophysical state of hydrophobic plasma (cell) membrane interior should be regarded as regulatory factor of DOR-signaling cascade.► Effect of cholesterol on the organization of plasma membranes and G protein activation. ► Cholesterol depletion increases membrane fluidity and mobility of plasma membranes. ► Cholesterol depletion does not alter the agonist binding site of δ-opioid receptor (DOR). ► Cholesterol depletion decreases the ability of DOR agonist to activate G proteins. ► Indication that biophysical state of membrane is a regulatory factor of DOR-signaling cascade.

Keywords: Abbreviations; β-CDX; β-cyclodextrin; DADLE; D-Ala2-D-Leu5 enkephalin; DMEM; Dulbecco's modified Eagle's medium; DOR; δ-opioid receptor; DOR-G; _i; 1α cells; HEK293 cells stably expressing DOR-G; _i; 1α (Cys; ³⁵¹; –Ile; ³⁵¹; ) fusion protein; DPH; 1,6-diphenyl-1,3,5-hexatriene; DRMs; detergent-resistant membrane domains; GPCR; G protein-coupled receptor; G proteins; heterotrimeric guanine nucleotide-binding regulatory proteins; G; _i; /G; _o; G proteins inhibiting adenylyl cyclase activity in pertussis toxin-sensitive manner; HEK; human embryonic kidney; PBS; phosphate-buffered saline; PM; plasma (cell) membrane; PMSF; phenylmethylsulfonyl fluoride; PNS; post-nuclear fraction; PTX; pertussis toxin; TBS; Tris-buffered saline; TRH; thyrotropin-releasing hormone; TRH-R; thyrotropin-releasing hormone receptor; TCSPC; Time Correlated Single Photon CountingPlasma membrane; Cholesterol depletion; Fluorescence spectroscopy studies; Hydrophobic membrane interior; δ-opioid receptor (DOR); G protein coupling; ±; PTX

Compatible solutes: Ectoine and hydroxyectoine improve functional nanostructures in artificial lung surfactants by Rakesh Kumar Harishchandra; Amit Kumar Sachan; Andreas Kerth; Georg Lentzen; Thorsten Neuhaus; Hans-Joachim Galla (pp. 2830-2840).

Ectoine and hydroxyectoine belong to the family of compatible solutes and are among the most abundant osmolytes in nature. These compatible solutes protect biomolecules from extreme conditions and maintain their native function. In the present study, we have investigated the effect of ectoine and hydroxyectoine on the domain structures of artificial lung surfactant films consisting of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and the lung surfactant specific surfactant protein C (SP-C) in a molar ratio of 80:20:0.4. The pressure–area isotherms are found to be almost unchanged by both compatible solutes. The topology of the fluid domains shown by scanning force microscopy, which is thought to be responsible for the biophysical behavior under compression, however, is modified giving rise to the assumption that ectoine and hydroxyectoine are favorable for a proper lung surfactant function. This is further evidenced by the analysis of the insertion kinetics of lipid vesicles into the lipid–peptide monolayer, which is clearly enhanced in the presence of both compatible solutes. Thus, we could show that ectoine and hydroxyectoine enhance the function of lung surfactant in a simple model system, which might provide an additional rationale to inhalative therapy.►Ectoine and hydroxyectoine are produced by bacteria and have water binding property. ►They act on line tension of lipid–protein membranes and alter the domain organization. ►They enhance the protrusion formation in artificial LS favoring the breathing process. ►Vesicle insertion into the surfactant monolayer is accelerated by compatible solutes.

Keywords: Abbreviations; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DPPG; 1,2-dipalmitoyl-; sn-; glycero-3-phosphoglycerol; AFM; atomic force microscopy; LB; Langmuir–Blodgett; LC; liquid condensed; LE; liquid expanded; LUV; large unilamellar vesicle; IRRAS; Infrared Reflection Absorption SpectroscopyLangmuir film balance; Domain structures; Surfactant protein C; Height profile; Vesicle insertion; Infrared Reflection Absorption Spectroscopy

Membrane properties of and cholesterol's interactions with a biologically relevant three-chain sphingomyelin: 3 O-palmitoyl- N-palmitoyl-D -erythro-sphingomyelin by Christian Sergelius; J. Peter Slotte (pp. 2841-2848).

Sphingomyelins (SMs) are order-imposing phospholipids in cell membranes which interact favorably with cholesterol. The hydrophobic part of SM constitutes a long-chain base with an amide-linked acyl chain, whereas the polar head group is phosphocholine. The long-chain base has a free hydroxyl group in position 3, which is an important donor/acceptor in hydrogen bonding. In newborn mammals, a SM in which a palmitic acid is esterified to the 3-OH has been reported. We have synthesized this SM analog (3 O-P-PSM) and studied its properties in bilayer membranes, and also determined its interactions with cholesterol. Fully hydrated 3 O-P-PSM bilayers underwent a gel-to-liquid crystalline phase transition at 55.5°C (ΔH 8kcal/mol), which is about 15°C higher than the phase transition temperature of PSM. The 3 O-P-PSM displayed rather poor miscibility with PSM in mixed bilayers, suggesting that the third acyl chain interfered significantly with lateral interactions. Bilayers made from 3 O-P-PSM were much more resistant to detergent-induced solubilization than bilayers made from PSM. In binary bilayers, cholesterol was able to destabilize the gel phase, and order the fluid phase of 3 O-P-PSM, in a concentration-dependent manner. Cholesterol was also able to form sterol-enriched ordered domains with 3 O-P-PSM in fluid POPC bilayers. The interaction between cholesterol and 3 O-P-PSM was not, however, as favorable as the interaction between cholesterol and PSM. It is unclear what physiological role 3 O-P-PSM could play in newborn mammalian membranes. However, it is clear that 3O-P-PSM will form more highly ordered domains than PSM while still having a limited ability to interact with cholesterol.► Newborn mammals have saturated 3 O-acyl sphingomyelins in their plasma. ► We made 3 O-palmitoyl palmitoylsphingomyelin and studied its membrane properties. ► The SM analog formed highly ordered domains in fluid bilayers. ► Cholesterol was able to interact with the SM analog, and formed liquid-ordered phase. ► We speculate that 3 O-acyl SM is important in membranes as a super-ordered lipid.

Keywords: Abbreviations; 3; O; -P-PSM; 3; O; -palmitoyl-; N; -palmitoyl-D; -erythro; -sphingomyelin; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; CTL; cholesta-5,7 (11)-trien-3-beta-ol; DMAP; 4-dimethylaminopyridine; DPH; 1,6-diphenyl-1,3,5-hexatriene; DSC; differential scanning calorimetry; mβCD; methylβcyclodextrin; PC; phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; N; -palmitoyl-D; -erythro; -sphingomyelin; SM; sphingomyelin; tPA; trans; parinaric acid; TX100; Triton X-100Lateral domain; Fluorescence spectroscopy; Differential scanning calorimetry; Cholestatrienol; Laurdan; Detergent solubility

Correlation between the ripple phase and stripe domains in membranes by Uffe Bernchou; Henrik Midtiby; John Hjort Ipsen; Adam Cohen Simonsen (pp. 2849-2858).

We investigate the relationship between stripe domains and the ripple phase in membranes. These have previously been observed separately without being linked explicitly. Past results have demonstrated that solid and ripple phases exhibit rich textural patterns related to the orientational order of tilted lipids and the orientation of ripple corrugations. Here we reveal a highly complex network pattern of ripple and solid domains in DLPC, DPPC bilayers with structures covering length scales from 10nm to 100μm. Using spincoated double supported membranes we investigate domains by correlated AFM and fluorescence microscopy. Cooling experiments demonstrate the mode of nucleation and growth of stripe domains enriched in the fluorescent probe. Concurrent AFM imaging reveals that these stripe domains have a one-to-one correspondence with a rippled morphology running parallel to the stripe direction. Both thin and thick stripe domains are observed having ripple periods of 13.5±0.2nm and 27.4±0.6nm respectively. These are equivalent to previously observed asymmetric/equilibrium and symmetric/metastable ripple phases, respectively. Thin stripes grow from small solid domains and grow predominantly in length with a speed of ~3 times that of the thick stripes. Thick stripes grow by templating on the sides of thinner stripes or can emerge directly from the fluid phase. Bending and branching angles of stripes are in accordance with an underlying six fold lattice. We discuss mechanisms for the nucleation and growth of ripples and discuss a generic phase diagram that may partly rationalize the coexistence of metastable and stable phases.► We study ripple and stripe domains in lipid bilayer membranes. ► We use combined AFM and fluorescence microscopy. ► Stripe domains consist of ripples running parallel to the stripes. ► Thick and thin stripes correspond to long and short ripple periods respectively. ► Thin stripes nucleate on gel domains.

Keywords: Supported lipid bilayer; Ripple phase; Stripe phase; Solid phase; AFM; Fluorescence microscopy

Expression, purification, electron microscopy, N-glycosylation mutagenesis and molecular modeling of human P2X4 and Dictyostelium discoideum P2XA by Maria Valente; Summer J. Watterson; Mark D. Parker; Robert C. Ford; Mark T. Young (pp. 2859-2866).

The recent publication of the apo-, closed-state 3D crystal structure of zebrafish (zf) P2X4.1 has not only revolutionized the P2X research field, but also highlighted the need for further crystal structures, of receptors in different activation states, so that we can gain a complete molecular understanding of ion channel function. zfP2X4.1 was selected as a 3D-crystallization candidate because of its ability to form stable trimers in detergent solution, and purified from over-expression in baculovirus-infected Spodoptera frugiperda (Sf9) insect cells. In this work, we have used a similar approach to express both human P2X4 (hP2X4) and Dictyostelium discoideum P2XA (DdP2XA) in Sf9 cells. Although hP2X4 did not form stable trimers in detergent solution, both receptors bound to ATP-coupled resins, indicating that their extracellular domains were folded correctly. DdP2XA formed strong trimers in detergent solution, and we were able to selectively purify trimers using preparative electrophoresis, and build a 21Å-resolution 3D structure using transmission electron microscopy and single particle analysis. Although the structure of DdP2XA possessed similar dimensions to those of the previously determined low-resolution hP2X4 structure and the zfP2X4.1 crystal structure, N-glycosylation mutagenesis and molecular modeling indicated differences between N-glycan usage and predicted accessibility in models of DdP2XA based on the zfP2X4.1 crystal structure. Our data demonstrate that DdP2XA expressed in insect cells retains ATP-binding capacity after detergent solubilization, is an ideal candidate for structural study, and possesses a significantly different 3D structure to that of both hP2X4 and zfP2X4.1.► We expressed human P2X4 and Dictyostelium discoideum (Dd) P2XA in Sf9 insect cells. ► Both receptors were capable of binding to ATP-coupled sepharose beads. ► Preparative electrophoresis was used to purify DdP2XA trimers. ► We determined a 21Å-resolution structure of DdP2XA using single particle analysis. ► DdP2XA cannot be modeled accurately using the zebrafish P2X4 crystal structure.

Keywords: Abbreviations; β-OG; n-octyl-β-; d; -glucoside; C; ₁₂; E; ₈; octaethylene glycol monododecyl ether; DDM; n-dodecyl-β-; d; -maltoside; FC-12; n-dodecylphosphocholine; LDAO; n-dodecyl-N,N-dimethylamine-N-oxide; PBS; phosphate-buffered saline; SDS; sodium dodecyl sulfate; Sf9 cells; Spodoptera frugiperda; cell-line derived from pupal ovaries; TEM; transmission electron microscopy, TX-100, Triton-X-100; zf; zebrafishP2X; Sf9; TEM; Single particle analysis; N-glycosylation; Modeller

Competing interactions for antimicrobial selectivity based on charge complementarity by Carola I.E. von Deuster; Volker Knecht (pp. 2867-2876).

Antimicrobial peptides (AMPs) are an evolutionary conserved component of the innate immune system and possible templates for the development of new antibiotics. An important property of antimicrobial peptides is their ability to discriminate bacterial from eucaryotic cells which is attributed to the difference in lipid composition of the outer leaflet of the plasma membrane between the two types of cells. Whereas eucaryotic cells usually expose zwitterionic lipids, procaryotic cells expose also anionic lipids which bind the cationic antimicrobial peptides electrostatically. An example is the antimicrobial peptide NK-2 which is highly cationic and favors binding to anionic membranes. In the present study, the difference in binding affinity of NK-2 for palmitoyl-oleoyl-phosphatidyl-glycerol (POPG) and palmitoyl-oleoyl-phosphatidyl-choline (POPC) is studied using molecular dynamics simulations in conjunction with a coarse grained model and thermodynamic integration, by computing the change in free energy and its components upon the transfer of NK-2 from POPC to POPG. The transfer is indeed found to be highly favorable. Interestingly, the favorable contribution from the electrostatic interaction between the peptide and the anionic lipids is overcompensated by an unfavorable contribution from the change in lipid–cation interactions due to the release of counterions from the lipids. The increase in entropy due to the release of the cations is compensated by other entropic components. The largest favorable contribution arises from the solvation of the counterions. Overall the interaction between NK-2 and POPG is not determined by a single driving force but a subtle balance of competing interactions.Display Omitted► The selectivity of the antimicrobial peptide NK-2 to procaryotes is studied. ► MD simulations using thermodynamic integration prove stronger binding to procaryotes. ► Electrostatic attraction between peptide and anionic lipids is compensated. ► Release of counterions has major impact on binding affinity. ► Competing interactions are listed.

Keywords: Abbreviations; POPG; palmitoyl-oleoyl-phosphatidyl-glycerol; POPC; palmitoyl-oleoyl-phosphatidyl-choline; MD; molecular dynamics; AMPs; antimicrobial peptides; PG; phosphatidyl-glycerol; PE; phosphatidyl-ethanolamine; PC; phosphatidyl-choline; DPPC; diplamitoyl-PC; DPPG; diplamitoyl-PG; DPPE; diplamitoyl-PE; LJ; Lennard–Jones; LINCS; linear constraint solver for molecular simulations; NT; N-terminus; NC3; choline; PO; ₄; phosphateAntimicrobial peptide; MD simulation; Selectivity; Membrane; Thermodynamic integration

Self-assembled, cation-selective ion channels from an oligo(ethylene glycol) derivative of benzothiazole aniline by Panchika Prangkio; Divya K. Rao; Kevin D. Lance; Mark Rubinshtein; Jerry Yang; Michael Mayer (pp. 2877-2885).

This paper describes the spontaneous formation of well-defined pores in planar lipid bilayers from the self-assembly of a small synthetic molecule that contains a benzothiazole aniline (BTA) group attached to a tetra-ethylene glycol (EG₄) moiety. Macroscopic and single-channel current recordings suggest that these pores are formed by the assembly of four BTA-EG₄ monomers with an open pore diameter that appears similar to the one of gramicidin pores (~0.4nm). The single-channel conductance of these pores is modulated by the pH of the electrolyte and has a minimum at pH ~3. Self-assembled pores from BTA-EG₄ are selective for monovalent cations and have long open channel lifetimes on the order of seconds. BTA-EG₄ monomers in these pores appear to be arranged symmetrically across both leaflets of the bilayer, and spectroscopy studies suggest that the fluorescent BTA group is localized inside the lipid bilayers. In terms of biological activity, BTA-EG₄ molecules inhibited growth of gram-positive Bacillus subtilis bacteria (IC₅₀ ~50μM) and human neuroblastoma SH-SY5Y cells (IC₅₀ ~60μM), while they were not toxic to gram-negative Escherichia coli bacteria at a concentration up to 500μM. Based on these properties, this drug-like, synthetic, pore-forming molecule with a molecular weight below 500gmol−¹ might be appealing as a starting material for development of antibiotics or membrane-permeating moieties for drug delivery. From a biophysical point of view, long-lived, well-defined ion-selective pores from BTA-EG₄ molecules offer an example of a self-assembled synthetic supramolecule with biological function.Display Omitted► We present a simple and small molecule (BTA-EG₄), which forms remarkably well-defined ion channels in lipid bilayers. ► This small molecule is composed of a benzothiazole aniline (BTA) group attached to tetra(ethylene glycol). ► These pores are formed by the self-assembly of approximately four monomers of BTA-EG₄. ► The BTA-EG₄ pores are selective for monovalent cations in the following sequence: H⁺>>Cs⁺>K⁺>Na⁺>Li⁺. ► BTA-EG₄ exhibits antibiotic activity against gram-positive bacteria and cytotoxicity against human neuroblastoma cells.

Keywords: Abbreviations; BTA; benzothiazole aniline; EG; ₄; tetra-ethylene glycol; PEO; poly(ethylene oxide); OEG; oligo(ethylene glycol); DiPhyPC; 1,2-diphytanoyl-; sn; -glycero-3-phosphatidylcholine; DiPhyPG; 1,2-diphytanoyl-; sn; -glycero-3-phosphatidylglycerol; DPPE; 1,2-dipalmitoyl-; sn; -glycero-3-phosphatidylethanolamine; gA; gramicidin ASelf-assembled ion channel; Membrane permeable; Benzothiazole aniline; Oligo(ethylene glycol); Biosensors; Antibiotics

Effects of bile salts on glucosylceramide containing membranes by Josefin Halin; Peter Mattjus (pp. 2886-2893).

The glycolipid transfer protein (GLTP) is capable of transporting glycolipids from a donor membrane, through the aqueous environment, to an acceptor membrane. The GLTP mediated glycolipid transfer from sphingomyelin membranes is very slow. In contrast, the transfer is fast from membranes composed of phosphatidylcholine. The lateral glycolipid membrane organization is known to be driven by their tendency to mix non-randomly with different membrane lipids. Consequently, the properties of the membrane lipids surrounding the glycolipids play an important role in the ability of GLTP to bind and transfer its substrates. Since GLTP transfer of glycolipids is almost nonexistent from sphingomyelin membranes, we have used this exceptionality to investigate if membrane intercalators can alter the membrane packing and induce glycolipid transfer. We found that the bile salts cholate, deoxycholate, taurocholate and taurodeoxycholate, cause glucosylceramide to become transferrable by GLTP. Other compounds, such as single chain lipids, ceramide and nonionic surfactants, that have membrane-perturbing effects, did not affect the transfer capability of GLTP. We speculate that the strong hydrogen bonding network formed in the interfacial region of glycosphingolipid–sphingomyelin membranes is disrupted by the membrane partition of the bile salts causing the glycosphingolipid to become transferrable.► The glycolipid transfer protein can be used to probe changes in lipid packing. ► Bile salts are clearly capable of affecting glycolipid lateral organization. ► Glycolipids could on a membrane level regulate bile salt partition and uptake.

Keywords: Glycolipid; Glucosylceramide; Glycolipid transfer protein; Hydrogen bond; Lipid lateral packing; BODIPY-labeled lipid

The structure of complexes between phosphatidylethanolamine and glucosylceramide: A matrix for membrane rafts by Peter J. Quinn (pp. 2894-2904).

Interaction between membrane lipids creates lateral domains within which essential membrane processes like trans-membrane signaling, differentiation etc. take place. Attention has focused on liquid-ordered phases formed by sphingomyelin and cholesterol but formation of ordered domains on the cytoplasmic membrane surfaces has largely been neglected. Synchrotron X-ray powder diffraction methods were used to investigate the interaction between two components of the cytoplasmic leaflet of the plasma membrane, phosphatidylethanolamine and glucosylceramide. Multilamellar dispersions of binary mixtures of different molecular species of phosphatidylethanolamine and glucosylceramide were examined. Stoichiometric complexes are formed when the phosphatidylethanolamine has at least one unsaturated fatty acid. The stoichiometry of the complexes was 2.0 fluid phospholipids per glucosylceramide with C22/24 N-acyl chains and 1.8 with C-12 chains. Saturated molecular species of phosphatidylethanolamines were immiscible with glucosylceramide. The complexes formed with unsaturated phosphatidylethanolamines and glucosylceramide are stable above physiological temperatures. A putative role of these matrices in membrane rafts is considered.► Only unsaturated phosphatidylethanolamines and glucosylceramide form stoichiometric complexes. ► The stoichiometry is 2:1 phospholipid:glucosylceramide when the phospholipid is in the fluid phase and about 1:5 in gel phase. ► The complexes are stable at temperatures well above 37°C. ► Complexes form with glucosylceramide with both long (C-22–24) and short (C-12) N-acyl fatty acids.

Keywords: Abbreviations; PE; phosphatidylethanolamine; PC; phosphatidylcholine; GlcCer; glucosylceramide; SAXS; small-angle X-ray scattering; WAXS; wide-angle X-ray scattering; L; _β; lamellar gel phase; L; _α; lamellar liquid-crystal phaseMembrane lipid; Raft structure; Lipid complex; Bilayer lipid; Model membrane; X-ray scattering

Expression of salinarum halorhodopsin in Escherichia coli cells: Solubilization in the presence of retinal yields the natural state by Yasutaka Yamashita; Takashi Kikukawa; Takashi Tsukamoto; Masakatsu Kamiya; Tomoyasu Aizawa; Keiichi Kawano; Seiji Miyauchi; Naoki Kamo; Makoto Demura (pp. 2905-2912).

Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all- trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3–4mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes. ► Salinarum halorhodopsin was expressed as a colorless pigment in E. coli membrane. ► It became colored after the membrane solubilization in the presence of retinal. ► The purified HR showed almost the native behaviors and Cl^--pumping activity.

Keywords: Abbreviations; HsHR; halorhodopsin from; Halobacterium salinarum; HR; halorhodopsin; NpHR; HR from; Natronomonas pharaonis; BR; bacteriorhodopsin; NpSRII; sensory rhodopsin II from; Natronomonas pharaonis; IPTG; isopropyl-β-D-thiogalactopyranoside; DDM; n-dodecyl β-D-maltopyranoside; MES; 2-(N-morpholino)ethanesulfonic acid; CCCP; carbonyl cyanide m-chlorophenylhydrazoneArchaeal rhodopsin; Halorhodopsin; Light-driven chloride pump; Photocycle

The effect of anions on the human P2X7 receptor by Christoph Kubick; Günther Schmalzing; Fritz Markwardt (pp. 2913-2922).

P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration–response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na⁺-based media and that external anions allosterically affect ion channel opening in the fully ATP⁴-liganded P2X7R through an extracellular anion binding site.► Extracellular organic/inorganic anions increase/decrease P2X7 receptor activation. ► Extracellular anions modify gating of P2X7 receptors via an allosteric binding site. ► Anions change open probability of P2X7R channels by affecting their shut times. ► Anions do not change P2X7R pore properties. ►The P2X7R is not anion permeable.

Keywords: Abbreviations; ATP; ^4-; free form of ATP, not bound to cations; hP2X7R; human purinergic P2X7 receptorP2 purinergic receptor; P2X receptor; P2X7 receptor; Patch clamp; Anion; Single channel

Membrane oligo- and polysialic acids by Teresa Janas; Tadeusz Janas (pp. 2923-2932).

Polysialic acid (polySia) and oligosialic acid (oligoSia) chains are linear polysaccharides composed of sialic acid monomers. The majority of biological poly/oligoSia chains are bound to membranes. There is a large diversity of membrane poly/oligoSia in terms of chain length, occurrence, biological function, and the mode of membrane attachment. Poly/oligoSia can be anchored to a membrane via a phospholipid (polySia in bacteria), a glycosphingolipid (oligoSia in gangliosides), an integral membrane glycoprotein, or a glycoprotein attached to a membrane via glycosylphosphatidylinositol. In eukaryotic cells, the attachment of a poly/oligoSia chain to the membrane anchor is usually through α-2,3-glycosidic linkage to a galactose. In prokaryotic cells this attachment is proposed to occur through glycosidic linkage to the phosphate group of a phospholipid. Both long polySia chains attached to membrane proteins and short oligoSia attached to glycosphingolipids or membrane proteins are frequently found in neural membranes. In humans, poly/oligoSia is involved in development and plasticity of the brain, pathophysiology of schizophrenic brains, cancer metastasis, neuroinvasive potential of pathogenic bacterial strains, and the immune response. Biological roles of poly/oligoSia are based on its ability to modulate repulsive and attractive interactions between two molecules, and its ability to modulate membrane surface charge density, pH at the membrane surface, and membrane potentials.► The majority of polySia is membrane-bound. ► There is a large diversity of membrane polySia in relation to the mode of the membrane attachment. ► PolySia can modulate membrane surface charge density, pH at the membrane surface and the membrane potentials. ► Mechanisms of polySia biological functions are based on intermolecular and membrane interactions.

Keywords: Polysialic acid; NCAM; Capsular polysaccharide; Ganglioside; Membrane potential; Surface pH

Bi-stability, hysteresis, and memory of voltage-gated lysenin channels by Daniel Fologea; Eric Krueger; Yuriy I. Mazur; Christine Stith; Yui Okuyama; Ralph Henry; Greg J. Salamo (pp. 2933-2939).

Lysenin, a 297 amino acid pore-forming protein extracted from the coelomic fluid of the earthworm E. foetida, inserts constitutively open large conductance channels in natural and artificial lipid membranes containing sphingomyelin. The inserted channels show voltage regulation and slowly close at positive applied voltages. We report on the consequences of slow voltage-induced gating of lysenin channels inserted into a planar Bilayer Lipid Membrane (BLM), and demonstrate that these pore-forming proteins constitute memory elements that manifest gating bi-stability in response to variable external voltages. The hysteresis in macroscopic currents dynamically changes when the time scale of the voltage variation is smaller or comparable to the characteristic conformational equilibration time, and unexpectedly persists for extremely slow-changing external voltage stimuli. The assay performed on a single lysenin channel reveals that hysteresis is a fundamental feature of the individual channel unit and an intrinsic component of the gating mechanism. The investigation conducted at different temperatures reveals a thermally stable reopening process, suggesting that major changes in the energy landscape and kinetics diagram accompany the conformational transitions of the channels. Our work offers new insights on the dynamics of pore-forming proteins and provides an understanding of how channel proteins may form an immediate record of the molecular history which then determines their future response to various stimuli. Such new functionalities may uncover a link between molecular events and macroscopic processing and transmission of information in cells, and may lead to applications such as high density biologically-compatible memories and learning networks.► The slow equilibration of lysenin channels yields hysteresis in conductance. ► The hysteresis and bi-stability persist at unusually large time scales. ► The hysteresis and bi-stability manifest at the single channel level. ► Temperature studies indicate a stable reopening pathway as the hysteresis source. ► Bi-stability in channels' functioning may explain the memory of individual cells.

Keywords: Abbreviations; BLM; Bilayer Lipid Membrane; I–V; current–voltage; P; _open; open probability; PFP; pore-forming protein; PFT; pore-forming toxin; V; _1/2; midway voltage of activationLysenin; Voltage-gated channels; Hysteresis; Memory; Pore-forming toxins

The correlation between multidomain enzymes and multiple activation mechanisms— The case of phospholipase Cβ and its membrane interactions by Harel Weinstein; Suzanne Scarlata (pp. 2940-2947).

Phospholipase Cβ2 (PLCβ2) is a large, multidomain enzyme that catalyzes the hydrolysis of the signaling lipid phosphoinositol 4,5 bisphosphate (PIP₂) to promote mitogenic and proliferative changes in the cell. PLCβ2 is activated by Gα and Gβγ subunits of heterotrimeric G proteins, as well as small G proteins and specific peptides. Activation depends on the nature of the membrane surface. Recent crystal structures suggest one model of activation involving the movement of a small autoinhibitory loop upon membrane binding of the enzyme. Additionally, solution studies indicate multiple levels of activation that involve changes in the membrane orientation as well as interdomain movement. Here, we review the wealth of biochemical studies of PLCβ2-G protein activation and propose a comprehensive model that accounts for both the crystallographic and solution results.► We review the domain organization of phospholipase Cb and relate this to modes of activation. ► We suggest how binding of PLCβ2 at the membrane surface through its PH domain induces activation. ► We discuss the role of the insertion loop as a model for activation. ► We present a comprehensive structure-based model of PLCβ2 activation states at the membrane. ► We discuss future challenges in the regulation of PLCβ enzymes in cellular settings.

Keywords: Phospholipase C; Protein domains; Protein activation; Protein–membrane interactions; Protein domains; Interdomain movement

Multi-Tox: Application of the ToxR-transcriptional reporter assay to the study of multi-pass protein transmembrane domain oligomerization by Catherine Joce; Alyssa A. Wiener; Hang Yin (pp. 2948-2953).

ToxR-based transcriptional reporter assays allow the strength of transmembrane helix interactions in biological membranes to be measured. Previously, these assays have only been used to study single-pass transmembrane systems. To facilitate investigation of polytopic transmembrane domain (TMD) oligomerization, we applied the ToxR methodology to the study of multi-pass TMD oligomerization to give ‘Multi-Tox’. Association propensities of the viral oncoprotein, latent membrane protein-1 (LMP-1), and the E. coli membrane-integral diacylglycerol kinase (DAGK) were studied by Multi-Tox, highlighting residues of particular mechanistic importance. Both homo- and hetero-oligomerizations were studied.► Multi-Tox studies multi-pass TMD oligomerization by ToxR reporter assay. ► Multi-Tox was applied to study homo- and hetero-oligomerizations. ► LMP-1 oligomerization is driven by a buried aspartic acid in helix five. ► K94 was identified as playing a key role in DAGK trimerization.

Keywords: Abbreviations; DAGK; diacylglycerol kinase; DN; dominant-negative; EBV; Epstein-Barr Virus; GpA; Glycophorin A; GPCR; G protein-coupled receptors; LMP-1; latent membrane protein-1; MBP; maltose binding protein; ONPG; ortho; -nitrophenyl β-galactoside; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; TMD; transmembrane domainMembrane proteins; Transcriptional reporter; ß-galactosidase; Latent membrane protein-1; Diacylglycerol kinase

ATP hydrolysis at one of the two sites in ABC transporters initiates transport related conformational transitions by Gergely Gyimesi; Srinivas Ramachandran; Pradeep Kota; Nikolay V. Dokholyan; Balázs Sarkadi; Hegedus Tamás Hegedűs (pp. 2954-2964).

ABC transporters play important roles in all types of organisms by participating in physiological and pathological processes. In order to modulate the function of ABC transporters, detailed knowledge regarding their structure and dynamics is necessary. Available structures of ABC proteins indicate three major conformations, a nucleotide-bound “bottom-closed” state with the two nucleotide binding domains (NBDs) tightly closed, and two nucleotide-free conformations, the “bottom-closed” and the “bottom-open”, which differ in the extent of separation of the NBDs. However, it remains a question how the widely open conformation should be interpreted, and whether hydrolysis at one of the sites can drive conformational transitions while the NBDs remain in contact. To extend our knowledge, we have investigated the dynamic properties of the Sav1866 transporter using molecular dynamics (MD) simulations. We demonstrate that the replacement of one ATP by ADP alters the correlated motion patterns of the NBDs and the transmembrane domains (TMD). The results suggest that the hydrolysis of a single nucleotide could lead to extracellular closure, driving the transport cycle. Essential dynamics analysis of simulations suggests that single nucleotide hydrolysis can drive the system toward a “bottom-closed” apo conformation similar to that observed in the structure of the MsbA transporter. We also found significant structural instability of the “bottom-open” form of the transporters in simulations. Our results suggest that ATP hydrolysis at one of the sites promotes transport related conformational changes leading to the “bottom-closed” apo conformation, which could thus be physiologically more relevant for describing the structure of the apo state.► We perform molecular dynamics simulations with two conformations of ABC transporters. ► Dynamics of the ATP/ATP and ATP/ADP bound outward-facing conformations differs. ► Outward-facing conformation can switch to MsbA-like bottom-closed apo conformation. ► The inward-facing structure may be a short-living transient conformation.

Keywords: Abbreviations; ABC; ATP Binding Cassette; NBD; nucleotide binding domain; TMD; transmembrane domain; ED; essential dynamics; ASA; accessible surface areaABC transporter; Molecular dynamic; Membrane bilayer; Multi-drug resistance; Constant contact model

Photo-dynamic induction of oxidative stress within cholesterol-containing membranes: Shape transitions and permeabilization by Rachid Kerdous; Julien Heuvingh; Stéphanie Bonneau (pp. 2965-2972).

Photochemical internalization is a drug delivery technology employing a photo-destabilization of the endosomes and the photo-controlled release of endocyted macromolecules into the cytosol. This effect is based on the ability of some photosensitizers to interact with endosomal membranes and to photo-induce damages leading to its breakdown. The permeabilization efficiency is not quantitatively related to the importance of the damages, but to their asymmetric repartition within the leaflets. Using unilamellar vesicles and a chlorin, we studied the effect of the membrane's cholesterol content on its photo-permeabilization. First, the affinity of the chlorin for membranes was studied. Then, we asymmetrically oxidized the membranes. For DOPC/CHOL GUVs, we observed different shape transitions, in accordance with an increase followed by a decrease of the membrane effective curvature. These modifications are delayed by the cholesterol. Finally, the photo-permeabilization of GUVs occurs, corresponding to a pore formation due to the membrane tension, resulting from vesicles buddings. Cholesterol-rich GUVs permeabilization occurs after a lag, and is less important. These results are interpreted regarding both (i) the cholesterol-induced tightening of the lipids, its consequences on physical parameters of the membrane and on oxidation rate and (ii) the suggested ability of cholesterol to flip rapidly and then to relax the differential density-based stress accumulated during membrane bending.Display Omitted► The cholesterol content of membranes influences their photo-induced permeabilization. ► Permeabilization efficiency is decreased by cholesterol-induced stress relaxation. ► We report a direct observation of the dynamics response of biomimetic vesicles. ► We propose the first quantitative analysis and physico-chemical interpretation. ► The role of cholesterol in membrane dynamics and permeabilization is clarified.

Keywords: Photosensitizer; Cholesterol; Curvature; Permeabilization; Photochemical Internalization; Liposome

The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ by Ophélie Fadel; Karim El Kirat; Sandrine Morandat (pp. 2973-2980).

Exogenous molecules from dietary sources such as polyphenols are very efficient in preventing the alteration of lipid membranes by oxidative stress. Among the polyphenols, we have chosen to study rosmarinic acid (RA). We investigated the efficiency of RA in preventing lipid peroxidation and in interacting with lipids. We used liposomes of 1,2-dilinoleoyl- sn-glycero-3-phosphocholine (DLPC) to show that RA was an efficient antioxidant. By HPLC, we determined that the maximum amount of RA associated with the lipids was ~1 mol%. Moreover, by using Langmuir monolayers, we evidenced that cholesterol decreases the penetration of RA. The investigation of transferred lipid/RA monolayers by atomic force microscopy revealed that 1 mol% of RA in the membrane was not sufficient to alter the membrane structure at the nanoscale. By fluorescence, we observed no significant modification of membrane permeability and fluidity caused by the interaction with RA. We also deduced that RA molecules were mainly located among the polar headgroups of the lipids. Finally, we prepared DLPC/RA vesicles to evidence for the first time that up to 1 mol% of RA inserts spontaneously in the membrane, which is high enough to fully prevent lipid peroxidation without any noticeable alteration of the membrane structure due to RA insertion.► Rosmarinic acid protects lipids efficiently against hydrophilic radicals. ► 1 mol% of rosmarinic acid inserts spontaneously into preformed membranes. ► Rosmarinic acid does not alter the membrane structure and properties. ► Rosmarinic acid resides preferentially among the polar headgroups of lipids. ► 1 mol% of membrane-inserted rosmarinic acid fully inhibits lipid peroxidation in situ.

Keywords: Biomimetic membrane; Oxidative stress; Rosmarinic acid; Antioxidant; Polyphenol; Lipid peroxidation

Protein palmitoylation and subcellular trafficking by Clara Aicart-Ramos; Ruth Ana Valero; Ignacio Rodriguez-Crespo (pp. 2981-2994).

Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16-carbon fatty acid palmitate, is the most common acylation of proteins in eukaryotic cells. This post-translational modification provides an important mechanism for regulating protein subcellular localization, stability, trafficking, translocation to lipid rafts, aggregation, interaction with effectors and other aspects of protein function. In addition, N-terminal myristoylation and C-terminal prenylation, two well-studied post-translational modifications, frequently precede protein S-palmitoylation at a nearby spot of the polypeptide chain. Whereas N-myristoylation and prenylation are considered essentially irreversible attachments, S-palmitoylation is a tightly regulated, reversible modification. In addition, the unique reversibility of protein palmitoylation also allows proteins to rapidly shuttle between intracellular membrane compartments in a process controlled, in some cases, by the DHHC family of palmitoyl transferases. Recent cotransfection experiments using the DHHC family of protein palmitoyl transferases as well as RNA interference results have revealed that these enzymes, frequently localized to the Golgi apparatus, tightly control subcellular trafficking of acylated proteins. In this article we will give an overview of how protein palmitoylation regulates protein trafficking and subcellular localization.► We describe herein the palmitoylation and trafficking of every small GTPase of the Ras and Rho families. ► Golgi-localized palmitoyl transferases of the DHHC family regulate palmitoylation cycles. ► Palmitoylation of eNOS and iNOS regulates their subcellular traffic. ► Surfactant protein C requires palmitoylation for activity but not for sorting.

Keywords: Acylation; Palmitoylation; Isoprenylation; Myristoylation; Subcellular traffic; Raft/caveola

Thermal, dynamic and structural properties of drug AT₁ antagonist olmesartan in lipid bilayers by Dimitrios Ntountaniotis; Gregor Mali; Simona Golic Grdadolnik; Halabalaki Maria; Alexios-Leandros Skaltsounis; Constantinos Potamitis; Eleni Siapi; Petros Chatzigeorgiou; Michael Rappolt; Thomas Mavromoustakos (pp. 2995-3006).

It is proposed that AT₁ antagonists (ARBs) exert their biological action by inserting into the lipid membrane and then diffuse to the active site of AT₁ receptor. Thus, lipid bilayers are expected to be actively involved and play a critical role in drug action. For this reason, the thermal, dynamic and structural effects of olmesartan alone and together with cholesterol were studied using differential scanning calorimetry (DSC),¹³C magic-angle spinning (MAS) nuclear magnetic resonance (NMR), cross-polarization (CP) MAS NMR, and Raman spectroscopy as well as small- and wide angle X-ray scattering (SAXS and WAXS) on dipalmitoyl-phosphatidylcholine (DPPC) multilamellar vesicles.¹³C CP/MAS spectra provided direct evidence for the incorporation of olmesartan and cholesterol in lipid bilayers. Raman and X-ray data revealed how both molecules modify the bilayer's properties. Olmesartan locates itself at the head-group region and upper segment of the lipid bilayers as¹³C CP/MAS spectra show that its presence causes significant chemical shift changes mainly in the A ring of the steroidal part of cholesterol. The influence of olmesartan on DPPC/cholesterol bilayers is less pronounced. Although, olmesartan and cholesterol are residing at the same region of the lipid bilayers, due to their different sizes, display distinct impacts on the bilayer's properties. Cholesterol broadens significantly the main transition, abolishes the pre-transition, and decreases the membrane fluidity above the main transition. Olmesartan is the only so far studied ARB that increases the gauche: trans ratio in the liquid crystalline phase. These significant differences of olmesartan may in part explain its distinct pharmacological profile.► Role of olmesartan/lipid bilayers interactions in AT₁ antagonism. ► Comparison of olmesartan properties in lipid bilayers with the important biological compound cholesterol. ► Topographical position of olmesartan in lipid bilayers.

Keywords: Olmesartan; Dipalmitoyl-phosphatidylcholine bilayer; Small-angle and wide angle X-ray scattering; Raman spectroscopy; Differential scanning calorimetry; Solid state NMR spectroscopy

Enforcing the positive charge of N-termini enhances membrane interaction and antitumor activity of bovine seminal ribonuclease by Gerardino D'Errico; Carmine Ercole; Marisa Lista; Elio Pizzo; Annarita Falanga; Stefania Galdiero; Roberta Spadaccini; Delia Picone (pp. 3007-3015).

Binding to cell membrane, followed by translocation into the cytosol and RNA degradation, is a necessary requirement to convert a ribonuclease into a cytotoxin for malignant tumor cells. In this paper, we investigate the membrane binding attitude of bovine seminal ribonuclease (BS-RNase) and its variant G38K-BS-RNase, bearing an enforced cluster of positive charges at the N-termini surface. By using a combination of biophysical techniques, including CD, SPR and ESR, we find for the two proteins a common, two-step mechanism of interaction with synthetic liposomes, an initial binding to the bilayer surface, driven by electrostatic interactions, followed by a shallow penetration in the lipid core. Protein binding effectively perturbs lipid packing and dynamics. Remarkably, the higher G38K-BS-RNase membrane interacting capability well correlates with its increased cytotoxicity for tumor cells. Overall, these studies shed light on the mechanism of membrane binding and perturbation, proving definitely the importance of electrostatic interactions in the cytotoxic activity of BS-RNase, and provide a rational basis to design proteins with anticancer potential.► Ribonucleases can be cytotoxic for tumor cells. ► Ribonuclease cytotoxicity depends on membrane interaction. ► The ribonuclease-membrane interaction is a two-step process. ► The G38K variant of bovine seminal-ribonuclease has a stronger membrane interaction. ► Membrane interaction capability of ribonucleases correlates with their cytotoxicity.

Keywords: Abbreviations; CD; circular dichroism; DLPG; dilauroyl phosphatidylglycerol; DMEM; Dulbecco's Modified Eagle's medium; DMPG; dimyristoyl phosphatidylglycerol; EIE; electrostatic interaction energy; ESR; electron spin resonance; LUVs; large unilamellar vesicles; MLVs; multilamellar vesicles; MTT; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; n; -PCSL; spin-labeled phosphatidylcholines with the nitroxide group at different positions; n; , in the; sn; -2 acyl chain; PBS; phosphate buffered saline; SPR; surface plasmon resonance; SUVs; small unilamellar vesicles; T; _m; melting temperatureMembrane interaction; Cytotoxic ribonuclease; ESR; SPR

Differential expression of human riboflavin transporters -1, -2, and -3 in polarized epithelia: A key role for hRFT-2 in intestinal riboflavin uptake by Veedamali S. Subramanian; Sandeep B. Subramanya; Laramie Rapp; Jonathan S. Marchant; Thomas Y. Ma; Hamid M. Said (pp. 3016-3021).

Transport of riboflavin (RF) across both the brush border membrane (BBM) and basolateral membrane (BLM) of the polarized enterocyte occurs via specific carrier-mediated mechanisms. Although, three human riboflavin transporters (hRFTs), i.e., hRFT-1, hRFT-2 and hRFT-3 are expressed in the intestine, little is known about the cell surface domain(s) at which these specific hRFTs are expressed. Here, we used live cell confocal imaging of intestinal epithelial Caco-2 and renal MDCK cells to show that the hRFT-1 is mainly expressed at the BLM, hRFT-2 is exclusively expressed at the apical membrane, while hRFT-3 is mostly localized inside intracellular vesicular structures (with some expression at the BLM). Further the level of hRFT-2 mRNA expression in Caco-2 cells and in native human intestine is significantly higher than that of hRFT-1 and -3; hRFT-2 was also more efficient in transporting³H-RF than hRFT-1 and -3. These findings implied an important role for hRFT-2 in intestinal RF uptake, a conclusion that was further supported by findings of hRFT-2 gene-specific siRNA knockdown investigation. These results show that members of the hRFT family are differentially expressed in polarized epithelia, and that the apically expressed hRFT-2 plays a key role in intestinal RF accumulation.► This study provides evidence for the importance of hRFT-2 in normal intestinal riboflavin absorption. ► The study also identifies which membrane domain of polarized epithelia the hRFT-1, -2 and -3 transporters are expressed. ► hRFT-2 is the most active and predominantly expressed RFT in normal intestine.

Keywords: Abbreviations; LAMP1; lysosome associated membrane protein1Transport; Intestine; Epithelia; Polarity; Targeting

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BBA - Biomembranes (v.1808, #12)