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BBA - Biomembranes (v.1788, #6)

Editorial Board (pp. i).

Aquaporins are multifunctional water and solute transporters highly divergent in living organisms by D. Gomes; A. Agasse ⁎; P. Thiébaud; S. Delrot; H. Gerós; F. Chaumont (pp. 1213-1228).

Aquaporins (AQPs) are ubiquitous membrane proteins whose identification, pioneered by Peter Agre's team in the early nineties, provided a molecular basis for transmembrane water transport, which was previously thought to occur only by free diffusion. AQPs are members of the Major Intrinsic Protein (MIP) family and often referred to as water channels. In mammals and plants they are present in almost all organs and tissues and their function is mostly associated to water molecule movement. However, recent studies have pointed out a wider range of substrates for these proteins as well as complex regulation levels and pathways. Although their relative abundance in plants and mammals makes it difficult to investigate the role of a particular AQP, the use of knock-out and mutagenesis techniques is now bringing important clues regarding the direct implication of specific AQPs in animal pathologies or plant deficiencies. The present paper gives an overview about AQP structure, function and regulation in a broad range of living organisms. Emphasis will be given on plant AQPs where the high number and diversity of these transport proteins, together with some emerging aspects of their functionalities, make them behave more like multifunctional, highly adapted channels rather than simple water pores.

Keywords: Aquaporin; Aquaglyceroporin; Aquaporin specificity; MIP; NPA; Plant aquaporin

Macroscopic domain formation during cooling in the platelet plasma membrane: An issue of low cholesterol content by Rachna Bali; Laura Savino; Diego A. Ramirez; Nelly M. Tsvetkova; Luis Bagatolli; Fern Tablin; John H. Crowe; Chad Leidy ⁎ (pp. 1229-1237).

There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of DiI18:0 at 24 °C, but not at 37 °C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets (∼15 mol%), appears to be crucial for the formation of large domains during cooling.

Keywords: Lipid membrane thermodynamics; Platelet; Lipid domain formation; Liquid-ordered phase; Cholesterol; FTIR; Lipid raft; Lipid platform

Genetic evidence for the requirement of the endocytic pathway in the uptake of coenzyme Q₆ in Saccharomyces cerevisiae by Sergio Padilla-López; María Jiménez-Hidalgo; Alejandro Martín-Montalvo; Catherine F. Clarke; Plácido Navas; Carlos Santos-Ocaña ⁎ (pp. 1238-1248).

Coenzyme Q is an isoprenylated benzoquinone lipid that functions in respiratory electron transport and as a lipid antioxidant. Dietary supplementation with Q is increasingly used as a therapeutic for treatment of mitochondrial and neurodegenerative diseases, yet little is known regarding the mechanism of its uptake. As opposed to other yeast backgrounds, EG103 strains are unable to import exogenous Q₆ to the mitochondria. Furthermore, the distribution of exogenous Q₆ among endomembranes suggests an impairment of the membrane traffic at the level of the endocytic pathway. This fact was confirmed after the detection of defects in the incorporation of FM4-64 marker and CPY delivery to the vacuole. A similar effect was demonstrated in double mutant strains in Q₆ synthesis and several steps of endocytic process; those cells are unable to uptake exogenous Q₆ to the mitochondria and restore the growth on non-fermentable carbon sources. Additional data about the positive effect of peptone presence for exogenous Q₆ uptake support the hypothesis that Q₆ is transported to mitochondria through an endocytic-based system.

Keywords: Abbreviations; DIGs; detergent-insoluble glycolipid-enriched complexes; CPY; carboxypeptidase Y; MAM; mitochondria-associated microsomes; Q; coenzyme Q or ubiquinone; RER; rough ER; SM; Sec1p-like/Munc-18; SNARE; soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TGN; trans-Golgi network; t-SNARE; target SNARECoenzyme Q; Ubiquinone; Endomembrane; Endocytosis; Q uptake; Mitochondrial disease

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis by Radovan Fišer; Ivo Konopásek ⁎ (pp. 1249-1254).

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage ( α=0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage ( α~15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K+E516K) exhibited much lower selectivity ( α∼6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.

Keywords: Abbreviations; ANTS; 8-Aminonaphthalene-1,3,6-trisulfonic acid; BLM; black lipid membranes; CyaA; adenylate cyclase toxin from; Bordetella pertussis; DPX; p; -xylene-bis-pyridinium bromide; FITC; fluorescein-5-isothiocyanate; HlyA; αhemolysin from; Escherichia coli; LUV; ₁₀₀; LUV; ₄₀₀; , LUV; ₁₀₀₀; , large unilamellar vesicles extruded through filters of 100, 400 and 1000 nm pore-size, respectively; PolyB; Polymyxin B from; Bacillus polymyxaCyaA; HlyA; ANTS; DPX; Fluorescence requenching method; Liposome disruption

A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na⁺/Ca²⁺ exchanger by Graciela Berberián; Mariana Bollo; Guillermo Montich; Gretel Roberts; Joseph A. DeGiorgis; Reinaldo DiPolo; Luis Beaugé ⁎ (pp. 1255-1262).

Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na⁺/Ca²⁺ exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far-UV circular dichroism and infrared spectra suggest a majority of β-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten β-sheets and two short α-helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.

Keywords: Na; ⁺; /Ca; ²⁺; counter-transport; Transport regulation; Lipid binding protein; Membrane transporter; Squid nerve

IR spectroscopy as a new tool for evidencing antitumor drug signatures by Régis Gasper; Janique Dewelle; Robert Kiss; Tatjana Mijatovic; Erik Goormaghtigh ⁎ (pp. 1263-1270).

There is a growing interest for screening antitumor drugs for their mechanism of action on cancer cells. Yet, screening for “modes of action” presents a technical challenge that is beyond the capability of conventional methods used in cellular or molecular biology. Several studies have highlighted the advantages of using infrared spectroscopy for diagnostic purposes at the clinical level for identifying cell types. In the present work, we suggest that the Fourier Transform Infrared (FTIR) spectrum of cells exposed to anti-cancer drugs could offer a unique opportunity to obtain a fingerprint of all molecules present in the cells and to observe, with a high sensitivity, the metabolic changes induced by potential anti-cancer drugs. Ouabain is one of the most potent cardenolides, which acts by inhibiting sodium pump activity. Cardenolides represent a class of compounds that are intended to soon enter clinical trials in oncology. In order to evaluate the potential of infrared spectroscopy to yield a signature for ouabain action on cancer cells, human prostate cancer PC-3 cells were treated with 36 nM ouabain, a sub-lethal concentration. Using ouabain as a model, we have thus demonstrated the possibility of using IR spectroscopy in the assessment of the global effects of an investigational compound on the cell constituents, thus contributing to setting up a new method for screening for novel anti-cancer agents in general, and potential anti-cancer cardenolides in particular. The most spectacular data obtained strongly suggest a modification in the nature of the cell lipids.

Keywords: Abbreviations; IR; infrared; FTIR; Fourier Transform infrared; CS; cardiotonic steroid; PCA; principal component analysis; PS; phosphatidylserine; GGR; global growth ratio; CCD; charge coupled deviceIR spectroscopy; Ouabain; Cancer; Sodium pump

Lipid bilayer disruption by oligomeric α-synuclein depends on bilayer charge and accessibility of the hydrophobic core by Bart D. van Rooijen; Mireille M.A.E. Claessens; Vinod Subramaniam (pp. 1271-1278).

Soluble oligomeric aggregates of α-synuclein have been implicated to play a central role in the pathogenesis of Parkinson's disease. Disruption and permeabilization of lipid bilayers by α-synuclein oligomers is postulated as a toxic mechanism, but the molecular details controlling the oligomer–membrane interaction are still unknown. Here we show that membrane disruption strongly depends on the accessibility of the hydrophobic membrane core and that charge interactions play an important but complex role. We systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomers using a dye release assay. Varying the lipid headgroup composition revealed that membrane disruption only occurs for negatively charged bilayers. Furthermore, the electrostatic repulsion between the negatively charged α-synuclein and the negative surface charge of the bilayer inhibits vesicle disruption at low ionic strength. The disruption of negatively charged vesicles further depends on lipid packing parameters. Bilayer composition changes that result in an increased lipid headgroup spacing make vesicles more prone to disruption, suggesting that the accessibility of the bilayer hydrocarbon core modulates oligomer–membrane interaction. These data shed important new insights into the driving forces governing the highly debated process of oligomer–membrane interactions.

Keywords: Amyloid; Synuclein; Lipid interaction; Pore; Membrane

Thermal and chemical unfolding and refolding of a eukaryotic sodium channel by Kalypso Charalambous; A.O. O'Reilly; Per A. Bullough; B.A. Wallace ⁎ (pp. 1279-1286).

Voltage-gated sodium channels are dynamic membrane proteins essential for signaling in nervous and muscular systems. They undergo substantial conformational changes associated with the closed, open and inactivated states. However, little information is available regarding their conformational stability. In this study circular dichroism spectroscopy was used to investigate the changes in secondary structure accompanying chemical and thermal denaturation of detergent-solubilised sodium channels isolated from Electrophorus electricus electroplax. The proteins appear to be remarkably resistant to either type of treatment, with “denatured” channels, retaining significant helical secondary structure even at 77 °C or in 10% SDS. Further retention of helical secondary structure at high temperature was observed in the presence of the channel-blocking tetrodotoxin. It was possible to refold the thermally-denatured (but not chemically-denatured) channels in vitro. The correctly refolded channels were capable of undergoing the toxin-induced conformational change indicative of ligand binding. In addition, flux measurements in liposomes showed that the thermally-denatured (but not chemically-denatured) proteins were able to re-adopt native, active conformations. These studies suggest that whilst sodium channels must be sufficiently flexible to undergo major conformational changes during their functional cycle, the proteins are highly resistant to unfolding, a feature that is important for maintaining structural integrity during dynamic processes.

Keywords: Abbreviations; CD; circular dichroism; DDM; dodecyl maltoside; TTX; tetrodotoxin; VGSC; voltage-gated sodium channel; cmc; critical micelle concentrationVoltage-gated sodium channel; Protein folding; Membrane protein; Secondary structure; Circular dichroism spectroscopy; Toxin binding

Lipid-mediated preferential localization of hypericin in lipid membranes by Yunn-Fang Ho; Ming-Huang Wu; Bor-Hen Cheng; Yar-Wen Chen; Ming-Chih Shih ⁎ (pp. 1287-1295).

Subcellular localization of a photosensitizer is critical to its therapeutic outcome during photodynamic therapy (PDT). We delineated the distribution of hypericin, a new generation photosensitizer, in model membrane systems to identify the operating principles of its subcellular accumulation. Results from fluorescence microscopy indicated preferential incorporation of hypericin in lipid of giant unilamellar vesicles. Monolayer fluorescence measurements further identified cholesterol as the key determinant for the observed selectivity of hypericin. The emission spectra of hypericin in lipid monolayers varied in a lipid-dependent manner and Stoke's shift behavior suggests that hypericin may form closely packed structure with cholesterol. Overall, our data lead to the conclusion that cholesterol is the major origin of the selectivity for hypericin in membrane systems. A hypothetical model depicting the intracellular and intravascular co-transport of hypericin and cholesterol because of their high affinity is presented.

Keywords: Hypericin; Cholesterol; Giant unilamellar vesicle; Phase separation; Subcellular distribution; Photodynamic therapy

The membrane-activity of Ibuprofen, Diclofenac, and Naproxen: A physico-chemical study with lecithin phospholipids by Marcela Manrique Moreno; Patrick Garidel; Mario Suwalsky; Jörg Howe; Klaus Brandenburg ⁎ (pp. 1296-1303).

Nonsteroidal anti-inflammatory drugs (NSAIDs) represent non-specific inhibitors of the cycloxygenase pathway of inflammation, and therefore an understanding of the interaction process of the drugs with membrane phospholipids is of high relevance. We have studied the interaction of the NSAIDs with phospholipid membranes made from dimyristoylphosphatidylcholine (DMPC) by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). FTIR data obtained via attenuated total reflectance (ATR) show that the interaction between DMPC and NSAIDs is limited to a strong interaction of the drugs with the phosphate region of the lipid head group. The FTIR transmission data furthermore are indicative of a strong effect of the drugs on the hydrocarbon chains inducing a reduction of the chain–chain interactions, i.e., a fluidization effect. Parallel to this, from the DSC data beside the decrease of T_m a reduction of the peak height of the melting endotherm connected with its broadening is observed, but leaving the overall phase transition enthalpy constant. Additionally, phase separation is observed, inducing the formation of a NSAID-rich and a NSAID-poor phase. This is especially pronounced for Diclofenac. Despite the strong influence of the drugs on the acyl chain moiety, FRET data do not reveal any evidence for drug incorporation into the lipid matrix, and ITC measurements performed do not exhibit any heat production due to drug binding. This implies that the interaction process is governed by only entropic reactions at the lipid/water interface.

Keywords: Abbreviations; NSAIDs; Nonsteroidal anti-inflammatory drugs; DMPC; dimyristoylphosphatidylcholine; FTIR; Fourier-transform infrared spectroscopy; ATR; attenuated total reflection; ITC; isothermal titration calorimetry; FRET; Förster resonance energy transfer spectroscopy; DSC; differential scanning calorimetry; T; _m; temperature of main phase transition; COX; cyclooxygenase; Pβ; ripple phase; Lα; liquid crystalline phaseDrug–membrane interaction; Nonsteroidal anti-inflammatory drug; Phospholipid membrane; Cyclooxygenase inhibitor; Entropic reaction

DNA alters the bilayer structure of cationic lipid diC14-amidine: A spin label study by Carlos R. Benatti; Rafael P. Barroso; Caroline Lonez; Jean-Marie Ruysschaert; M. Teresa Lamy ⁎ (pp. 1304-1309).

Cationic lipids–DNA complexes (lipoplexes) have been used for delivery of nucleic acids into cells in vitro and in vivo. Despite the fact that, over the last decade, significant progress in the understanding of the cellular pathways and mechanisms involved in lipoplexes-mediated gene transfection have been achieved, a convincing relationship between the structure of lipoplexes and their in vivo and in vitro transfection activity is still missing. How does DNA affect the lipid packing and what are the consequences for transfection efficiency is the point we want to address here. We investigated the bilayer organization in cationic liposomes by electron spin resonance (ESR). Phospholipids spin labeled at the 5th and 16th carbon atoms were incorporated into the DNA/diC14-amidine complex. Our data demonstrate that electrostatic interactions involved in the formation of DNA–cationic lipid complex modify the packing of the cationic lipid membrane. DNA rigidifies the amidine fluid bilayer and fluidizes the amidine rigid bilayer just below the gel–fluid transition temperature. These effects were not observed with single nucleotides and are clearly related to the repetitive charged motif present in the DNA chain and not to a charge–charge interaction. These modifications of the initial lipid packing of the cationic lipid may reorient its cellular pathway towards different routes. A better knowledge of the cationic lipid packing before and after interaction with DNA may therefore contribute to the design of lipoplexes capable to reach specific cellular targets.

Keywords: diC14-amidine; Cationic liposome; DNA interaction; Spin label; Structural property

Thermotropic behavior and lateral distribution of very long chain sphingolipids by Y. Jenny E. Björkqvist; Jonathan Brewer; Luis A. Bagatolli; J. Peter Slotte; Bodil Westerlund ⁎ (pp. 1310-1320).

Sphingolipids containing very long acyl chains are abundant in certain specialized tissues and minor components of plasma membranes in most mammalian cells. There are cellular processes in which these sphingolipids are required, and the function seems to be mediated through sphingolipid-rich membrane domains. This study was conducted to explore how very long acyl chains of sphingolipids influence their lateral distribution in membranes. Differential scanning calorimetry showed that 24:0- and 24:1-sphingomyelins, galactosylceramides and glucosylceramides exhibited complex thermotropic behavior and partial miscibility with palmitoyl sphingomyelin. The T_m was decreased by about 20 °C for all 24:1-sphingolipids compared to the corresponding 24:0-sphingolipids. The ability to pack tightly with ordered and extended acyl chains is a necessity for membrane lipids to partition into ordered domains in membranes and thus the 24:1-sphingolipids appeared less likely to do so. Fluorescence quenching measurements showed that the 24:0-sphingolipids formed ordered domains in multicomponent membranes, both as the only sphingolipid and mixed with palmitoyl sphingomyelin. These domains had a high packing density which appeared to hinder the partitioning of sterols into them, as reported by the fluorescent cholesterol analog cholestatrienol. 24:0-SM was, however, better able to accommodate sterol than the glycosphingolipids. The 24:1-sphingolipids could, depending on head group structure, either stabilize or disrupt ordered sphingolipid/cholesterol domains. We conclude that very long chain sphingolipids, when present in biological membranes, may affect the physical properties of or the distribution of sterols between lateral domains. It was also evident that not only the very long acyl chain but also the specific molecular structure of the sphingolipids was of importance for their membrane properties.

Keywords: Abbreviations; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; CTL; cholestatrienol; DPH; diphenylhexatriene; GalCer; galactosylceramide; GlcCer; glucosylceramide; GSL; glycosphingolipid; GUV; giant unilamellar vesicle; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; d; -; erythro; -; N; -palmitoyl-sphingomyelin; SL; sphingolipid; SM; sphingomyelin; T; _m; gel-to-liquid phase transition temperature; tPA; trans; -parinaric acid; VLC; very long chainCholesterol; Galactosylceramide; Glucosylceramide; Sphingomyelin; Differential scanning calorimetry; Fluorescence spectroscopy

Polar residues in transmembrane helices can decrease electrophoretic mobility in polyacrylamide gels without causing helix dimerization by William F. Walkenhorst; Mikhail Merzlyakov; Kalina Hristova; William C. Wimley (pp. 1321-1331).

There are only a few available methods to study lateral interactions and self assembly of transmembrane helices. One of the most frequently used methods is sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) which can report on strong interactions between peptides in SDS solution. Here we offer a cautionary tale about studying the folding and assembly of membrane proteins using peptides and SDS-PAGE experiments as a membrane mimetic system. At least for the specific peptide and detergent systems studied here, we show that a polar asparagine residue in the 12th position of an otherwise hydrophobic helical segment of 20 amino acids causes a peptide to migrate on SDS-PAGE gels with an apparent molecular weight that is twice its true molecular weight, suggesting dimerization. However when examined carefully in SDS solutions and in situ in the polyacrylamide gel itself using Forster resonance energy transfer no interaction can be detected. Instead we show evidence suggesting that differential interactions between peptide and detergent drive the differences in electrophoretic mobility without any interaction between peptides. These results emphasize the need to apply multiple independent techniques to the study of membrane protein folding, and they highlight the usefulness of studying folding and structure of membrane proteins in lipid membranes rather than in detergents.

Keywords: Transmembrane; Asparagine; MS1; SDS-PAGE; Dimerization; Membrane protein

A giant liposome for single-molecule observation of conformational changes in membrane proteins by Yasuhiro Onoue; Toshiharu Suzuki; Max Davidson; Mattias Karlsson; Owe Orwar; Masasuke Yoshida; Kazuhiko Kinosita Jr. ⁎ (pp. 1332-1340).

We present an experimental system that allows visualization of conformational changes in membrane proteins at the single-molecule level. The target membrane protein is reconstituted in a giant liposome for independent control of the aqueous environments on the two sides of the membrane. For direct observation of conformational changes, an extra-liposomal site(s) of the target protein is bound to a glass surface, and a probe that is easily visible under a microscope, such as a micron-sized plastic bead, is attached to another site on the intra-liposomal side. A conformational change, or an angular motion in the tiny protein molecule, would manifest as a visible motion of the probe. The attachment of the protein on the glass surface also immobilizes the liposome, greatly facilitating its manipulation such as the probe injection. As a model system, we reconstituted ATP synthase (F_OF₁) in liposomes tens of μm in size, attached the protein specifically to a glass surface, and demonstrated its ATP-driven rotation in the membrane through the motion of a submicron bead.

Keywords: Single-molecule; Giant liposome; Membrane protein; Conformational change; ATP synthase

Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A by Tamás Hegedűs; Andrei Aleksandrov; April Mengos; Liying Cui; Timothy J. Jensen; John R. Riordan ⁎ (pp. 1341-1349).

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in transcellular ion transport and when defective, results in the genetic disease cystic fibrosis. CFTR is novel in the ATP-binding cassette superfamily as an ion channel that is enabled by a unique unstructured regulatory domain. This R domain contains multiple protein kinase A sites, which when phosphorylated allow channel gating. Most of the sites have been indicated to stimulate channel activity, while two of them have been suggested to be inhibitory. It is unknown whether individual sites act coordinately or distinctly. To address this issue, we raised monoclonal antibodies recognizing the unphosphorylated, but not the phosphorylated states of four functionally relevant sites (700, 737, 768, and 813). This enabled simultaneous monitoring of their phosphorylation and dephosphorylation and revealed that both processes occurred rapidly at the first three sites, but more slowly at the fourth. The parallel phosphorylation rates of the stimulatory 700 and the putative inhibitory 737 and 768 sites prompted us to reexamine the role of the latter two. With serines 737 and 768 reintroduced individually into a PKA insensitive variant, in which serines at 15 sites had been replaced by alanines, a level of channel activation by PKA was restored, showing that these sites can mediate stimulation. Thus, we have provided new tools to study the CFTR regulation by phosphorylation and found that sites proposed to inhibit channel activity can also participate in stimulation.

Keywords: Abbreviations; CFTR; Cystic Fibrosis Conductance Transmembrane Regulator; PKA; protein kinase A; PP1, PP2A, PP2B; protein phosphatases; NBD; nucleotide binding domain; SA; serine to alanine mutationCFTR; R domain; PKA; Phosphorylation-sensitive antibody; Individual phosphorylation site; ABC protein

Towards an interpretation of¹³C chemical shifts in bathorhodopsin, a functional intermediate of a G-protein coupled receptor by Axel Gansmüller; Maria Concistrè; Neville McLean; Ole G. Johannessen; Ildefonso Marín-Montesinos; Petra H.M. Bovee-Geurts; Peter Verdegem; Johan Lugtenburg; Richard C.D. Brown; Willem J. DeGrip; Malcolm H. Levitt ⁎ (pp. 1350-1357).

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired¹³C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the¹³C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the¹³C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.

Keywords: Double-quantum solid-state NMR; Bathorhodopsin; Retinylidene PSB; ¹³; C chemical shift; Energy storage; Bathochromic shift

Cytochrome c induces lipid demixing in weakly charged phosphatidylcholine/phosphatidylglycerol model membranes as evidenced by resonance energy transfer by Galyna P. Gorbenko ⁎; Valeriya M. Trusova; Julian G. Molotkovsky; Paavo K.J. Kinnunen (pp. 1358-1365).

Resonance energy transfer (RET) between anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as donors and the heme groups of cytochrome c (cyt c) as acceptors was examined in PC/PG model membranes containing 10, 20 or 40 mol% PG with an emphasis on evaluating lipid demixing caused by this protein. The differences between AV-PC and AV-PG RET profiles observed at PG content 10 mol% were attributed to cyt c ability to produce segregation of acidic lipids into lateral domains. The radius of lipid domains recovered using Monte-Carlo simulation approach was found not to exceed 4 nm pointing to the local character of cyt c-induced lipid demixing. Increase of the membrane PG content to 20 or 40 mol% resulted in domain dissipation as evidenced by the absence of any RET enhancement while recruiting AV-PG instead of AV-PC.

Keywords: Resonance energy transfer; Cytochrome; c; Protein–lipid interactions; Heme bilayer location; Lipid demixing

Membrane surface charge modulates lipoprotein complex forming capability of peptides derived from the C-terminal domain of apolipoprotein E by Abhay H. Pande ⁎; Rajan K. Tripathy; Sunil A. Nankar (pp. 1366-1376).

Apolipoprotein E (apoE) plays a major role in the transport and metabolism of lipid by acting as a ligand for low density lipoprotein-receptors. The amphipathic helical regions of its C-terminal domain are necessary for the lipoprotein binding and assembly of nascent lipoprotein particles. Lipoproteins in the plasma are known to possess a net negative charge, determined by both its protein and lipid components, which regulates the metabolism of lipoproteins. The role of membrane surface charge on the interaction of apoE has not been studied previously. Also the importance of individual amphipathic helical regions of its C-terminal domain in binding to negatively charged lipid membrane is not addressed. In this study we have compared the interaction of four peptide segments of apoE C-terminal domain (apoE_(202–223), apoE_(223–244), apoE_(245–266), and apoE_(268–289)) with zwitterionic and negatively charged model membranes by employing UV–visible and fluorescence spectroscopy, circular dichroism, and native PAGE analysis. Our results show that the peptide sequence 202–223, 245–266 and 268–289 of apoE has higher affinity towards negatively charged lipid membrane and are independently capable of forming lipoprotein particles of 17±2 nm Stokes diameter. The results suggest that surface charge of lipoprotein regulates its metabolism possibly by modulating the recruitment of apoE on its surface.

Keywords: Amphipathic peptide; Lipoprotein particle; Anionic lipid; Circular dichroism; Fluorescence quenching; α-helix; Turbidity clearance

Polyelectrolyte and unfolded protein pore entrance depends on the pore geometry by Manuela Pastoriza-Gallego; Gabriel Gibrat; Bénédicte Thiebot; Jean-Michel Betton; Juan Pelta ⁎ (pp. 1377-1386).

We determined the ability of Maltose Binding Protein and the polyelectrolyte dextran sulfate to enter into and interact with channels formed by Staphylococcus aureus α-hemolysin. The entry of either macromolecule in the channel pore causes transient, but well-defined decreases in the single-channel ionic current. The protein and polyelectrolyte were more likely to enter the pore mouth at the channel's cap domain than at the stem side. When the cap domain was denatured in the presence of 4 M urea, the probability that either the denatured protein or polyelectrolyte entered the pore from the cap-domain side decreased. For channels in their native conformation, the polyelectrolyte-induced current blockades were characterized by two mean residence times that were independent of the side of entry. For channels with a denaturated cap domain, the mean polyelectrolyte residence times for relatively long-lived blockades decreased, while that for short-lived blockades were unchanged. For denatured protein, we also observed 2 characteristic residence times that were relatively fast. Only the relatively short-lived blockades were observed with native channels. When the α-hemolysin monomers in aqueous solution were incubated in 4 M urea before channel formation, the two characteristic residence times were greater than those for pre-formed pores that were subsequently perturbed by urea. These times might correspond to the interactions between the unfolded protein and the partially unfolded channel.

Keywords: α-hemolysin; Pore forming toxin; Single-channel current recording; Polyelectrolyte and protein transport; Protein unfolding

Alamethicin in lipid bilayers: Combined use of X-ray scattering and MD simulations by Jianjun Pan; D. Peter Tieleman; John F. Nagle; Norbert Kučerka; Stephanie Tristram-Nagle ⁎ (pp. 1387-1397).

We study fully hydrated bilayers of two di-monounsaturated phospholipids diC18:1PC (DOPC) and diC22:1PC with varying amounts of alamethicin (Alm). We combine the use of X-ray diffuse scattering and molecular dynamics simulations to determine the orientation of alamethicin in model lipids. Comparison of the experimental and simulated form factors shows that Alm helices are inserted transmembrane at high humidity and high concentrations, in agreement with earlier results. The X-ray scattering data and the MD simulations agree that membrane thickness changes very little up to 1/10 Alm/DOPC. In contrast, the X-ray data indicate that the thicker diC22:1PC membrane thins with added Alm, a total decrease in thickness of 4 Å at 1/10 Alm/diC22:1PC. The different effect of Alm on the thickness changes of the two bilayers is consistent with Alm having a hydrophobic thickness close to the hydrophobic thickness of 27 Å for DOPC; Alm is then mismatched with the 7 Å thicker diC22:1PC bilayer. The X-ray data indicate that Alm decreases the bending modulus ( K_C) by a factor of ∼2 in DOPC and a factor of ∼10 in diC22:1PC membranes (P/L ∼1/10). The van der Waals and fluctuational interactions between bilayers are also evaluated through determination of the anisotropic B compressibility modulus.

Keywords: Lipid bilayer; Peptide; X-ray; Structure; MD simulation; Alamethicin

Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation by Yehudi Self-Medlin ⁎; Jungsoo Byun; Robert F. Jacob; Yoshiko Mizuno; R. Preston Mason (pp. 1398-1403).

Oxidative damage to vascular cell membrane phospholipids causes physicochemical changes in membrane structure and lipid organization, contributing to atherogenesis. Oxidative stress combined with hyperglycemia has been shown to further increase the risk of vascular and metabolic diseases. In this study, the effects of glucose on oxidative stress-induced cholesterol domain formation were tested in model membranes containing polyunsaturated fatty acids and physiologic levels of cholesterol. Membrane structural changes, including cholesterol domain formation, were characterized by small angle X-ray scattering (SAXS) analysis and correlated with spectrophotometrically-determined lipid hydroperoxide levels. Glucose treatment resulted in a concentration-dependent increase in lipid hydroperoxide formation, which correlated with the formation of highly-ordered cholesterol crystalline domains (unit cell periodicity of 34 Å) as well as a decrease in overall membrane bilayer width. The effect of glucose on lipid peroxidation was further enhanced by increased levels of cholesterol. Treatment with free radical-scavenging agents inhibited the biochemical and structural effects of glucose, even at elevated cholesterol levels. These data demonstrate that glucose promotes changes in membrane organization, including cholesterol crystal formation, through lipid peroxidation.

Keywords: Membrane bilayer; Hyperglycemia; Glucose; Peroxidation; Cholesterol domain; Polyunsaturated fatty acid

Computational studies of gramicidin permeation: An entryway sulfonate enhances cation occupancy at entry sites by Morad Mustafa ⁎; Douglas J. Henderson; David D. Busath (pp. 1404-1412).

The impact on the cation-transport free-energy profile of replacing the C-terminal ethanolamine in the gramicidin A channel with a taurine residue is studied using molecular dynamics simulations of gramicidin A (1JNO) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl saline solution. The potential of mean force for ion transport is obtained by umbrella sampling. The presence of a negatively charged sulfonate group at the entrance of the gramicidin channel affects the depth and the location of the binding sites, producing a strong attraction for the cations in the bulk. The potential of mean force by the sulfonate acting directly through electrostatics and van der Waals interactions on the test ion is highly modulated by indirect effects (i.e., sulfonate effects on other components of the system that, in turn, affect the ion free-energy profile in the channel). Because the “entry” sites are located symmetrically at both entry and exit of the channel, the deeper free-energy wells should inhibit exit. Given that the channel has increased conductance experimentally, the simulation results suggest that the channel conductance is normally entry limited.

Keywords: Ion channel; Potential of mean force; Sulfonate parametrization; Water layering; Molecular dynamics simulation

Corrigendum to “Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols” [Biochim. Biophys. Acta 1778 (2008) 2244–2257] by Barry W. Walker; Nathan Manhanke; Keith J. Stine ⁎ (pp. 1413-1413).

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BBA - Biomembranes (v.1788, #6)

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BBA - Biomembranes (v.1788, #6)