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BBA - Biomembranes (v.1808, #6)

Editorial Board (pp. i).

Mutational analysis of calnexin by Jody Groenendyk; Monika Dabrowska; Marek Michalak (pp. 1435-1440).

Calnexin is a type I endoplasmic reticulum lectin-like chaperone protein. In this study, we have used site-specific mutagenesis to investigate the functional importance of glutamate E³⁵¹ found at the tip of the P-domain of calnexin, and tryptophan W⁴²⁸ found in the carbohydrate binding region of the globular domain of the protein. The E³⁵¹ and W⁴²⁸ calnexin mutants lost the ability to inhibit aggregation of IgY (glycosylated substrate). The E³⁵¹ mutation led to slightly enhanced ERp57 binding to calnexin, whereas W⁴²⁸ greatly enhanced binding of ERp57 to calnexin. These findings indicate that modification of a residue(s) in the carbohydrate binding region may have a profound effect on the structural and functional properties of the P-domain and consequently on association of calnexin with the folding enzyme ERp57.

Keywords: Abbreviations; ER; endoplasmic reticulum; MDH; malate dehydrogenase; RU; resonance units; SPR; surface Plasmon resonanceEndoplasmic reticulum; Protein folding; Calnexin; Chaperone; Mutagenesis

Identification and functional characterization of uric acid transporter Urat1 ( Slc22a12) in rats by Masanobu Sato; Tomohiko Wakayama; Hideaki Mamada; Yoshiyuki Shirasaka; Takeo Nakanishi; Ikumi Tamai (pp. 1441-1447).

Uric acid transporter URAT1 contributes significantly to reabsorption of uric acid in humans to maintain a constant serum uric acid (SUA) level. Since alteration of SUA level is associated with various diseases, it is important to clarify the mechanism of change in SUA. However, although expression of mRNA of an ortholog of URAT1 ( rUrat1) in rats has been reported, functional analysis and localization have not been done. Therefore, rat rUrat1 was functionally analyzed using gene expression systems and isolated brush-border membrane vesicles (BBMVs) prepared from rat kidney, and its localization in kidney was examined immunohistochemically. Uric acid transport by rUrat1 was chloride (Cl⁻) susceptible with a Km of 1773μM. It was inhibited by benzbromarone and trans-stimulated by lactate and pyrazinecarboxylic acid (PZA). Cl⁻ gradient-susceptible uric acid transport by BBMVs showed similar characteristics to those of uric acid transport by rUrat1. Moreover, rUrat1 was localized at the apical membrane in proximal tubular epithelial cells in rat kidney. Accordingly, rUrat1 is considered to be involved in uric acid reabsorption in rats in the same manner as URAT1 in humans. Therefore, rUrat1 may be a useful model to study issues related to the role of human URAT1.► Rat uric acid transporter rUrat1 ( Slc22a12) functions at apical membrane in proximal tubular cells. ► Characteristics of rUrat1 are comparable to that of human URAT1 and mouse Rst. ► Some uricosuric and antiuricosuric drugs alter the transport activity of rUrat1.

Keywords: Urate; Kidney; Excretion; Reabsorption; Secretion

Secondary structure, dynamics, and architecture of the p7 membrane protein from hepatitis C virus by NMR spectroscopy by Gabriel A. Cook; Stanley J. Opella (pp. 1448-1453).

P7 is a small membrane protein that is essential for the infectivity of hepatitis C virus. Solution-state NMR experiments on p7 in DHPC micelles, including hydrogen/deuterium exchange, paramagnetic relaxation enhancement and bicelle ‘ q-titration,’ demonstrate that the protein has a range of dynamic properties and distinct structural segments. These data along with residual dipolar couplings yield a secondary structure model of p7. We were able to confirm previous proposals that the protein has two transmembrane segments with a short interhelical loop containing the two basic residues K33 and R35. The 63-amino acid protein has a remarkably complex structure made up of seven identifiable sections, four of which are helical segments with different tilt angles and dynamics. A solid-state NMR two-dimensional separated local field spectrum of p7 aligned in phospholipid bilayers provided the tilt angles of two of these segments. A preliminary structural model of p7 derived from these NMR data is presented.

Keywords: Abbreviations; HCV; hepatitis C virus; NMR; nuclear magnetic resonance; DHPC; (1,2-dihexyl-1-; sn; -glycero-3-phosphocholine); NOE; nuclear Overhauser effect; PRE; paramagnetic relaxation enhancement; RDC; residual dipolar couplings; SDS; sodium dodecyl sulfate; D; ₂; O; deuterium oxide; EDTA; ethylenediaminetetraacetic acid; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; 6-O-PC; 1,2-di-; O; -hexyl-; sn; -glycero-3-phosphocholine; IPAP; in-phase/anti-phase; CSI; chemical shift index; PISA; polar index slant angle; ER; endoplasmic reticulumHepatitis C virus; p7; Membrane protein; Secondary structure; Nuclear magnetic resonance

Transmembrane helix 7 in the Na⁺/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function by Ana M. Pajor; Nina N. Sun; Aditya D. Joshi; Kathleen M. Randolph (pp. 1454-1461).

Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na⁺/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased V_max for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer.► TM7 of the Na+/dicarboxylate cotransporter 1 is likely to be an outer helix. ► Gly-356 was inhibited by MTSET but could not be labeled by the bulkier MTSEA-biotin. ► Phe-336 and Phe-339 are important for substrate turnover or selectivity. ► TM7 contains functionally important residues; cysteine mutants had reduced activity independent of protein expression.

Keywords: Abbreviations; DASS; divalent anion sodium symporter family; MTSEA-biotin; N; -biotinaminoethyl methanethiosulfonate; MTSET; [2-(trimethylammonium)ethyl]-methanethiosulfonate; NaDC1; sodium dicarboxylate cotransporter 1; PMSF; phenylmethylsulfonyl fluoride; rb; rabbit; SLC13; solute carrier family 13; TM; transmembrane helixCysteine substitution; Methanethiosulfonate reagents; Site-directed mutagenesis; SLC13 family; Sodium; Succinate

Solution- and solid-state NMR studies of GPCRs and their ligands by Satita Tapaneeyakorn; Alan D. Goddard; Joanne Oates; Christine L. Willis; Anthony Watts (pp. 1462-1475).

G protein-coupled receptors (GPCRs) represent one of the major targets of new drugs on the market given their roles as key membrane receptors in many cellular signalling pathways. Structure-based drug design has potential to be the most reliable method for novel drug discovery. Unfortunately, GPCR-ligand crystallisation for X-ray diffraction studies is very difficult to achieve. However, solution- and solid-state NMR approaches have been developed and have provided new insights, particularly focussing on the study of protein-ligand interactions which are vital for drug discovery. This review provides an introduction for new investigators of GPCRs/ligand interactions using NMR spectroscopy. The guidelines for choosing a system for efficient isotope labelling of GPCRs and their ligands for NMR studies will be presented, along with an overview of the different sample environments suitable for generation of high resolution structural information from NMR spectra.

Keywords: Abbreviations; β; ₂; AR; β; ₂; adrenergic receptor; CP; cross polarisation; CXCR1; chemokine receptor; DAGK; diacylglycerol kinase; GPCR; G protein-coupled receptor; MAS; magic angle spinning; MBP; maltose binding protein; NOE; nuclear overhauser effect; NTS1; neurotensin receptor 1; OmpG; outer membrane protein G; REDOR; rotational echo double resonance; RR; rotational resonance; solNMR; solution-state NMR; SSNMR; solid-state NMR; trNOE; transferred nuclear overhauser effect; SPPS; solid-phase peptide synthesis; τ; _c; −; ¹; tumbling rateSolution-state NMR; Solid-state NMR; GPCR; Protein-ligand interaction; Expression system; Sample environment

Mode of action of parasporin-4, a cytocidal protein from Bacillus thuringiensis by Shiro Okumura; Hiroyuki Saitoh; Tomoyuki Ishikawa; Kuniyo Inouye; Eiichi Mizuki (pp. 1476-1482).

Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines, CACO-2, Sawano, and MOLT-4 cells, in particular. When cells were administrated with PS4, cell swelling and nuclear shrinkage were induced, and, the ballooned cells burst within 24h. PSI-BLAST search showed that the protein shared homology not only with B. thuringiensis Cry toxins but also with aerolysin-type β-pore-forming toxins. Circular dichroism measurements suggested that PS4 was a β-sheet-rich protein. PS4 aggregated into oligomers on the plasma membrane of PS4-susceptible CACO-2 cells, but not on that of PS4-resistant HeLa cells. Leakage of lactate dehydrogenase and influx of extracellular FITC-dextrans were observed only in susceptible cells. The activation of effectors caspase 3 and/or 7 was not observed in PS4-treated CACO-2 cells. It was shown that cytotoxicity of the PS4 against CACO-2 cells was exhibited when treated by cyclodextrin which induces cholesterol depletion. These results suggest that PS4 is a unique β-pore-forming toxin with a cholesterol-independent activity.► Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillus thuringiensis strain. ► PS4 exhibits specific cytotoxicity against human cancer cell lines, but not against normal tissue cells. ► PS4 induces plasma membrane damage to the PS4-susceptible cells. ► PS4 aggregates into oligomers only on the plasma membrane of PS4-susceptible cells. ► PS4 is a unique β-pore-forming toxin with a cholesterol-independent activity.

Keywords: Abbreviations; β-PFT; β-pore-forming toxin; BSA; bovine serum albumin; BSA-PBS; PBS containing 1% BSA, 0.05% sodium azide; CD; circular dichroism; CDC; cholesterol-dependent cytolysin; LDH; lactate dehydrogenase; MTT; 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2; H; -tetrazolium bromide; PSI-BLAST; position-specific iterated-BLAST; proPS4; pro-parasporin-4; PS4; parasporin-4; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis Bacillus thuringiensis; β-pore-forming toxin; Cytotoxic activity; Parasporin

Structural and functional analyses of PpENA1 provide insights into cation binding by type IID P-type ATPases in lower plants and fungi by Damian P. Drew; Maria Hrmova; Christina Lunde; Andrew K. Jacobs; Mark Tester; Geoffrey B. Fincher (pp. 1483-1492).

PpENA1 is a membrane-spanning transporter from the moss Physcomitrella patens, and is the first type IID P-type ATPase to be reported in the plant kingdom. In Physcomitrella, PpENA1 is essential for normal growth under moderate salt stress, while in yeast, type IID ATPases provide a vital efflux mechanism for cells under high salt conditions by selectively transporting Na⁺ or K⁺ across the plasma membrane. To investigate the structural basis for cation-binding within the type IID ATPase subfamily, we used homology modeling to identify a highly conserved cation-binding pocket between membrane helix (MH) 4 and MH 6 of the membrane-spanning pore of PpENA1. Mutation of specific charged and polar residues on MHs 4–6 resulted in a decrease or loss of protein activity as measured by complementation assays in yeast. The E298S mutation on MH 4 of PpENA1 had the most significant effect on activity despite the presence of a serine at this position in fungal type IID ATPases. Activity was partially restored in an inactivated PpENA1 mutant by the insertion of two additional serine residues on MH 4 and one on MH 6 based on the presence of these residues in fungal type IID ATPases. Our results suggest that the residues responsible for cation-binding in PpENA1 are distinct from those in fungal type IID ATPases, and that a fungal-type cation binding site can be successfully engineered into the moss protein.►Amino acids on MHs 4, 6 and 8 of PpENA1 contribute to cation binding. ►Cation binding sites of PpENA1 differ from those of fungal homologs. ►Comparative modeling enables the engineering of cation binding residues.

Keywords: Abbreviations; SERCA; sarco-endoplasmic reticulum Ca; ²⁺; ATPase; MH; membrane helix; HCA; hydrophobic cluster analysis; HA; hemagglutininPpENA1; Type IID ATPase; Physcomitrella; Homology modeling; Na; ⁺; pump

Towards a structural understanding of the smallest known oncoprotein: Investigation of the bovine papillomavirus E5 protein using solution-state NMR by Gavin King; Joanne Oates; Dharmesh Patel; Hugo A. van den Berg; Ann M. Dixon (pp. 1493-1501).

The homodimeric E5 protein from bovine papillomavirus activates the platelet-derived growth factor β receptor through transmembrane (TM) helix–helix interactions leading to uncontrolled cell growth. Detailed structural information for the E5 dimer is essential if we are to uncover its unique mechanism of action. In vivo mutagenesis has been used to identify residues in the TM domain critical for dimerization, and we previously reported that a truncated synthetic E5 peptide forms dimers via TM domain interactions. Here we extend this work with the first application of high-resolution solution-state NMR to the study of the E5 TM domain in SDS micelles. Using selectively¹⁵N-labelled peptides, we first probe sample homogeneity revealing two predominate species, which we interpret to be monomer and dimer. The equilibrium between the two states is shown to be dependent on detergent concentration, revealed by intensity shifts between two sets of peaks in¹⁵N-¹H HSQC experiments, highlighting the importance of sample preparation when working with these types of proteins. This information is used to estimate a free energy of association (Δ G_x°=−3.05kcal mol−¹) for the dimerization of E5 in SDS micelles. In addition, chemical shift changes have been observed that indicate a more pronounced change in chemical environment for those residues expected to be at the dimer interface in vivo versus those that are not. Thus we are able to demonstrate our in vitro dimer is comparable to that defined in vivo, validating the biological significance of our synthetic peptide and providing a solid foundation upon which to base further structural studies. Using detergent concentration to modulate oligomeric state and map interfacial residues by NMR could prove useful in the study of other homo-oligomeric transmembrane proteins.► This is the first application of solution-state NMR to study the E5 oncoprotein TM domain in detergent micelles. ► This study reveals monomer dimer equilibrium in SDS micelles. ► Structure of in vitro dimer is comparable to that of wild-type dimer found in vivo.

Keywords: Bovine papillomavirus E5 protein; Oncogene; Solution-state NMR; Transmembrane domain; Dimer

Decrease in transient receptor potential melastatin 6 mRNA stability caused by rapamycin in renal tubular epithelial cells by Akira Ikari; Ayumi Sanada; Hayato Sawada; Chiaki Okude; Chie Tonegawa; Junko Sugatani (pp. 1502-1508).

Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), is used in treatments for transplantation and cancer. Rapamycin causes hypomagnesemia, although precisely how has not been examined. Here, we investigated the effect of rapamycin on the expression of transient receptor potential melastatin 6 (TRPM6), a Mg²⁺ channel. Rapamycin and LY-294002, an inhibitor of phosphatidilinositol-3 kinase (PI3K) located upstream of mTOR, inhibited epidermal growth factor (EGF)-induced expression of the TRPM6 protein without affecting TRPM7 expression in rat renal NRK-52E epithelial cells. Both rapamycin and LY-294002 decreased EGF-induced Mg²⁺ influx. U0126, a MEK inhibitor, inhibited EGF-induced increases in c-Fos, p-ERK, and TRPM6 levels. In contrast, neither rapamycin nor LY-294002 inhibited EGF-induced increases in p-ERK and c-Fos levels. EGF increased p-Akt level, an effect inhibited by LY-294002 and 1L-6-hydroxymethyl- chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (Akt inhibitor). Akt inhibitor decreased TRPM6 level similar to rapamycin and LY-294002. These results suggest that a PI3K/Akt/mTOR pathway is involved in the regulation of TRPM6 expression. Rapamycin inhibited the EGF-induced increase in TRPM6 mRNA but did not inhibit human TRPM6 promoter activity. In the presence of actinomycin D, a transcriptional inhibitor, rapamycin accelerated the decrease in TRPM6 mRNA. Rapamycin decreased the expression and activity of a luciferase linked with the 3′-untranslated region of human TRPM6 mRNA. These results suggest that TRPM6 expression is up-regulated by a PI3K/Akt/mTOR pathway and rapamycin reduces TRPM6 mRNA stability, resulting in a decrease in the reabsorption of Mg²⁺.► Rapamycin inhibits epidermal growth factor-induced elevation of TRPM6 mRNA. ► Rapamycin decreases epidermal growth factor-induced Mg²⁺ influx. ► Rapamycin does not affect the human TRPM6 promoter activity. ► A PI3K/Akt/mTOR pathway is involved in the regulation of TRPM6 mRNA stability.

Keywords: Abbreviations; EGF; epidermal growth factor; [Mg; ²⁺; ]; _i; intracellular free magnesium concentration; mTOR; mammalian target of rapamycin; TRPM6; transient receptor potential melastatin 6; PI3K; phosphatidylinositol 3-kinaseHypomagnesemia; Kidney; Magnesium; Mammalian target of rapamycin; Transient receptor potential melastatin 6

A glycosyltransferase-enriched reconstituted membrane system for the synthesis of branched O-linked glycans in vitro by Sandrine Susini; Charlotte Jeanneau; Sylvie Mathieu; Sylvie Carmona; Assou El-Battari (pp. 1509-1519).

Mimicking the biochemical reactions that take place in cell organelles is becoming one of the most important challenges in biological chemistry. In particular, reproducing the Golgi glycosylation system in vitro would allow the synthesis of bioactive glycan polymers and glycoconjugates for many future applications including treatments of numerous pathologies. In the present study, we reconstituted a membrane system enriched in glycosyltransferases obtained by combining the properties of the wheat germ lectin with the dialysable detergent n-octylglucoside. When applied to cells engineered to express the O-glycan branching enzyme core2 beta (1,6)-N-acetylglucosaminyltransferase (C2GnT-I), this combination led to the reconstitution of lipid vesicles exhibiting an enzyme activity 11 times higher than that found in microsomal membranes. The enzyme also showed a slightly higher affinity than its soluble counterpart toward the acceptor substrate. Moreover, the use of either the detergent re-solubilization, glycoprotein substrates or N-glycanase digestion suggests that most of the reconstituted glycosyltransferases have their catalytic domains in an extravesicular orientation. Using the disaccharide substrate Galβ1-3GalNAc-O-p-nitrophenyl as a primer, we performed sequential glycosylation reactions and compared the recovered oligosaccharides to those synthesized by cultured parental cells. After three successive glycosylation reactions using a single batch of the reconstituted vesicles and without changing the buffer, the acceptor was transformed into an O-glycan with chromatographic properties similar to glycans produced by C2GnT-I-expressing cells. Therefore, this new and efficient approach would greatly improve the synthesis of bioactive carbohydrates and glycoconjugates in vitro and could be easily adapted for the study of other reactions naturally occurring in the Golgi apparatus such as N-glycosylation or sulfation.►Concentration/reconstitution of Golgi glycosyltransferases into (endogenous)lipid vesicles. ►High enzyme activity compared to microsomes. ►Reconstituted glycosyltransferases having their catalytic domains in an extravesicular orientation. ►One-pot and multistep reactions for rapid synthesis of core2-based O-glycans.

Keywords: Abbreviations; GEMS; glycosyltransferase-enriched membrane system; WGA; wheat germ agglutinin; OG; n-octyl-β-; d; -glucopyranoside; C2GnT-I; core2 ß1,6-N-acetylglucosaminyltransferase-I; EGFP; green fluorescent protein; α2FT; α(1,2)-fucosyltransferase-I (FucT-I); FucT-VII; α(1,3)-fucosyltransferase-VII; α3ST; α(2,3)-sialyltransferases; α6ST; α(2,6)-sialyltransferase-I (ST6Gal-I); NeuAc; N-acetyl-neuraminic acid (sialic acid); Gal; galactose; GlcNAc; N-acetylglucosamine; GalNAc; N-aceylgalactosamine; RITC; rhodamine isothiocyanate; MES; 2-(N-morphilino)ethanesulfonic acid; TBS; Tris-buffered saline; FucT-III; α(1,3/4)-fucosyltransferase-III; FucT-VI; α(1,3)-fucosyltransferase-VI; FucT-IX; α(1,3)-fucosyltransferase-IXMembrane reconstitution; Golgi; Glycosyltransferase; Glycosylation; O; -glycans

Nature of sterols affects plasma membrane behavior and yeast survival during dehydration by Sebastien Dupont; Laurent Beney; Thierry Ferreira; Patrick Gervais (pp. 1520-1528).

The plasma membrane (PM) is a main site of injury during osmotic perturbation. Sterols, major lipids of the PM structure in eukaryotes, are thought to play a role in ensuring the stability of the lipid bilayer during physicochemical perturbations. Here, we investigated the relationship between the nature of PM sterols and resistance of the yeast Saccharomyces cerevisiae to hyperosmotic treatment. We compared the responses to osmotic dehydration (viability, sterol quantification, ultrastructure, cell volume, and membrane permeability) in the wild-type (WT) strain and the ergosterol mutant erg6Δ strain. Our main results suggest that the nature of membrane sterols governs the mechanical behavior of the PM during hyperosmotic perturbation. The mutant strain, which accumulates ergosterol precursors, was more sensitive to osmotic fluctuations than the WT, which accumulates ergosterol. The hypersensitivity of erg6Δ was linked to modifications of the membrane properties, such as stretching resistance and deformation, which led to PM permeabilization during the volume variation during the dehydration–rehydration cycles. Anaerobic growth of erg6Δ strain with ergosterol supplementation restored resistance to osmotic treatment. These results suggest a relationship between hydric stress resistance and the nature of PM sterols. We discuss this relationship in the context of the evolution of the ergosterol biosynthetic pathway.► Yeast mutant in sterol synthetic pathway ( erg6Δ) is sensitive to hydric stress. ► Sterol nature affects plasma membrane integrity during hydric perturbation. ► Sterol structure influences plasma membrane deformation and resistance to stretching. ► Ergosterol complementation restores osmotic resistance in a sensitive mutant ( erg6Δ). ► Ergosterol is involved in survival of wild-type yeast during hydric fluctuations.

Keywords: Dehydration; Osmotic stress; Cell survival; Plasma membrane; Ergosterol; Sterol evolution

Is an orthorhombic lateral packing and a proper lamellar organization important for the skin barrier function? by Daniël Groen; Dana S. Poole; Gert S. Gooris; Joke A. Bouwstra (pp. 1529-1537).

The lipid organization in the stratum corneum (SC), plays an important role in the barrier function of the skin. SC lipids form two lamellar phases with a predominantly orthorhombic packing. In previous publications a lipid model was presented, referred to as the stratum corneum substitute (SCS), that closely mimics the SC lipid organization and barrier function. Therefore, the SCS serves as a unique tool to relate lipid organization with barrier function. In the present study we examined the effect of the orthorhombic to hexagonal phase transition on the barrier function of human SC and SCS. In addition, the SCS was modified by changing the free fatty acid composition, resulting in a hexagonal packing and perturbed lamellar organization. By measuring the permeability to benzoic acid as function of temperature, Arrhenius plots were constructed from which activation energies were calculated. The results suggest that the change from orthorhombic to hexagonal packing in human SC and SCS, does not have an effect on the permeability. However, the modified SCS revealed an increased permeability to benzoic acid, which we related to its perturbed lamellar organization. Thus, a proper lamellar organization is more crucial for a competent barrier function than the presence of an orthorhombic lateral packing.►The SCS mimics the lipid organization and barrier function of human SC. ►The orthorhombic lateral packing is less crucial for the SC barrier function. ►A proper lamellar organization is vital for a competent SC barrier function.

Keywords: Stratum corneum; Lipid; Hexagonal; Orthorhombic; Lamellar phase; Arrhenius

Electromediated formation of DNA complexes with cell membranes and its consequences for gene delivery by Jean-Michel Escoffre; Thomas Portet; Cyril Favard; Teissie Justin Teissié; David S. Dean; Marie-Pierre Rols (pp. 1538-1543).

Electroporation is a physical method to induce the uptake of therapeutic drugs and DNA, by eukaryotic cells and tissues. The phenomena behind electro-mediated membrane permeabilization to plasmid DNA have been shown to be significantly more complex than those for small molecules. Small molecules cross the permeabilized membrane by diffusion whereas plasmid DNA first interacts with the electropermeabilized part of the cell surface, forming localized aggregates. The dynamics of this process is still poorly understood because direct observations have been limited to scales of the order of seconds. Here, cells are electropermeabilized in the presence of plasmid DNA and monitored with a temporal resolution of 2ms. This allows us to show that during the first pulse application, plasmid complexes, or aggregates, start to form at distinct sites on the cell membrane. FRAP measurements show that the positions of these sites are remarkably immobile during the application of further pluses. A theoretical model is proposed to explain the appearance of distinct interaction sites, the quantitative increase in DNA and also their immobility leading to a tentative explanation for the success of electro-mediated gene delivery.► DNA/membrane interaction occurs during cell electrotransfection. ► This interaction is imaged at unprecedent time resolution (ms). ► Observations are consistent with our theoretical model. ► DNA aggregates are formed in the vicinity of or in the plasma membrane. ► Successive electric pulses always lead to accumulation of DNA exactly at the same location than after the first pulse.

Keywords: Electroporation; DNA transfer; Modeling; Fluorescence microscopy

Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity by Tina Holm; Raagel Helin Räägel; Samir EL Andaloussi; Margot Hein; Mae Maarja Mäe; Margus Pooga; Ülo Langel (pp. 1544-1551).

Cell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being “retro-inversion” (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast tol-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20μM peptide concentration within 4h while parentl-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides.Display Omitted►Retro-inversion of some cell-penetrating peptides induces trypsin insensitivity. ►Retro-inversion of some cell-penetrating peptides causes cytotoxicity. ►The cytotoxicity is caused by disrupted mitochondrial membrane potential. ►Peptide length and hydrophobicity are important for the observed toxicity.

Keywords: Abbreviations; CPP; cell-penetrating peptide; RI; retro inverso; PNA; peptide nucleic acid; PI; propidium iodide; LDH; lactate dehydrogenase; FA; focal adhesionCell-penetrating peptide; Retro-inverso; Cytotoxicity; Hydrophobicity; Apoptosis

Molecular analysis of antimicrobial agent translocation through the membrane porin BpsOmp38 from an ultraresistant Burkholderia pseudomallei strain by Wipa Suginta; Kozhinjampara R. Mahendran; Watcharin Chumjan; Eric Hajjar; Albert Schulte; Mathias Winterhalter; Helge Weingart (pp. 1552-1559).

Burkholderia pseudomallei ( Bps) is a Gram-negative bacterium that causes melioidosis, an infectious disease of animals and humans common in northern and north-eastern parts of Thailand. Successful treatment of melioidosis is difficult due to intrinsic resistance of Bps to various antibacterial agents. It has been suggested that the antimicrobial resistance of this organism may result from poor permeability of the active compounds through porin channels located in the outer membrane (OM) of the bacterium. In previous work, a 38-kDa protein, named “ BpsOmp38”, was isolated from the OM of Bps. A topology prediction and liposome-swelling assay suggested that BpsOmp38 comprises a β-barrel structure and acts as a general diffusion porin. The present study employed black lipid membrane (BLM) reconstitution to demonstrate the single-channel conductance of the trimeric BpsOmp38 to be 2.7±0.3nS in 1M KCl. High-time resolution BLM measurements displayed ion current blockages of seven antimicrobial agents in a concentration-dependent manner with the translocation on-rate ( k_on) following the order: norfloxacin≫ertapenem>ceftazidime>cefepime>imipenem>meropenem>penicillin G. The dwell time of a selected antimicrobial agent (ertapenem) decayed exponentially with increasing temperature. The energy barrier for the ertapenem binding to the affinity site inside the BpsOmp38 channel was estimated from the Arrhenius plot to be 12kT and for the ertapenem release to be 13kT at +100mV. The BLM data obtained from this study provide the first insight into antimicrobial agent translocation through the BpsOmp38 channel. ► BpsOmp38 of B. pseudomallei was reconstituted into lipid bilayers for single-channel recordings. ► The trimeric BpsOmp38 formed an ion channel with the conductance of 2.7±0.3nS in 1M KCl. ► Flux of seven antibiotics through BpsOmp38 was analyzed and translocation on/off rates were assessed. ► The dwell time of the antibiotic ertapenem decayed exponentially with increases in temperature. ► Energy barriers obtained from Arrhenius plots suggested one affinity site inside the Omp pores.

Keywords: Black lipid membrane reconstitution; Burkholderia pseudomallei; Melioidosis; Outer membrane protein; Porin

hERG channel blockade by externally applied quaternary ammonium derivatives by Kee-Hyun Choi; Chiman Song; Dongyun Shin; Sungnam Park (pp. 1560-1566).

The human ether-à-go-go related gene potassium channel is a key player in cardiac rhythm regulation, thus being an important subject for a cardiac toxicity test. Ever since human ether-à-go-go related gene channel inhibition-related cardiac arrest was proven to be fatal, numerous numbers of data on human ether-à-go-go related gene channel inhibition have been piled up. However, there has been no quantitative study on human ether-à-go-go related gene channel inhibition by quaternary ammonium derivatives, well-known potassium channel blockers. Here, we present human ether-à-go-go related gene channel blockade by externally applied quaternary ammonium derivatives using automated whole-cell patch-clamp recordings as well as ab initio quantum calculations. The inhibitory constants and the relative binding energies for human ether-à-go-go related gene channel inhibition were obtained from quaternary ammoniums with systematically varied head and tail groups, indicating that more hydrophobic quaternary ammoniums have higher affinity blockade while cation-π interactions or size effects are not a deterministic factor for human ether-à-go-go related gene channel inhibition by quaternary ammoniums. Further studies on the effect of quaternary ammoniums on human ether-à-go-go related gene channel inactivation implied that hydrophobic quaternary ammoniums either with a longer tail group or with a bigger head group than tetraethylammonium permeate the cell membrane to easily access the high-affinity internal binding site in human ether-à-go-go related gene channel and exert stronger blockade. These results may be informative for the rational drug design to avoid cardiac toxicity.► More hydrophobic blockers have higher affinity hERG channel blockade. ► Hydrophobic blockers permeate the membrane to access the high-affinity internal binding site. ► Cation-π interactions or size effects are not a deterministic factor for blockade in the internal site.

Keywords: hERG channel; Quaternary ammonium; Hydrophobicity; Cation-π interaction; Inactivation

Study of the inhibition capacity of an 18-mer peptide domain of GBV-C virus on gp41-FP HIV-1 activity by I. Haro; Gomara M.J. Gómara; R. Galatola; Domenech O. Domènech; J. Prat; V. Girona; M.A. Busquets (pp. 1567-1573).

The peptide sequence (175–192) RFPFHRCGAGPKLTKDLE (P59) of the E2 envelope protein of GB virus C (GBV-C) has been proved to decrease cellular membrane fusion and interfere with the HIV-1 infectivity in a dose-dependent manner. Based on these previous results, the main objective of this study was to deepen in the physicochemical aspects involved in this interaction. First, we analyzed the surface activity of P59 at the air–water interface as well as its interaction with zwitterionic or negatively charged lipid monolayers. Then we performed the same experiments with mixtures of P59/gp41-FP. Studies on lipid monolayers helped us to understand the lipid–peptide interaction and the influence of phospholipids on peptide penetration into lipid media. On another hand, studies with lipid bilayers showed that P59 decreased gp41-FP binding to anionic Large Unilamellar Vesicles. Results can be attributed to the differences in morphology of the peptides, as observed by Atomic Force Microscopy. When P59 and gp41-FP were incubated together, annular structures of about 200nm in diameter appeared on the mica surface, thus indicating a peptide–peptide interaction. All these results confirm the gp41-FP–P59 interaction and thus support the hypothesis that gp41-FP is inhibited by P59.► A peptide sequence of E2 GBV-C (P59) decreases the surface activity of gp41 HIV-1. ► P59 modifies gp41 penetration into lipid monolayers. ► P59 decreases the binding of gp41 HIV-1 to anionic lipid bilayers. ► AFM of a mixture P59/gp41 shows annular structures of about 200nm of diameter.

Keywords: Abbreviations; LUVs; Large Unilamellar Vesicles; P59; Peptide sequence (175–192): RFPFHRCGAGPKLTKDLE, of the E2 envelope protein of GBV; gp41 FP; HIV-1 FP; PC; l; -α-phosphatidylcholine (egg); PS; l; -α-phosphatidylserine (bovine); π; _sat; saturation pressure; π; _max; maximum insertion pressure; π; _o; initial surface pressure; Δ; π; pressure increase; TRIS; Tris hydroxymethylaminomethane; GBV-C; GB virus CKey words; HIV-1; GBV-C; AFM; Lipid monolayer; Peptide–peptide binding

Small-angle neutron scattering studies of the effects of amphotericin B on phospholipid and phospholipid–sterol membrane structure by F. Foglia; A.F. Drake; A.E. Terry; S.E. Rogers; M.J. Lawrence; D.J. Barlow (pp. 1574-1580).

Small-angle neutron scattering (SANS) studies have been performed to study the structural changes induced in the membranes of vesicles prepared (by thin film evaporation) from phospholipid and mixed phospholipid–sterol mixtures, in the presence of different concentrations and different aggregation states of the anti-fungal drug, amphotericin B (AmB). In the majority of the experiments reported, the lipid vesicles were prepared with the drug added directly to the lipid dispersions dissolved in solvents favouring either AmB monomers or aggregates, and the vesicles then sonicated to a mean size of ~100nm. Experiments were also performed, however, in which micellar dispersions of the drug were added to pre-formed lipid and lipid–sterol vesicles. The vesicles were prepared using the phospholipid palmitoyloleoylphosphatidylcholine (POPC), or mixtures of this lipid with either 30mol% cholesterol or 30mol% ergosterol. Analyses of the SANS data show that irrespective of the AmB concentration or aggregation state, there is an increase in the membrane thickness of both the pure POPC and the mixed POPC-sterol vesicles—in all cases amounting to ~4Å. The structural changes induced by the drug's insertion into the model fungal cell membranes (as mimicked by POPC-ergosterol vesicles) are thus the same as those resulting from its insertion into the model mammalian cell membranes (as mimicked by POPC-cholesterol vesicles). It is concluded that the specificity of AmB for fungal versus human cells does not arise because of (static) structural differences between lipid–cholesterol-AmB and lipid–ergosterol-AmB membranes, but more likely results from differences in the kinetics of their transmembrane pore formation and/or because of enthalpic differences between the two types of sterol-AmB complexes.Display Omitted►Aamphotericin (AmB) causes an increase in thickness of POPC, and POPC-sterol membranes. ►The increase in membrane thickness is independent of AmB concentration and aggregation state. ►AmB appears to have the same effect on POPC-ergosterol and POPC-cholesterol membranes.

Keywords: Phospholipid; POPC; Cholesterol; Ergosterol; Amphotericin; Lipid vesicles; Small-angle neutron scattering; Anti-fungal drugs; Circular dichroism

Water permeation dynamics of AqpZ: A tale of two states by Lin Xin; Haibin Su; Claus Helix Nielsen; Chuyang Tang; Jaume Torres; Yuguang Mu (pp. 1581-1586).

Molecular dynamics simulations of aquaporin Z homotetramer which is a membrane protein facilitating rapid water movement through the plasma membrane of Escherichia coli were performed. Initial configurations were taken from the open and closed states of crystal structures separately. The resulting water osmotic permeability ( p_f) and diffusive permeability ( p_d) displayed distinct features. Consistent with previous studies, the side chain conformation of arginine189 was found to mediate the water permeability. A potential of mean force (PMF) as a function of the distance between NH1 of R189 and carbonyl oxygen of A117 was constructed based on the umbrella sampling technique. There are multiple local minima and transition states on the PMF. The assignment of the open or closed state was supported by the permeability p_f, calculated within trajectories in umbrella sampling simulations. Our study disclosed a detailed mechanism of the gated water transport.►Dual permeation states of AqpZ revealed by MD simulations. ►Free energy profiles of AqpZ open and closed states obtained using umbrella sampling technique. ►Detailed structure–permeability correlation.

Keywords: AqpZ dual permeation states; PMF; Structure–permeability relation; MD simulations

Functional analysis of the conserved hydrophobic gate region of the magnesium transporter CorA by Svidova Soňa Svidová; Gerhard Sponder; Rudolf J. Schweyen; Kristina Djinović-Carugo (pp. 1587-1591).

The Leu294 residue in the cytoplasmic neck of Thermotoga maritima CorA is considered to be the main gate for Mg²⁺ transport. We created three site-directed mutants at this position: in the Leu294Asp and Leu294Gly mutants we observed a defect in closing of the pore, while in the Leu294Arg mutant not only gating, but also the regulation of Mg²⁺ uptake was affected. Our results confirmed the importance of the Leu294 for gating of Mg²⁺ transport and in addition revealed the influence of the charge and structural features of the amino acid residues on the gating mechanism.► Mutational study of the ion gate of Mg²⁺ transporter CorA. ► Effect of exchange of Leu294 with a positively charged residue on gating of CorA (Leu294Arg mutant). ► Effect of exchange of Leu294 with a negatively charged residue on gating of CorA (Leu294Asp mutant). ► Effect of removal of side chain at position 294 on gating of CorA (Leu294Gly mutant).

Keywords: CorA; Magnesium transport; Hydrophobic gate; Thermotoga maritima

Investigating the effects of L- to D-amino acid substitution and deamidation on the activity and membrane interactions of antimicrobial peptide anoplin by Amy Won; Mourin Khan; Sorin Gustin; Akuvi Akpawu; Deeptee Seebun; Tyler J. Avis; Bonnie O. Leung; Adam P. Hitchcock; Anatoli Ianoul (pp. 1592-1600).

Isolated from the venom sac of solitary spider wasp, Anoplius samariensis, anoplin is the smallest linear α-helical antimicrobial peptide found naturally with broad spectrum activity against both Gram-positive and Gram-negative bacteria, and little hemolytic activity toward human erythrocytes. Deamidation was found to decrease the peptide's antibacterial properties. In the present work, interactions of amidated (Ano-NH₂) and deamidated (Ano-OH) forms of anoplin as well as Ano-NH₂ composed of all D-amino acids (D-Ano-NH₂) with model cell membranes were investigated by means of Langmuir Blodgett (LB) technique, atomic force microscopy (AFM), X-ray photoemission electron microscopy (X-PEEM) and carboxyfluorescein leakage assay in order to gain a better understanding of the effect of these peptide modifications on membrane binding and lytic properties. According to LB, all three peptides form stable monolayers at the air/water interface with Ano-NH₂ occupying a slightly greater area per molecule than Ano-OH. All three forms of the peptide interact preferentially with anionic 1,2-dipalmitoyl- sn-glycero-3-[phospho- rac-(1-glycerol)] (DPPG), rather than zwitterionic 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) lipid monolayer. Peptides form nanoscale clusters in zwitterionic but not in anionic monolayers. Finally, membrane lytic activity of all derivatives was found to depend strongly on membrane composition and lipid/peptide ratio. The results suggest that amidated forms of peptides are likely to possess higher membrane binding affinity due to the increased charge.Display Omitted► D-anoplin has the same antibacterial activity as L-anoplin. ► Deamidated anoplin occupies smaller area in a monolayer. ► Anoplin preferentially interacts with anionic phospholipid and forms clusters in zwitterionic monolayers. ► Lytic activity of anoplin depends on the membrane lipid composition.

Keywords: Antimicrobial peptide; Phospholipid; Monolayer; Atomic force microscopy; Model cell membrane; X-ray photoemission electron microscopy

Hetero-oligomeric cell wall channels (porins) of Nocardia farcinica by Klackta Christian Kläckta; Knorzer Philipp Knörzer; Riess Franziska Rieß; Roland Benz (pp. 1601-1610).

The cell wall of Nocardia farcinica contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of N. farcinica for negatively charged antibiotics. Based on partial sequencing of the protein responsible for channel formation derived from N. farcinica ATTC 3318 we were able to identify the corresponding genes ( nfa15890 and nfa15900) within the known genome of N. farcinica IFM 10152. The corresponding genes of N. farcinica ATTC 3318 were separately expressed in the Escherichia coli BL21DE3Omp8 strain and the N-terminal His₁₀-tagged proteins were purified to homogeneity using immobilized metal affinity chromatography. The pure proteins were designated NfpA_NHis and NfpB_NHis, for N. farcinica porin A and N. farcinica porin B. The two proteins were checked separately for channel formation in lipid bilayers. Our results clearly indicate that the proteins NfpA_NHis and NfpB_NHis expressed in E. coli could only together form a channel in lipid bilayer membranes. This means that the cell wall channel of N. farcinica is formed by a heterooligomer. NfpA and NfpB form together a channel that may structurally be related to MspA of Mycobacterium smegmatis based on amino acid comparison and renaturation procedure.Display OmittedProspective structure of the NfpA/NfpB oligomeric cell wall channel of Nocardia farcinica. It is assumed that four NfpA–NfpB subunits (shown in the four colors) form the cell wall channel. The channel is seen from the surface side.Side view of the prospective structure of the NfpA/NfpB oligomeric cell wall channel of Nocardia farcinica. The four colors correspond to the four NfpA–NfpB subunits of the cell wall channel. The upper side of the channel structure faces the surface of the Nocardia cell. The graphic was designed using rasmol.► The cell wall channel of the mycolata Nocardia farcinica ATTC 3318 was subjected to Edman-degradation. ► Based on BLAST analysis six genes were identified in genome of N. farcinica IFM 10152 coding for cell wall channel. ► The six genes are organized in tandems of two genes separated only by few base pairs from one another. ► The cell wall channel of N. farcinica ATTC 3318 is formed by a heteropolymer of two subunits NfpA and NfpB. ► NfpA and NfpB form together a channel structurally related to MspA of Mycobacterium smegmatis.

Keywords: Cell wall channel; Porin; MspA; Mycolic acid; Lipid bilayer membrane; N. farcinica

Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding by Kathryn A. Smith; John C. Conboy (pp. 1611-1617).

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule–membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl- sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl- sn-glycero-3-phospho-l-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule–membrane interactions in a high-throughput manner.►Lipid microarrays were successfully used to model small molecule–membrane binding. ► Merocyanine 540 binding to lipid microarrays was measured using fluorescence microscopy. ► The impact of cholesterol on dye binding was dependent on the primary lipid component. ► Negatively charged lipids electrostacially repel the monomeric form of the dye from fluid phase bilayers.

Keywords: Abbreviations; MC540; merocyanine 540; MLBAs; micropatterned lipid bilayer arrays; CFM; continuous flow microspotter; l.c.; liquid-crystalline; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; SOPC; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphocholine; DOPS; 1,2-dioleoyl-; sn-; glycero-3-phospho-; l; -serine (sodium salt); CHO; cholesterol; PSLBs; planar supported lipid bilayers; BSA; bovine serum albumin; PBS; phosphate-buffered saline; SUV; small unilamellar vesicle; PDMS; poly(dimethylsiloxane); PC; phosphatidylcholine; PS; phosphatidylserine, phase transition temperatureMC540; Fluorescence microscopy; Lipid bilayer; Microarray; Cholesterol

Spin-echo EPR of Na,K-ATPase unfolding by urea by Rita Guzzi; Mohammad Babavali; Rosa Bartucci; Luigi Sportelli; Mikael Esmann; Derek Marsh (pp. 1618-1628).

Denaturant-perturbation and pulsed EPR spectroscopy are combined to probe the folding of the membrane-bound Na,K-ATPase active transport system. The Na,K-ATPase enzymes from shark salt gland and pig kidney are covalently spin labelled on cysteine residues that either do not perturb or are essential to hydrolytic activity (Class I and Class II –SH groups, respectively). Urea increases the accessibility of water to the spin-labelled groups and increases their mutual separations, as recorded by D₂O interactions from ESEEM spectroscopy and instantaneous spin diffusion from echo-detected EPR spectra, respectively. The greater effects of urea are experienced by Class I groups, which indicates preferential unfolding of the extramembrane domains. Conformational heterogeneity induced by urea causes dispersion in spin-echo phase-memory times to persist to higher temperatures. Analysis of lineshapes from partially relaxed echo-detected EPR spectra indicates that perturbation by urea enhances the amplitude and rate of fluctuations between conformational substates, in the higher temperature regime, and also depresses the glasslike transition in the protein. These non-native substates that are promoted by urea lie off the enzymatic pathway and contribute to the loss of function.► Urea increases water accessibility of kidney Na,K-ATPase Class I groups relative to Class II groups. ► Urea increases conformational heterogeneity of protein substates in Na,K-ATPase. ► Urea enhances the amplitude and rate of fluctuations between substates and depresses the glasslike transition in the Na,K-ATPase.

Keywords: Denaturation; Spin label; SH groups; Echo-detected EPR; ESEEM; Librations; Solvation

Molecular cloning and characterization of a hamster Ca_v1.3 Ca²⁺ channel variant with a long carboxyl terminus by Ho-Won Kang; Jin-Yong Park; Jung-Ha Lee (pp. 1629-1638).

We have cloned a hamster Ca_v1.3 variant with a long carboxyl terminus. This differs from the first hamster Ca_v1.3 clone which has a short carboxyl terminus. When relative expression levels of the two variants were examined using quantitative RT-PCR, the long Ca_v1.3 transcripts were detected abundantly in the brain and testis, moderately in the heart, pancreas, and kidney, and weakly in the lung. Comparatively, the short Ca_v1.3 transcripts were detected less abundantly in most of the tissues. The two Ca_v1.3 variants were reconstituted in Xenopus oocytes and their electrophysiological properties were characterized using a two-electrode voltage clamping method. The long Ca_v1.3 variant was ~5-fold better expressed than the short Ca_v1.3 variant. When Ca²⁺ was used as a charge carrier, the long Ca_v1.3 variant containing an IQ (Ile-Gln) motif displayed strong calcium-dependent inactivation, while the short variant that was deficient of an IQ motif showed little calcium-dependent inactivation. Examination of other biophysical properties revealed that potentials for activation threshold, peak current, and half-activation and inactivation of the long Ca_v1.3 were significantly lower than those of the short Ca_v1.3. These findings suggest that the long carboxyl tail plays crucial roles in not only facilitating calcium-dependent inactivation, but also improving expression and negative shifting of the activation and inactivation properties.► We cloned a hamster Cav1.3 variant with a long carboxyl tail from hamster brain RNA using RT-PCR. ► The long Cav1.3 was better expressed than the short variant in current amplitude and surface density. ► The long carboxyl tail plays in crucial roles for CDI and negative shifting of the gating properties.

Keywords: Hamster Ca; _v; 1.3 Ca; ²⁺; channel variants; Carboxyl terminus; Voltage clamping; EGFP; qRT-PCR

Structure and metal ion binding of the first transmembrane domain of DMT1 by Dan Wang; Yuande Song; Jiantao Li; Chunyu Wang; Fei Li (pp. 1639-1644).

DMT1 is an integral membrane protein with 12 putative transmembrane domains. As a divalent metal ion transporter, it plays an important role in metal ion homeostasis from bacteria to human. Loss-function mutations at the conserved motif DPGN located within the first transmembrane domain (TMD1) of DMT1 indicate the significance of TMD1 in the biological function of the protein. In the present work, we study the structure, topology and metal ion binding of DMT1-TMD1 peptide by nuclear magnetic resonance using sodium dodecyl sulfate and dodecylphosphocholine micelles as membrane mimics. We find that the peptide forms an α-helix-extended segment–α-helix configuration in which the motif DPGN locates at the central flexible region. The N-terminal part of the peptide is deeply embedded in micelles, while the motif section and the C-terminal part are close to the surface of micelles. The peptide can bind to Mn²⁺ and Co²⁺ ions by the side chains of the negatively charged residues in the motif section and the C-terminal part of TMD1. The crucial role of the central flexible region and the C-terminal part of TMD1 in metal ion capture is confirmed by the binding of the N-terminal part truncated TMD1 to metal ions.► DMT1-TMD1 folds as an α-helix-extended segment–α-helix structure. ► The C-terminal part of DMT1-TMD1 is close to the surface of micelles and binds Mn²⁺ and Co²⁺. ► The negatively charged residues play an important role in metal ion binding.

Keywords: DMT1-TMD1; Structure; Topology; Metal ion binding; NMR

Thermostability of Rhodopseudomonas viridis and Rhodospirillum rubrum chromatophores reflecting physiological conditions by Takayuki Odahara; Noriyuki Ishii; Ayako Ooishi; Shinya Honda; Hatsuho Uedaira; Masayuki Hara; Jun Miyake (pp. 1645-1653).

Relationships between growth conditions and thermostability were examined for photosynthetic inner membranes (chromatophores) from Rhodopseudomonas viridis and Rhodospirillum rubrum of which morphology, lipid composition, and protein/lipid rate are rather mutually different. Signals observed by differential scanning calorimetry of the chromatophores were correlated with thermal state transitions of the membrane components by reference to temperature dependencies of circular dichroism and absorption spectra of the purified supramolecule comprising a photoreaction center and surrounding light-harvesting pigment–protein complexes that are the prominent proteins in both membranes. The differential scanning calorimetry curves of those chromatophores exhibited different dependencies on growth stages and environmental temperatures. The obtained result appeared to reflect the differences in the protein/lipid rate and protein–lipid specificity between the two chromatophores.►The dynamics of intact membranes is fundamental for understanding the functions. ►Effect of growth conditions on between dynamics was examined for two chromatophores. ►DSC of the two membranes exhibited different temperature dependence. ►The difference was evaluated well in terms of protein–lipid rate and specificity.

Keywords: Chromatophore; Differential scanning calorimetry; Photoreaction unit; Photosynthetic bacterium; Thermostability

Interaction of epicatechin gallate with phospholipid membranes as revealed by solid-state NMR spectroscopy by Yoshinori Uekusa; Miya Kamihira-Ishijima; Osamu Sugimoto; Takeshi Ishii; Shigenori Kumazawa; Kozo Nakamura; Ken-ichi Tanji; Akira Naito; Tsutomu Nakayama (pp. 1654-1660).

Epicatechin gallate (ECg), a green tea polyphenol, has various physiological effects. Our previous nuclear Overhauser effect spectroscopy (NOESY) study using solution NMR spectroscopy demonstrated that ECg strongly interacts with the surface of phospholipid bilayers. However, the dynamic behavior of ECg in the phospholipid bilayers has not been clarified, especially the dynamics and molecular arrangement of the galloyl moiety, which supposedly has an important interactive role. In this study, we synthesized [¹³C]-ECg, in which the carbonyl carbon of the galloyl moiety was labeled by¹³C isotope, and analyzed it by solid-state NMR spectroscopy. Solid-state³¹P NMR analysis indicated that ECg changes the gel-to-liquid-crystalline phase transition temperature of DMPC bilayers as well as the dynamics and mobility of the phospholipids. In the solid-state¹³C NMR analysis under static conditions, the carbonyl carbon signal of the [¹³C]-ECg exhibited an axially symmetric powder pattern. This indicates that the ECg molecules rotate about an axis tilting at a constant angle to the bilayer normal. The accurate intermolecular–interatomic distance between the labeled carbonyl carbon of [¹³C]-ECg and the phosphorus of the phospholipid was determined to be 5.3±0.1Å by¹³C–³¹P rotational echo double resonance (REDOR) measurements. These results suggest that the galloyl moiety contributes to increasing the hydrophobicity of catechin molecules, and consequently to high affinity of galloyl-type catechins for phospholipid membranes, as well as to stabilization of catechin molecules in the phospholipid membranes by cation–π interaction between the galloyl ring and quaternary amine of the phospholipid head-group.Display Omitted► ECg changes the T_m and dynamic behavior of DMPC bilayers. ► ECg molecules rotate about an axis tilting at a constant angle to the bilayer normal. ► The galloyl moiety contributes the stabilization of ECg molecules in the bilayers. ► Cation–π interaction is considered to be an important factor in this interaction.

Keywords: Abbreviations; ECg; epicatechin gallate; MLVs; multilamellar vesicles; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; NOESY; nuclear Overhauser effect spectroscopy; DD; dipolar decoupling; CP; cross polarization; MAS; magic angle spinning; REDOR; rotational echo double resonanceECg; Phospholipid membranes; Interaction; Solid-state NMR; REDOR; Cation–π interaction

Interactions between selected photosensitizers and model membranes: an NMR classification by Mattia Marzorati; Peter Bigler; Martina Vermathen (pp. 1661-1672).

Membrane interactions of porphyrinic photosensitizers (PSs) are known to play a crucial role for PS efficiency in photodynamic therapy (PDT). In the current paper, the interactions between 15 different porphyrinic PSs with various hydrophilic/lipophilic properties and phospholipid bilayers were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. PS-membrane interactions were deduced from analysis of the main DOPC¹H-NMR resonances (choline and lipid chain signals). Initial membrane adsorption of the PSs was indicated by induced changes to the DOPC choline signal, i.e. a split into inner and outer choline peaks. Based on this parameter, the PSs could be classified into two groups, Type-A PSs causing a split and the Type-B PSs causing no split. A further classification into two subgroups each, A1, A2 and B1, B2 was based on the observed time-dependent changes of the main DOPC NMR signals following initial PS adsorption. Four different time-correlated patterns were found indicating different levels and rates of PS penetration into the hydrophobic membrane interior. The type of interaction was mainly affected by the amphiphilicity and the overall lipophilicity of the applied PS structures. In conclusion, the NMR data provided valuable structural and dynamic insights into the PS-membrane interactions which allow deriving the structural constraints for high membrane affinity and high membrane penetration of a given PS.►¹H-NMR allows to study and model porphyrin membrane interactions. ► Porphyrin uptake by model membranes consists of two main steps. ► Porphyrin structural properties affect rate and amount of membrane uptake. ► High porphyrin lipophilicity and/or amphiphilicity yield high membrane loadings.

Keywords: Abbreviations; PSs; photosensitizers; PDT; photodynamic therapy; PCI; photochemical internalization; DOPC; dioleoyl-phosphatidyl-choline; CE; chlorin e6; RG7; rhodin G7; CEMED; chlorin e6 monoethylene diamine monoamide; m-CEMED; mesochlorin e6 monoethylene diamine amide; MACE; mono-; l; -aspartyl-chlorin e6; Arg-CE; arginine amide of chlorin e6; Tyr-CE; monotyrosine amide of chlorin e6; HPIX; hematoporphyrin IX; DPIX-DSME; deuteroporphyrin IX 2,4-disulfonic acid dimethyl ester; CPIII; coproporphyrin III; DPIX-DS; deuteroporphyrin IX 2,4-disulfonic acid; TCPhP; 5,10,15,20-Tetrakis-(4-carboxyphenyl)-21,23H-porphyrin; TSPhP; 5,10,15,20-Tetrakis(4-sulfonatophenyl)-21,23H-porphyrin; TMPyP; 5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin; THPhP; 5,10,15,20-Tetrakis-(3-hydroxyphenyl)-21,23H-porphyrin; PL; phospholipid; PBS; phosphate buffered salineNMR spectroscopy; Phospholipid bilayer; Membrane distribution; PS-membrane interaction; Chlorin; Porphyrin

On the role of anionic lipids in charged protein interactions with membranes by Igor Vorobyov; Toby W. Allen (pp. 1673-1683).

We investigate the role of anionic lipids in the binding to, and subsequent movement of charged protein groups in lipid membranes, to help understand the role of membrane composition in all membrane-active protein sequences. We demonstrate a small effect of phosphatidylglycerol (PG) lipids on the ability of an arginine (Arg) side chain to bind to, and cross a lipid membrane, despite possessing a neutralizing charge. We observe similar membrane deformations in lipid bilayers composed of phosphatidylcholine (PC) and PC/PG mixtures, with comparable numbers of water and lipid head groups pulled into the bilayer hydrocarbon core, and prohibitively large ~20kcal/mol barriers for Arg transfer across each bilayer, dropping by just 2–3kcal/mol due to the binding of PG lipids. We explore the causes of this small effect of introducing PG lipids and offer an explanation in terms of the limited membrane interaction for the choline groups of PC lipids bound to the translocating ion. Our calculations reveal a surprising lack of preference for Arg binding to PG lipids themselves, but a small increase in interfacial binding affinity for lipid bilayers containing PG lipids. These results help to explain the nature of competitive lipid binding to charged protein sequences, with implications for a wide range of membrane binding domains and cell perturbing peptides.► No preferential binding of anionic lipids to arginine at the bilayer interface. ► Bilayer perturbations and energy barriers for Arg translocation insensitive to PG. ► Balancing water and lipid phosphate contributions yield similar PMFs in PG bilayers. ► “Leading charge” electrostatic description explains insensitivity to PG binding.

Keywords: Abbreviations; PG; phosphatidylglycerol; PC; phosphatidylcholine; PE; phosphatidylethanolamine; DPPG; dipalmitoylphosphatidylglycerol; DPPC; dipalmitoylphosphatidylcholine; Arg; arginine; MD; molecular dynamics; PMF; potential of mean force; RDF; radial distribution function; COM; center of massProtein–lipid interaction; Membrane binding; Anionic lipid; Phosphatidylglycerol; Arginine; Cell penetrating peptide

Isolation and cloning of the K⁺-independent, ouabain-insensitive Na⁺-ATPase by Miguel A. Rocafull; Freddy J. Romero; Luz E. Thomas; Jesús R. del Castillo (pp. 1684-1700).

Primary Na⁺ transport has been essentially attributed to Na⁺/K⁺ pump. However, there are functional and biochemical evidences that suggest the existence of a K⁺-independent, ouabain-insensitive Na⁺ pump, associated to a Na⁺-ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na⁺-ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na⁺-ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg²⁺ dependence, specific Na⁺ stimulation, K⁺ independence, ouabain insensitivity and inhibition by furosemide (IC₅₀: 0.5mM) and vanadate (IC₅₀: 9.1μM). IgY antibodies against purified Na⁺-ATPase did not recognize Na⁺/K⁺-ATPase and vice versa. Analysis of purified Na⁺-ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (β) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na⁺/K⁺-ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148bp ( atna) was cloned, in addition to Na⁺/K⁺-ATPase α1-isoform cDNA (1283bp). In MDCK cells, which constitutively express Na⁺-ATPase, silencing of atna mRNA specifically suppressed Na⁺-ATPase α-subunit and ouabain-insensitive Na⁺-ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na⁺/K⁺-ATPase α1-isoform. These structural and biochemical evidences identify the K⁺-independent, ouabain-insensitive Na⁺-ATPase as a unique P-type ATPase.► K⁺-independent, ouabain-insensitive Na⁺-ATPase was purified from enterocytes. ► It is constituted by an α-subunit of 90kDa and a glycosylated β-subunit of 50kDa. ► Its corresponding cDNA ( atna) was cloned and encodes a protein with 811 amino acids. ► The atna cDNA codes for 6 transmembrane domains and the 9 P-type ATPase motifs. ► Na⁺- and Na⁺/K⁺-ATPase mRNAs seem to be independently transcribed from atp1a1 locus.

Keywords: atna; atp1a1; Intestine; Kidney; P-type ATPase; Sodium pump

Disrupting microtubule network immobilizes amoeboid chemotactic receptor in the plasma membrane by S. de Keijzer; J. Galloway; G.S. Harms; P.N. Devreotes; P.A. Iglesias (pp. 1701-1708).

Signaling cascades are initiated in the plasma membrane via activation of one molecule by another. The interaction depends on the mutual availability of the molecules to each other and this is determined by their localization and lateral diffusion in the cell membrane. The cytoskeleton plays a very important role in this process by enhancing or restricting the possibility of the signaling partners to meet in the plasma membrane. In this study we explored the mode of diffusion of the cAMP receptor, cAR1, in the plasma membrane of Dictyostelium discoideum cells and how this is regulated by the cytoskeleton. Single-particle tracking of fluorescently labeled cAR1 using Total Internal Reflection Microscopy showed that 70% of the cAR1 molecules were mobile. These receptors showed directed motion and we demonstrate that this is not because of tracking along the actin cytoskeleton. Instead, destabilization of the microtubules abolished cAR1 mobility in the plasma membrane and this was confirmed by Fluorescence Recovery after Photobleaching. As a result of microtubule stabilization, one of the first downstream signaling events, the jump of the PH domain of CRAC, was decreased. These results suggest a role for microtubules in cAR1 dynamics and in the ability of cAR1 molecules to interact with their signaling partners.► The GPCR cAR1 shows directed movement in the basal plasma membrane of D. discoideum. ► cAR1 tracking is independent of actin, although actin the speed of movement restricts. ► cAR1 mobility is abolished upon microtubule destabilization. ► Microtubule-dependent cAR1 immobilization impairs downstream signaling.

Keywords: Abbreviations; GPCR; G-protein coupled receptor; cAR1; cAMP receptor 1; TIRFM; Total Internal Reflection Microscopy; FRAP; Fluorescence Recovery after Photobleaching; PH; Pleckstrin Homology; TMR; Tetramethylrhodamine; MSD; Mean Square DisplacementSingle-molecule TIRF microscopy; G-protein coupled receptor; Directed diffusion; Microtubules; Actin; FRAP

L-selectin transmembrane and cytoplasmic domains are monomeric in membranes by Sankaranarayanan Srinivasan; Wei Deng; Renhao Li (pp. 1709-1715).

A recombinant protein termed CLS, which corresponds to the C-terminal portion of human L-selectin and contains its entire transmembrane and cytoplasmic domains (residues Ser473-Arg542), has been produced and its oligomeric state in detergents characterized. CLS migrates in the SDS polyacrylamide gel at a pace that is typically expected from a complex twice of its molecular weight. Additional studies revealed, however, that this is due to residues in the cytoplasmic domain, as mutations in this region or its deletion significantly increased the electrophoretic rate of CLS. Analytical ultracentrifugation and fluorescence resonance energy transfer studies indicated that CLS reconstituted in dodecylphosphocholine detergent micelles is monomeric. When the transmembrane domain of L-selectin is inserted into the inner membrane of Escherichia coli as a part of a chimeric protein in the TOXCAT assay, little oligomerization of the chimeric protein is observed. Overall, these results suggest that transmembrane and cytoplasmic domains of L-selectin lack the propensity to self-associate in membranes, in contrast to the previously documented dimerization of the transmembrane domain of closely related P-selectin. This study will provide constraints for future investigations on the interaction of L-selectin and its associating proteins.►CLS protein (C-terminal fragment of L-selectin, residues S473-R542) exhibits anomalously slow electrophoretic rate in SDS-PAGE. ►Basic-rich juxtamembrane region is responsible for the slow migration in SDS gel. ►CLS is monomeric in zwitterionic detergent micelles by FRET. ►L-selectin transmembrane domain lacks propensity to dimerize in a cell membrane.

Keywords: Abbreviations; CAT; chloramphenicol acetyl transferase; CD; circular dichroism; DPC; dodecylphosphocholine; FRET; fluorescence resonance energy transfer; GST; glutathione; S; -transferase; HFIP; hexafluoroisopropanol; HPLC; high performance liquid chromatography; PSGL-1; P-selectin glycoprotein ligand-1; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCEP–HCl; Tris(2-carboxyethyl) phosphine hydrochloride; TM; transmembrane; TMR; tetramethylrhodamineL-selectin; Transmembrane domain; Membrane protein; TOXCAT; Fluorescence resonance energy transfer; SDS-PAGE

The Pseudomonas aeruginosa membranes: A target for a new amphiphilic aminoglycoside derivative? by Myriam Ouberai; Farid El Garch; Antoine Bussiere; Mickael Riou; David Alsteens; Laurence Lins; Isabelle Baussanne; Dufrene Yves F. Dufrêne; Robert Brasseur; Jean-Luc Decout; Marie-Paule Mingeot-Leclercq (pp. 1716-1727).

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3′,4′,6-tri-2-naphtylmethylene neamine (3′,4′,6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3′,4′,6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3′,4′,6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3′,6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.► An amphiphilic neamine derivative active against sensitive and resistant bacteria. ►Neamine derivative interacts and destabilizes P. aeruginosa lipid membrane. ► Amphiphilic neamine derivative: from an intracellular to a membrane target activity. ►Small amphiphilic aminoglycosides targeting lipid membrane.

Keywords: Aminoglycosides; Naphthylmethylene Neamine derivatives; Pseudomonas aeruginosa; Lipid membrane

A patch clamp study on the electro-permeabilization of higher plant cells: Supra-physiological voltages induce a high-conductance, K⁺ selective state of the plasma membrane by Lars H. Wegner; Bianca Flickinger; Christian Eing; Berghofer Thomas Berghöfer; Petra Hohenberger; Wolfgang Frey; Peter Nick (pp. 1728-1736).

Permeabilization of biological membranes by pulsed electric fields (“electroporation”) is frequently used as a tool in biotechnology. However, the electrical properties of cellular membranes at supra-physiological voltages are still a topic of intensive research efforts. Here, the patch clamp technique in the whole cell and the outside out configuration was employed to monitor current–voltage relations of protoplasts derived from the tobacco culture cell line “Bright yellow-2”. Cells were exposed to a sequence of voltage pulses including supra-physiological voltages. A transition from a low-conductance (~0.1nS/pF) to a high-conductance state (~5nS/pF) was observed when the membrane was either hyperpolarized or depolarized beyond threshold values of around −250 to −300mV and +200 to +250mV, respectively. Current–voltage curves obtained with ramp protocols revealed that the electro-permeabilized membrane was 5–10 times more permeable to K⁺ than to gluconate. The K⁺ channel blocker tetraethylammonium (25mM) did not affect currents elicited by 10ms-pulses, suggesting that the electro-permeabilization was not caused by a non-physiological activation of K⁺ channels. Supra-physiological voltage pulses even reduced “regular” K⁺ channel activity, probably due to an increase of cytosolic Ca²⁺ that is known to inhibit outward-rectifying K⁺ channels in Bright yellow-2 cells. Our data are consistent with a reversible formation of aqueous membrane pores at supra-physiological voltages.► This is the first patch clamp study on higher plant cell membranes at extreme voltages that improves our understanding of electroporation in plant cells and beyond. ► Below -250 to -300mV and above 200to 250mV the conductance increased ~50fold and membranes were 5-10 times more permeable to K+ than to gluconate, but electro-permeabilization was not due to a non-physiological K+ channel activation.

Keywords: Abbreviations; BSA; bovine serum albumin; BTP; Bis–Tris propane; BY-2; Bright yellow-2; EGTA; ethylene glycol tetraacetic acid; E; _rev.; reversal potential; NTORK; Nicotiana tabacum; outward rectifying K; ⁺; channel; SD; standard deviation; SE; standard error; TEA; tetraethylammoniumElectroporation; Threshold potential; Patch clamp; Tobacco cell culture; Bright yellow-2 (BY-2)

Deciphering a mechanism of membrane permeabilization by α-hordothionin peptide by Svetlana V. Oard (pp. 1737-1745).

α-Hordothionin (αHTH) belongs to thionins, the plant antimicrobial peptides with membrane-permeabilizing activity which is associated with broad-range antimicrobial activity. Experimental data have revealed a phospholipid-binding site and indicated formation of ion channels as well as membrane disruption activity of thionin. However, the mechanism of membrane permeabilization by thionin remained unknown. Here it is shown that thionin is a small water-selective channel. Unbiased high-precision molecular modeling revealed formation of a water-selective pore running through the αHTH double α-helix core when the peptide interacted with anions. Anion-induced unfolding of the C-end of the α2-helix opened a pore mouth. The pore started at the α2 C-end between the hydrophilic and the hydrophobic regions of the peptide surface and ended in the middle of the unique hydrophobic region at the C-end of the α1-helix. Highly conserved residues including cysteines and tyrosine lined the pore walls. A large positive electrostatic potential accumulated inside the pore. The narrow pore was, nonetheless, sufficient to accommodate at least one water molecule along the channel except for two constriction sites. Both constriction sites were formed by residues participating in the phospholipid-binding site. The channel properties resembled that of aquaporins with two selectivity filters, one at the entrance, inside the α2 C-end cavity, and a second in the middle of the channel. It is proposed that the αHTH water channel delivers water molecules to the bilayer center that leads to local membrane disruption. The proposed mechanism of membrane permeabilization by thionins explains seemingly controversial experimental data.► Formation of a water-selective pore running through the thionin α-helix core. ► Unfolding of the C-end of the α2-helix opens a pore mouth. ► Highly conserved cysteines and tyrosine line the pore walls. ► Thionin is a small water channel with properties resembling that of aquaporins. ► It is proposed that the thionin water channel delivers water to the bilayer center.

Keywords: Antimicrobial peptides; Thionins; Membrane permeabilizing activity; Mechanism

Exploring the conformational dynamics and membrane interactions of PorB from C. glutamicum: A multi-scale molecular dynamics simulation study by Pineiro Ángel Piñeiro; Peter J. Bond; Syma Khalid (pp. 1746-1752).

Members of the gram-positive mycolata bacteria have unusual cell envelopes which help them to avoid the immune system and the effects of most antibiotics, whilst rendering them permeable to solutes of importance in industrial bioconversion. It is therefore of interest to understand the molecular mechanisms for this selective permeability. PorB is an unusual porin from the outer membrane (OM) of Corynebacterium glutamicum. It has been proposed as an atypical α-helical, symmetrical homo-pentameric architecture, with an unusual distribution of polar amino acids on its surface. The proposed structure is too short to traverse a typical phospholipid bilayer, in contrast with the β-barrel porins of Gram-negative bacteria. Nevertheless, it has been shown to form small anion-selective channels in membranes typical of Escherichia coli. To further understand its function, we have performed ~400ns of all-atom and ~270 μs of coarse-grained simulations of PorB in a range of membrane mimetic and phospholipid milieus. Our results suggest that PorB can undergo spontaneous conformational rearrangements that allow it to adapt to its local lipid environment. We speculate that the increased flexibility of this α-helical porin in comparison with rigid β-barrels may be an adaptation for the heterogeneous mycolic OM, and explains its demonstrated ability to form measurable pores with phospholipid membranes.► The employment of atomistic molecular dynamics simulations to identify a key conformational change that gives a better representation of the protein in a membrane environment compared to the x-ray structure. ► The demonstration that Ca2+ ions within the pore are essential for the stability of the model pentamer. ► The demonstration that the x-ray structure-derived model does not fit well into a phospholipid bilayer but that the modified model does. ► The identification of two types of lipid behaviour around the protein that is consistent with other membrane proteins. ► Determination of protein–lipid interaction lifetimes. These are quite short lived.Display Omitted

Keywords: Outer membrane protein; Porin; Molecular dynamics; Coarse-grained; Multiscale; C. glutamicum

Interactions of the AT₁ antagonist valsartan with dipalmitoyl-phosphatidylcholine bilayers by C. Potamitis; P. Chatzigeorgiou; E. Siapi; K. Viras; T. Mavromoustakos; A. Hodzic; G. Pabst; F. Cacho-Nerin; P. Laggner; M. Rappolt (pp. 1753-1763).

Valsartan is a marketed drug with high affinity to the type 1 angiotensin (AT₁) receptor. It has been reported that AT₁ antagonists may reach the receptor site by diffusion through the plasma membrane. For this reason we have applied a combination of differential scanning calorimetry (DSC), Raman spectroscopy and small and wide angle X-ray scattering (SAXS and WAXS) to investigate the interactions of valsartan with the model membrane of dipalmitoyl-phosphatidylcholine (DPPC). Hence, the thermal, dynamic and structural effects in bulk as well as local dynamic properties in the bilayers were studied with different valsartan concentrations ranging from 0 to 20mol%. The DSC experimental results showed that valsartan causes a lowering and broadening of the phase transition. A splitting of the main transition is observed at high drug concentrations. In addition, valsartan causes an increase in enthalpy change of the main transition, which can be related to the induction of interdigitation of the lipid bilayers in the gel phase. Raman spectroscopy revealed distinct interactions between valsartan with the lipid interface localizing it in the polar head group region and in the upper part of the hydrophobic core. This localization of the drug molecule in the lipid bilayers supports the interdigitation view. SAXS measurements confirm a monotonous bilayer thinning in the fluid phase, associated with a steady increase of the root mean square fluctuation of the bilayers as the valsartan concentration is increased. At high drug concentrations these fluctuations are mainly governed by the electrostatic repulsion of neighboring membranes. Finally, valsartans' complex thermal and structural effects on DPPC bilayers are illustrated and discussed on a molecular level.► Valsartan, an antihypertensive drug with high affinity to the AT1 receptor. ► A combination of DSC, Raman spectroscopy and SAXS/WAXS is applied. ► Interactions of valsartan (0 to 20 mol%) with a model membrane system are presented. ► The localization of the drug molecule in the membranes induces lipid interdigitation. ► Valsartans' thermal and structural effects are illustrated on a molecular level.

Keywords: Valsartan; Dipalmitoyl-phosphatidylcholine; Bilayer; Small-angle and wide-angle X-ray scattering; Raman spectroscopy; Differential scanning calorimetry

Microcin J25 membrane interaction: Selectivity toward gel phase by Fernando Dupuy; Roberto Morero (pp. 1764-1771).

The interaction of the tryptophan-containing variant of microcin J25, MccJ25 I13W, with phosphatidylcholine membranes was studied by fluorescence spectroscopy techniques. The peptide was able to interact with dimiristoylphophatidylcholine and dipalmitoylphosphatidylcholine liposomes only when the membranes were in gel phase, as was demonstrated by the blue shift of the intrinsic fluorescence of MccJ25 I13W. The binding isotherm showed a cooperative partition of the peptide toward the membrane and the binding constant increased as the temperature decreased and the order parameter increased. No interaction with liquid crystalline membranes was observed. Studies of dynamic quenching of the fluorescence indicated that the peptide penetrated the lipid bilayer and was located primarily in the interfacial region. Our results suggest that MccJ25 I13W interacts with gel phase phospholipids and increases both its own affinity for the bilayer and the membrane permeability of small ions.► The peptide MccJ25 I13W interacts with gel phase membranes at the interphacial zone. ► The binding isotherm of MccJ25 I13W shows a cooperative partition toward membrane. ► MccJ25 I13W stabilizes the gel phase and induces membrane permeabilization.

Keywords: Microcin; Membrane; Fluorescence quenching; Binding isotherm; Permeabilization

Extracellular K⁺ elevates outward currents through Kir2.1 channels by increasing single-channel conductance by Tai-An Liu; Hsueh-Kai Chang; Ru-Chi Shieh (pp. 1772-1778).

Outward currents through inward rectifier K⁺ channels (Kir) play a pivotal role in determining resting membrane potential and in controlling excitability in many cell types. Thus, the regulation of outward Kir current ( I_K1) is important for appropriate physiological functions. It is known that outward I_K1 increases with increasing extracellular K⁺ concentration ([ K⁺]_o), but the underlying mechanism is not fully understood. A “K⁺-activation of K⁺-channel” hypothesis and a “blocking-particle” model have been proposed to explain the [ K⁺]_o-dependence of outward I_K1. Yet, these mechanisms have not been examined at the single-channel level. In the present study, we explored the mechanisms that determine the amplitudes of outward I_K1 at constant driving forces [membrane potential ( V_m) minus reversal potential ( E_K)]. We found that increases in [ K⁺]_o elevated the single-channel current to the same extent as macroscopic I_K1 but did not affect the channel open probability at a constant driving force. In addition, spermine-binding kinetics remained unchanged when [ K⁺]_o ranged from 1 to 150mM at a constant driving force. We suggest the regulation of K⁺ permeation by [ K⁺]_o as a new mechanism for the [ K⁺]_o-dependence of outward I_K1.► We study the regulation of outward Kir2.1 currents by [K⁺]_o. ► [K⁺]_o up-regulates single-channel conductance but not open probability. ► [K⁺]_o-effect on outward Kir2.1 currents is not related to relief of channel block. ► [K⁺]_o directly modulates K⁺ permeation in Kir2.1 channels.

Keywords: Abbreviations; Kir; inward rectifier K; ⁺; channels; I; _K1; Kir2.1 currents; [; K; ⁺; ]; _o; extracellular K; ⁺; concentration; V; _m; membrane potential; k; ₁; rate constant for the transition from the closed to the open state; k; ₋₁; rate constant for the transition from the open to the closed state; μ; the association rate constant for spermine binding; λ; the dissociation rate constant for spermine binding; i; _K1; single-channel Kir2.1 currentsInward-rectification; Permeation; Kinetics; Excitability

Corrigendum to “A pyrrolidine-based specific inhibitor of cytosolic phospholipase A₂α blocks arachidonic acid release in a variety of mammalian cells.” [Biochem. Biophys. Acta 1513 (2001) 160–166] by Farideh Ghomashchi; Allison Stewart; Ying Hefner; Sasanka Ramanadham; John Turk; Christina C. Leslie; Michael H. Gelb (pp. 1779-1779).

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BBA - Biomembranes (v.1808, #6)