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BBA - Biomembranes (v.1778, #6)

Editorial Board (pp. ii).

Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding by Samuel Murail; Jean-Claude Robert; Coic Yves-Marie Coïc; Jean-Michel Neumann; Mariano A. Ostuni; Zhin-Xing Yao; Vassilios Papadopoulos; Nadège Jamin; Lacapere Jean-Jacques Lacapère (pp. 1375-1381).

Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from¹H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using¹H–¹⁵N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.

Keywords: Abbreviations; DPC; dodecyl phosphocholine; DIPEA; N; ,; N; -diisopropylethylamine; DSS; dimethylsilapentane-sulfonic acid, sodium salt; HATU; 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium hexafluorophosphate; HSQC; heteronuclear single quantum correlation; NOESY; nuclear Overhauser enhancement spectroscopy; PAL-PEG-PS; 5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid-polyethylene glycol-polystyrene; TOCSY; Total correlation spectroscopyTSPO; PBR; NMR and CD spectroscopy; Drug ligand binding

Intertissue regulation of carnitine palmitoyltransferase I (CPTI): Mitochondrial membrane properties and gene expression in rainbow trout ( Oncorhynchus mykiss) by Andrea J. Morash; Makiko Kajimura; Grant B. McClelland (pp. 1382-1389).

Carnitine palmitoyltransferase (CPT) I is regulated by several genetic and non-genetic factors including allosteric inhibition, mitochondrial membrane composition and/or fluidity and transcriptional regulation of enzyme content. To determine the intrinsic differences in these regulating factors that may result in differences between tissues in fatty acid oxidation ability, mitochondria were isolated from red, white and heart muscles and liver tissue from rainbow trout. Maximal activity ( V_max) for β-oxidation enzymes and citrate synthase per mg tissue protein as well as CPT I in isolated mitochondria followed a pattern across tissues of red muscle>heart>white muscle>liver suggesting both quantitative and qualitative differences in mitochondria. CPT I inhibition showed a similar pattern with the highest malonyl-CoA concentration to inhibit activity by 50% (IC₅₀) found in red muscle while liver had the lowest. Tissue malonyl-CoA content was highest in white muscle with no differences between the other tissues. Interestingly, the gene expression profiles did not follow the same pattern as the tissue enzyme activity. CPT I mRNA expression was greatest in heart>red muscle>white muscle>liver. In contrast, PPARα mRNA was greatest in the liver>red muscle>heart>white muscle. There were no significant differences in the mRNA expression of PPARβ between tissues. As well, no significant differences were found in the mitochondrial membrane composition between tissues, however, there was a tendency for red muscle to exhibit higher proportions of PUFAs as well as a decreased PC:PE ratio, both of which would indicate increased membrane fluidity. In fact, there were significant correlations between IC₅₀ of CPT I for malonyl-CoA and indicators of membrane fluidity across tissues. This supports the notion that sensitivity of CPT I to its allosteric regulator could be modulated by changes in mitochondrial membrane composition and/or fluidity.

Keywords: Carnitine palmitoyltransferase I; Enzyme inhibition; Mitochondria; Membrane composition; Malonyl-CoA

Binding and interactions of L-BABP to lipid membranes studied by molecular dynamic simulations by Marcos Ariel Villarreal; Massimiliano Perduca; Hugo L. Monaco; Guillermo G. Montich (pp. 1390-1397).

Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a β-barrel of ten strands capped with a short helix–loop–helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.

Keywords: Abbreviations; L-BABP; chicken liver bile acid-binding protein; FABP; fatty acid-binding protein; DLPS; 1-lauroyl-2-lauroyl-; sn; -glycero-3-phosphoserine; DPPC; 1-palmitoyl-2-palmitoyl-; sn; -glycero-3-phosphocholine; FT-IR; Fourier transform infrared; rmsd; root mean square deviationL-BABP; FABP; Molecular dynamic simulation; Lipid–protein interaction; Electric field; Conformational change

Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-β-cyclodextrin. Role of low-density caveolae-like membranes by Jorgelina Buschiazzo; Ida C. Bonini; Telma S. Alonso (pp. 1398-1406).

The invaginated structure of caveolae seems to provide an optimal environment for hormone binding leading to oocyte meiotic maturation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from Bufo arenarum oocytes and we modulated cellular cholesterol to further understand how these domains perform their regulatory functions in the amphibian system. Light membranes derive from the plasma membrane as suggested by the enrichment in the activity of 5′nucleotidase. Lipid analysis by chromatography techniques revealed that this fraction is enriched in phosphatidylserine and cholesterol and that it evidences an important level of sphingomyelin. The finding of a single 21 kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, c-Src is significantly associated to this fraction. Cholesterol content of oocytes treated with methyl-β-cyclodextrin (MβCD) decreased when compared to control oocytes. Drug treatment inhibited meiotic maturation in a dose-dependent manner and affected the localization of caveolin and c-Src among membrane fractions. Repletion of cholesterol showed a recovery of the ability of MβCD-treated oocytes to mature, particularly at the 25 mM concentration in which reversibility was close to the control level. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes.

Keywords: Abbreviations; ANOVA; analysis of variance; Chol; cholesterol; DPG; diphosphatidylglycerol; DRM; detergent-resistant membranes; ECL; enhanced chemiluminescence; EH; extra heavy membranes; ERK; extracellular signal-regulated kinase; FRET; fluorescence resonance energy transfer; GPI; glycosylphosphatidylinositol; GVBD; germinal vesicle breakdown; H; heavy membranes; HDM; high-density membrane; HRP; horseradish peroxidase; L; light membranes; LDM; low-density membrane; LPC; lysophosphatidylcholine; LPE; lysophosphatidylethanolamine; MβCD; methyl-β-cyclodextrin; Pi; inorganic phosphorus; PA; phosphatidic acid; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositol; PL; phospholipid; PS; phosphatidylserine; PDGF; platelet-derived growth factor; PKC; protein kinase C; PMSF; phenylmethanesulfonyl fluoride; PR; progesterone receptor; PVDF; polyvinylidene difluoride; SDS- PAGE; SDS-polyacrylamide gel electrophoresis; SM; sphingomyelin; SPT; single-particle tracking; TH; total homogenate; TLC; thin-layer chromatographyAmphibian oocytes; Meiotic maturation; Caveolae; Src kinases; Cholesterol; Phospholipids

N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter ( HsPCFT) by Ersin Selcuk Unal; Rongbao Zhao; Andong Qiu; I. David Goldman (pp. 1407-1414).

The human proton-coupled folate transporter ( HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~55 kDa), higher than predicted (~50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~47 kDa protein; substitution of both sites gave a smaller (~35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH₂ or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.

Keywords: Abbreviations; RFC; reduced folate carrier; PCFT; proton-coupled folate transporter; SLC; solute carrier family; TMDs; transmembrane domains; PNGaseF; peptide-N; ⁴; -(; N; -acetyl-β-; d; -glucosaminyl)asparagine amidase F; Endo H; endo-β-; N; -acetylglucosaminidase H; MTX; methotrexate; DTT; dithiothreitol; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; OMIM; Online Mendelian Inheritance in ManPCFT, proton-coupled folate transporter; HCP1; PCFT/HCP1; PCFT glycosylation; Folate transport; Intestinal folate absorption; PCFT secondary structure; Hereditary folate malabsorption (HFM); SLC46A1

pH and monovalent cations regulate cytosolic free Ca²⁺ in E. coli by Riffat Naseem; I. Barry Holland; Annick Jacq; Kenneth T. Wann; Anthony K. Campbell (pp. 1415-1422).

The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca²⁺ in E. coli through Ca²⁺ influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca²⁺ was 0.2–0.5 µM. In the presence of external Ca²⁺ (1 mM) at alkaline pH this rose to 4 µM, being reduced to 0.9 µM at acid pH. Removal of external Ca²⁺ caused an immediate decrease in cytosolic free Ca²⁺ at 50–100nM s−¹. Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca²⁺/H⁺exchanger, appeared not to be a major Ca²⁺-efflux pathway. In the absence of added Na⁺, but with 1 mM external Ca²⁺, cytosolic free Ca²⁺ rose to approximately 10 µM. The addition of Na⁺(half maximum 60 mM) largely blocked this increase and immediately stimulated Ca²⁺ efflux. However, this effect was not specific, since K⁺ also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca²⁺ efflux. The results were consistent with H⁺ and monovalent cations competing with Ca²⁺ for a non-selective ion influx channel. Ca²⁺ entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca²⁺ in Escherichia coli. The number of Ca²⁺ ions calculated to move per cell per second ranged from <1 to 100, depending on conditions. Yet a single eukaryote Ca²⁺ channel, conductance 100 pS, should conduct >6 million ions per second. This raises fundamental questions about the nature and regulation of Ca²⁺ transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.

Keywords: Calcium channels; Calcium efflux; Bacteria; Polyhydroxybutyrate; Aequorin; Lanthanum

Molecular dynamics simulation of structural changes of lipid bilayers induced by shock waves: Effects of incident angles by Kenichiro Koshiyama; Tetsuya Kodama; Takeru Yano; Shigeo Fujikawa (pp. 1423-1428).

Unsteady and nonequilibrium molecular dynamics simulations of the response of dipalmitoylphosphatidylcholine (DPPC) bilayers to the shock waves of various incident angles are presented. The action of an incident shock wave is modeled by adding a momentum in an oblique direction to water molecules adjacent to a bilayer. We thereby elucidate the effects of incident shock angles on (i) collapse and rebound of the bilayer, (ii) lateral displacement of headgroups, (iii) tilts of lipid molecules, (iv) water penetration into the hydrophobic region of the bilayer, and (v) momentum transfer across the bilayer. The number of water molecules delivered into the hydrophobic region is found to be insensitive to incident shock angles. The most important structural changes are the lateral displacement of headgroups and tilts of lipid molecules, which are observed only in the half of the bilayer directly exposed to a shock wave for all incident shock angles studied here. As a result, only the normal component of the added oblique momentum is substantially transferred across the bilayer. This also suggests that the irradiation by shock waves may induce a jet-like streaming of the cytoplasm toward the nucleus.

Keywords: Acoustic wave; Cell membrane permeabilization; Impulse; Shear force; Sonoporation; Ultrasound

Nectin-like molecule 1 is a glycoprotein with a single N-glycosylation site at N290KS which influences its adhesion activity by Jing Gao; Tao Chen; Guangyu Hu; Yanhua Gong; Boqin Qiang; Jiangang Yuan ⁎; Xiaozhong Peng ⁎ (pp. 1429-1435).

Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3, from now on referred to as NECL1, is a neural tissue-specific immunoglobulin-like cell–cell adhesion molecule which has Ca²⁺-independent homo- or heterophilic cell–cell adhesion activity and plays an important role in the formation of synapses, axon bundles and myelinated axons. Here we first detected the expression of NECL1 in human fetal and adult brains, and mouse brains at different developmental stages. The results indicated that two bands with molecular weights of about 62 kDa and 48 kDa were found in human fetal brain, while only one band with a molecular weight of about 48 kDa was found in human adult brain; two bands with molecular weights of about 62 kDa and 48 kDa whose expression level gradually increased were also found from mouse E16 to P14, while only one band with a molecular weight of about 48 kDa was found from P14. Bioinformatics analysis showed there were two putative N-glycosylation sites within human NECL1 at positions N25LS and N290KS and within mouse Necl1 at positions N23LS and N288KS, respectively. There was no O-glycosylation site in either human NECL1 or mouse Necl1. Based on the results of N-Glycosidase F treatment with human fetal brain tissue and lysates from transient transfection with human wild-type or glycosylation site mutant NECL1 in 293ET cells, we demonstrated that human NECL1 is an N-linked glycoprotein with a single glycosylation site at position N290KS. Cell aggregation assay further showed there was an increased adhesion activity after the glycosylation site mutation of NECL1 molecule.

Keywords: Abbreviations; E16; embryonic day 16; NCAM; neural cell adhesion molecule; NECL1; Nectin-like molecule 1; NECL2; Nectin-like molecule 2; NECL5; Nectin-like molecule 5; P7; postnatal day 7; P14; postnatal day 14; pAb; polyclonal antibody; PSA; polysialic acid; WT; wild-typeCell–cell adhesion; Nectin-like molecule 1(Necl1); N-linked glycoprotein

Binding of N-terminal fragments of anthrax edema factor (EF_N) and lethal factor (LF_N) to the protective antigen pore by Michael Leuber; Angelika Kronhardt; Fiorella Tonello; Federica Dal Molin; Roland Benz (pp. 1436-1443).

Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA₆₃, forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA₆₃-channels. Here, in experiments with artificial lipid bilayer membranes, EF_N and LF_N show block of PA₆₃-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF_N, increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA₆₃-channel.

Keywords: Anthrax toxin; Edema factor; Lethal factor; PA channel; N-terminal fragment; Lipid bilayer

Structure analysis of the fourth transmembrane domain of Nramp1 in model membranes by Rong Xue; Shuo Wang; Haiyan Qi; Yuande Song; Chunyu Wang; Fei Li (pp. 1444-1452).

Nramp1 (natural resistance-associated macrophage protein 1) is an integral membrane protein with 12 putative transmembrane domains. As a proton-coupled divalent metal cation transporter, it is involved in defense against intracellular pathogens. Disease-causing mutation in Nramp1 occurring at glycine 169 located within the fourth transmembrane domain (TM4) suggests functional importance of this domain. In this paper, we study the three-dimensional structures of a peptide, corresponding to the TM4 of the wild-type Nramp1, in SDS micelles and 2, 2, 2-trifluoroethanol solvent using CD and NMR spectroscopies. We have found that an α-helix is predominantly induced in membrane-mimetic environments and the folding of the C-terminal residues is regulated by pH in SDS micelles. The peptide is embedded in SDS micelles and self-associated by coiled-coil interactions. The helix of the peptide in TFE is lengthened towards the N-terminus compared with those in SDS micelles at acidic pH and the self-association of the peptide is also observed in TFE. The fact that Mn²⁺ ions are accessible to Asp-14 located in the interior of SDS micelles is found and the binding affinity is increased with increasing pH. The self-association of the peptide may provide a path by which Mn²⁺ ions pass through the membrane.

Keywords: Nramp1-TM4; Structure; Topology; Assembly; NMR; pH titration

Roles of integral protein in membrane permeabilization by amphidinols by Nagy Morsy; Keiichi Konoki; Toshihiro Houdai; Nobuaki Matsumori; Tohru Oishi; Michio Murata; Saburo Aimoto (pp. 1453-1459).

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 μM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.

Keywords: Abbreviations; PC; phosphatidylcholine; GpA; glycophorin A; AM; amphidinol; SDS; sodium dodecyl sulfate; PAGE; polyacrylamide gel electrophoresis; EDTA; ethylenediamine tetraacetic acid; NHS; N; -hydroxysuccinimide; EDC; 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride; CTAB; Cetyltrimethylammonium bromide; SUV; small unilamellar vesicles; LUV; large unilamellar vesicles; MLV; multilamellar vesicles; PBS; phosphate buffered saline; BSA; bovine serum albumin; GpA-TM; glycophorin A transmembrane peptide; NIES; National Institute of Environmental StudiesAntifungal; Amphidinol; Hemolysis; Glycophorin A

Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L by Rohit Sood; Paavo K.J. Kinnunen (pp. 1460-1466).

Sterols impart significant changes to the biophysical properties of lipid bilayers. In this regard the impact of cholesterol on membrane organization and dynamics is particularly well documented and serves for comparison with other sterols. However, the factors underlying the molecular evolution of cholesterol remain enigmatic. To this end, cholesterol attenuates membrane perturbation by the so-called antimicrobial peptides (AMPs), produced ubiquitously by eukaryotic cells to combat bacterial infections by compromising the permeability barrier function of the microbial target membranes. In the present study, we addressed the effects of cholesterol, ergosterol, and lanosterol on the membrane association of two structurally and functionally diverse AMPs viz. LL-37(F27W) and temporin L (TemL) using fluorescence spectroscopy. Interestingly, sterol concentration dependent effects on the membrane association of these peptides were observed. At X_Sterol=0.5 cholesterol was most effective in reducing the membrane intercalation of both LL-37(F27W) and TemL, the corresponding efficiencies of the three sterols decreasing as cholesterol>lanosterol≥ergosterol, and cholesterol>lanosterol>ergosterol. It is conceivable that part of the selection pressure for the chemical evolution of cholesterol may have derived from the ability to protect the AMP-secreting host cell from the membrane damaging action of the antimicrobial peptides.

Keywords: Abbreviations; AMPs; antimicrobial peptides; k; _q; bimolecular quenching constant; Chol; cholesterol; Ergo; ergosterol; EDTA; ethylenediaminetetraacetic acid; Lano; lanosterol; LL-37 (F27W); LL-37 with Phe27 replaced by Trp; LL-37; native LL-37; L/P; lipid/peptide ratio; LUV; large unilamellar vesicles; l; _o; liquid ordered phase; l; _d; liquid disordered phase; M; ₁; order parameter from NMR; PC; phosphatidylcholine; PG; phosphatidylglycerol; P/L; peptide/lipid ratio; Q; quencher; Spm; sphingomyelin; SOPC; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; S; _o; solid ordered phase; K; _SV; Stern–Volmer quenching constant; λ; _max; fluorescence emission maximum; TemL; temporin LAntimicrobial peptides; Sterols; Liposomes, Hydrophobicity; Fluorescence spectroscopy

Controlled release mechanisms of spontaneously forming unilamellar vesicles by Mu-Ping Nieh; John Katsaras; Xiaoyang Qi (pp. 1467-1471).

Spontaneously forming small unilamellar vesicles (SULVs) are easy to prepare and show great promise for use in delivering therapeutic payloads. We report of SULVs made up of the ternary phospholipid mixture, dimyristoyl-phosphatidylcholine (DMPC), dihexanoyl-phosphatidylcholine (DHPC) and dimyristoyl-phosphatidylglycerol (DMPG), which have been characterized by small angle neutron scattering (SANS). These low-polydispersity (0.14–0.19) SULVs range in size (i.e., radius) from 110 to 215 Å and are capable of entrapping, and subsequently releasing, hydrophilic molecules (e.g., fluorescent dyes and quenchers) in a controlled fashion over two different temperature ranges. The low-temperature release mechanism involves the SULVs transforming into discoidal micelles, with an onset temperature ( T_o) of ~32 °C, while the high-temperature release mechanism is more gradual, presumably the result of defects formed through the continuous dissolution of DHPC into solution. Both of these mechanisms differ from other, previously reported thermosensitive liposomes.

Keywords: Spontaneously forming unilamellar vesicles; Small angle neutron scattering; Thermosensitive; Controlled release; Phospholipid; DMPC; DHPC; DMPG; Low-polydispersity; Fluorescence

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely by Julia Caussin; Gert S. Gooris; Michelle Janssens; Joke A. Bouwstra (pp. 1472-1482).

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.

Keywords: Abbreviations; CER; ceramide; CHOL; cholesterol; FFA; free fatty acids; DFFA; perdeuterated free fatty acids; SAXD; small angle X-ray diffraction; WAXD; wide angle X-ray diffraction; FTIR; Fourier transform infrared (spectroscopy); LPP; long periodicity phase; SPP; short periodicity phaseFTIR; Stratum corneum; Lipid; Ceramide; Glycerol

Membrane partitioning of various δ-opioid receptor forms before and after agonist activations: The effect of cholesterol by Andre Aurore André; Gérald Gaibelet; Laurent Le Guyader; Michèle Welby; André Lopez; Chantal Lebrun (pp. 1483-1492).

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168–9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR⁎) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR⁎ is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR⁎ was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor–G-protein interaction.

Keywords: GPCR; hDOR; Lateral organization; Raft; Hydrophobic mismatch; Cholesterol

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins by Ulrich Terpitz; Daniel Raimunda; Markus Westhoff; Vladimir L. Sukhorukov; Beauge Luis Beaugé; Ernst Bamberg; Dirk Zimmermann (pp. 1493-1500).

Giant protoplasts of Saccharomyces cerevisiae of 10–35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1–12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66±0.07 µF/cm²) and conductance (23–44 µS/cm²) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.

Keywords: Patch clamp; Electrofusion; Yeast; Saccharomyces cerevisiae; Giant protoplast; Channelrhodopsin-2

Importance of the phosphocholine linkage on sphingomyelin molecular properties and interactions with cholesterol; a study with phosphate oxygen modified sphingomyelin-analogues by Bjorkbom Anders Björkbom; Tetsuya Yamamoto; Satoshi Kaji; Shuji Harada; Shigeo Katsumura; J. Peter Slotte (pp. 1501-1507).

We have characterized the molecular properties and membrane behavior of synthetically modified sphingomyelin analogues, modified on the oxygen connecting the phosphocholine group to the ceramide backbone. The oxygen was replaced with an S-atom (S-PSM), an NH-group (NH-PSM) or a CH₂-group (CH₂-PSM). Diphenylhexatriene and Laurdan anisotropy experiments showed that an S-linkage increased and NH- and CH₂-linkages decreased the stability of PSM-analogue bilayer membranes as compared to PSM. When the polarity of the interface was probed using Laurdan, S-PSM appeared to have a lower polarity as compared to PSM whereas NH-PSM and CH₂-PSM had higher polarities of their respective interfaces. Fluorescence quenching-studies with cholestatrienol showed that all compounds formed SM/cholesterol-rich domains. The S-PSM/cholesterol and PSM/cholesterol domains displayed a similar thermostability, whereas NH-PSM/cholesterol and CH₂-PSM/cholesterol domains were less thermostable. DSC on vesicles containing the PSM-analogues showed a more complex melting behavior as compared to PSM, whereas equimolar mixtures of the PSM-analogues and PSM showed almost ideal mixing with PSM for NH- and S-PSM. Our data show that the properties of the bond linking the phosphocholine head group to the 1-hydroxyl on the ceramide molecule is important for the stability of SM/SM and SM/cholesterol interactions.

Keywords: Abbreviations; 7SLPC; 1-palmitoyl-2-stearoyl-(7-DOXYL)-; sn; -glycero-3-phosphocholine; CH; ₂; -PSM; PSM with its proximal esteric oxygen replaced with a CH; ₂; -group; CTL; cholesta-5,7,9(11)-trien-3-beta-ol; DHSM; d; -; erythro-N; -palmitoyl-dihydrosphingomyelin; DPH; 1,6-diphenyl-1,3,5-hexatriene; DSC; differential scanning calorimetry; GP; generalized polarization; Laurdan; 6-lauroyl-2-(; N; ,; N; -dimethylamino)naphthalene; l; _d; liquid disordered-phase; l; _o; liquid ordered-phase; NH-PSM; PSM with its proximal esteric oxygen replaced with an NH-group; PCPE; N; -palmitoyl ceramide phosphoethanolamine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; d; -; erythro-N; -palmitoyl-sphingomyelin; SM; sphingomyelin; S-PSM; PSM with its proximal esteric oxygen replaced with a S-atom; T; _m; mid temperature of the transition between gel and l; _d; -phaseSphingomyelin interactions; Fluorescence quenching; Anisotropy; Phosphocholine linkage; Membrane–water interface; Differential scanning calorimetry

Signaling by the human serotonin_1A receptor is impaired in cellular model of Smith–Lemli–Opitz Syndrome by Yamuna Devi Paila; Mamidanna R.V.S. Murty; Mariappanadar Vairamani; Amitabha Chattopadhyay (pp. 1508-1516).

The Smith–Lemli–Opitz Syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. SLOS is clinically diagnosed by reduced plasma levels of cholesterol along with elevated levels of 7-dehydrocholesterol (and its positional isomer 8-dehydrocholesterol) and the ratio of their concentrations to that of cholesterol. Since SLOS is associated with neurological deformities and malfunction, exploring the function of neuronal receptors and their interaction with membrane cholesterol under these conditions assumes significance. We have earlier shown the requirement of membrane cholesterol for the ligand binding function of an important neurotransmitter G-protein coupled receptor, the serotonin_1A receptor. In the present work, we have generated a cellular model of SLOS using CHO cells stably expressing the human serotonin_1A receptor. This was achieved by metabolically inhibiting the biosynthesis of cholesterol, utilizing a specific inhibitor (AY 9944) of the enzyme required in the final step of cholesterol biosynthesis. We utilized this cellular model to monitor the function of the human serotonin_1A receptor under SLOS-like condition. Our results show that ligand binding activity, G-protein coupling and downstream signaling of serotonin_1A receptors are impaired in SLOS-like condition, although the membrane receptor level does not exhibit any reduction. Importantly, metabolic replenishment of cholesterol using serum partially restored the ligand binding activity of the serotonin_1A receptor. These results are potentially useful in developing strategies for the future treatment of the disease since intake of dietary cholesterol is the only feasible treatment for SLOS patients.

Keywords: Abbreviations; 5-HT; _1A; receptor; 5-hydroxytryptamine-1A receptor; 5-HT; _1A; R-EYFP; 5-hydroxytryptamine-1A receptor tagged to enhanced yellow fluorescent protein; 7-DHC; 7-dehydrocholesterol; 8-DHC; 8-dehydrocholesterol; 8-OH-DPAT; 8-hydroxy-2(di-; N; -propylamino)tetralin; 7-DHCR; 3β-hydroxy-steroid-Δ; ⁷; -reductase; AY 9944; trans; -1,4-bis(2-dichlorobenzylaminomethyl)cyclohexane dihydrochloride; BCA; bicinchoninic acid; DMPC; dimyristoyl-; sn; -glycero-3-phosphocholine; DPH; 1,6-diphenyl-1,3,5-hexatriene; EYFP; enhanced yellow fluorescent protein; GFP; green fluorescent protein; GPCR; G-protein coupled receptor; GTP-γ-S; guanosine-5′-; O; -(3-thiotriphosphate); IBMX; 3-isobutyl-1-methylxanthine; MβCD; methyl-β-cyclodextrin; PMSF; phenylmethylsulfonyl fluoride; SLOS; Smith–Lemli–Opitz SyndromeCholesterol; 7-dehydrocholesterol; Serotonin; _1A; receptor; G-protein coupling; Downstream signaling; Smith–Lemli–Opitz Syndrome; Cholesterol replenishment

FTIR studies show lipophilic moisturizers to interact with stratum corneum lipids, rendering the more densely packed by Julia Caussin; Gert S. Gooris; Joke A. Bouwstra (pp. 1517-1524).

Lipophilic moisturizers are widely used to treat dry skin. However, their interaction with the lipids in the upper layer of the skin, the stratum corneum (SC), is largely unknown. In the present study this interaction of three moisturizers, isostearyl isostearate (ISIS), isopropyl isostearate (IPIS) and glycerol monoisostearate (GMIS), has been elucidated using lipid mixtures containing isolated ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA), mimicking the lipid composition and organization in SC. The conformational ordering and the lateral packing of the lipid mixtures were examined by Fourier transformed infrared spectroscopy. Equimolar CER:CHOL:FFA mixtures show an orthorhombic to hexagonal phase transition between 22 and 30 °C and an ordered–disordered phase transition between 46 and 64 °C. Addition of 20% m/m ISIS or IPIS increased the thermotropic stability of the orthorhombic lateral packing, while GMIS had no influence. Furthermore, small amounts of all three moisturizers are incorporated into the CER:CHOL:FFA lattice, while the majority of the moisturizer exists in separate domains. Especially the thermotropic stabilization of the orthorhombic lateral packing, which might reduce water loss from the skin, is considered to contribute to the moisturizing effect of IPIS and ISIS in stratum corneum.

Keywords: Abbreviations; SC; stratum corneum; CER; ceramides; CHOL; cholesterol; FFA; free fatty acids; DFFA; perdeuterated free fatty acids; ISIS; isostearyl isostearate; IPIS; isopropyl isostearate; GMIS; glycerol monoisostearateStratum corneum; Dry skin; FTIR; Moisturizer; Ceramide

Corrigendum to “Aquaporin-11 containing a divergent NPA motif has normal water channel activity” [Biochim. Biophys. Acta 1768 (2007) 688–693] by Kaya Yakata; Yoko Hiroaki; Kenichi Ishibashi; Eisei Sohara; Sei Sasaki; Kaoru Mitsuoka; Yoshinori Fujiyoshi (pp. 1525-1525).

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BBA - Biomembranes (v.1778, #6)