Check out our New Publishers' Select for Free Articles

BBA - Biomembranes (v.1818, #3)

Editorial Board (pp. i).

Lipid–protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: Comparison with detergent micelles, bicelles and liposomes by E.N. Lyukmanova; Z.O. Shenkarev; N.F. Khabibullina; G.S. Kopeina; M.A. Shulepko; A.S. Paramonov; K.S. Mineev; R.V. Tikhonov; L.N. Shingarova; L.E. Petrovskaya; D.A. Dolgikh; A.S. Arseniev; M.P. Kirpichnikov (pp. 349-358).

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid–protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K⁺ channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of¹⁵N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.Display Omitted► Membrane mimicking additives promote cell-free synthesis of soluble membrane proteins. ► Lipid–protein nanodiscs were compared with detergent micelles, bicelles and liposomes. ► Nanodiscs facilitate the correct folding of synthesized membrane proteins. ► Partial disintegration of nanodiscs composed of unsaturated lipids was observed. ► NMR studies of proteins co-translationally incorporated into nanodiscs are possible.

Keywords: Abbreviations; Brij-35; dodecylpolyoxyethylene 35; Brij-58; hexadecylpolyoxyethylene 58; Brij-78; octadecylpolyoxyethylene 78; Brij-98; octadecylpolyoxyethylene 98; CF; cell-free; DC7PC; 1,2-diheptanoyl-sn-glycero-3-phosphocholine; DHPC; 1,2-dihexanoyl-sn-glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; DOPG; 1,2-dioleoyl-sn-glycero-3-phosphoglycerol; DPC; dodecylphosphocholine, FOS-12; DTSP; 3,3′-dithiodipropionic acid di(N-hydroxysuccinimide ester); ESR; bacteriorhodopsin from; Exiguobacterium sibiricum; FM; feeding mixture; FOS-14; tetradecylphosphocholine; HSQC; heteronuclear single quantum correlation; IMP; integral membrane protein; LDAO; n-dodecyl-N,N-dimethylamine-N-oxide; LPN; lipid–protein nanodisc; LS; lauryl sarcosine; MSP; membrane scaffold protein; PC; phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RM; reaction mixture; R; _St; hydrodynamic radius of a particle, Stokes radius; shTM-ErbB3; shortened transmembrane domain of human receptor tyrosine kinase ErbB3; SDS; sodium dodecyl sulfate; TM; transmembrane; TM-ErbB3; transmembrane domain of human receptor tyrosine kinase ErbB3; TROSY; transverse relaxation-optimized spectroscopy; Triton X-100; polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether; Tween-20; polyoxyethylene(20)sorbitan monolaurate; VSD; voltage-sensing domain of KvAP channel from; Aeropyrum pernixMembrane protein; Membrane mimetics; Nanodisc; Cell-free expression; NMR; Protein folding

Conformational changes of the betaine transporter BetP from Corynebacterium glutamicum studied by pulse EPR spectroscopy by S.C.T. Nicklisch; D. Wunnicke; I.V. Borovykh; S. Morbach; J.P. Klare; H.-J. Steinhoff; Kramer R. Krämer (pp. 359-366).

The betaine transporter BetP from Corynebacterium glutamicum is activated by hyperosmotic stress critically depending on the presence and integrity of its sensory C-terminal domain. The conformational properties of the trimeric BetP reconstituted in liposomes in the inactive state and during osmotic activation were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Comparison of intra- and intermolecular inter spin distance distributions obtained by double electron–electron resonance (DEER) EPR with the crystal structure of BetP by means of a rotamer library analysis suggest a rotation of BetP protomers within the trimer by about 15° as compared to the X-ray structure. Furthermore, we observed conformational changes upon activation of BetP, which are reflected in changes of the distances between positions 545 and 589 of different protomers in the trimer. Introduction of proline at positions 550 and 572, both leading to BetP variants with a permanent (low level) transport activity, caused changes of the DEER data similar to those observed for the activated and inactivated state, respectively. This indicates that not only displacements of the C-terminal domain in general but also concomitant interactions of its primary structure with surrounding protein domains and/or lipids are crucial for the activity regulation of BetP.► Spin labels at C-terminus exhibit high mobility indicative of a flexible C-domain. ► Spin labels in the core region show low mobility and a distinct distance. ► Rotamer analysis claims 15° protomer rotation in comparison to crystal structure. ► BetP activation alters interspin distance revealing conformational changes. ► Permanent C-domain displacement shows DEER data similar to that of activated BetP.

Keywords: Abbreviations; Cw-EPR; continuous wave EPR; DEER; double electron–electron resonance; EPR; electron paramagnetic resonance; RLA; rotamer library analysis; SDSL; site-directed spin labeling; TM; transmembrane; DDM; n-dodecyl-β-; d; -maltoside; MTSSL; methanethiosulfonate spin labelSite-directed spin labeling; Electron paramagnetic resonance (EPR) spectroscopy; Double electron–electron resonance; Osmotic stress; Sodium/glycine betaine symporter

Voltage-dependent inactivation of the plasmodial surface anion channel via a cleavable cytoplasmic component by Abdulnaser Alkhalil; Liang Hong; Wang Nguitragool; Sanjay A. Desai (pp. 367-374).

Erythrocytes infected with malaria parasites have increased permeability to ions and various nutrient solutes, mediated by a parasite ion channel known as the plasmodial surface anion channel (PSAC). The parasite clag3 gene family encodes PSAC activity, but there may also be additional unidentified components of this channel. Consistent with a lack of clag3 homology to genes of other ion channels, PSAC has a number of unusual functional properties. Here, we report that PSAC exhibits an unusual form of voltage-dependent inactivation. Inactivation was readily detected in the whole-cell patch-clamp configuration after steps to negative membrane potentials. The fraction of current that inactivates, its kinetics, and the rate of recovery were all voltage-dependent, though with a modest effective valence (0.7±0.1 elementary charges). These properties were not affected by solution composition or charge carrier, suggesting inactivation intrinsic to the channel protein. Intriguingly, inactivation was absent in cell-attached recordings and took several minutes to appear after obtaining the whole-cell configuration, suggesting interactions with soluble cytosolic components. Inactivation could also be largely abolished by application of intracellular, but not extracellular protease. The findings implicate inactivation via a charged cytoplasmic channel domain. This domain may be tethered to one or more soluble intracellular components under physiological conditions.► The plasmodial surface anion channel exhibits voltage-dependent inactivation. ► Both inactivation and recovery are voltage-dependent with a small effective valence. ► Inactivation requires washout of cytosol and is affected by intracellular protease. ► A model based on charged domains of the channel protein is presented.

Keywords: Abbreviations; PSAC; plasmodial surface anion channel; clag; cytoadherence linked antigen; V; _m; membrane potential; z; _g; effective valence of inactivating particle; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidPSAC; Voltage-dependent inactivation; Patch-clamp; Malaria; Antimalarial drug targets; Clag genes

Dipole potential as a driving force for the membrane insertion of polyacrylic acid in slightly acidic milieu by Anna K. Berkovich; Eugeny P. Lukashev; Nickolay S. Melik-Nubarov (pp. 375-383).

In this work, we report on the interaction of polyacrylic acid with phosphatidylcholine bilayers and monolayers in slightly acidic medium. We found that adsorption of polyacrylic acid on liposomes composed of egg lecithin at pH 4.2 results in the formation of small pores permeable for low molecular weight solutes. However, the pores were impermeable for trypsin indicating that no solubilization of liposomes occurred. The pores were permeable for both positively charged trypsin substrate N-benzoyl-l-arginine ethyl ester and negatively charged pH-indicator pyranine. Two lines of evidence were obtained confirming the involvement of the membrane dipole potential in the insertion of polyacrylic acid into lipid bilayer. (i) Addition of phloretin, a molecule which is known to decrease dipole potential of lipid bilayer, reduced the rate of a polyacrylic acid induced leakage of pyranine from liposomes. (ii) Direct measurements of air/lipid monolayer/water interface surface potential using Kelvin probe showed that adsorption of polyacrylic acid at pH 4.2 induced a decrease in both boundary and dipole potential by 37 and 62mV for ester lipid dioleoylphosphatidylcholine (DOPC). Replacement of DOPC by ether lipid 1,2-di-O-oleyl- sn-glycero-3-phosphocholine (DiOOPC) which is known to form monolayers and bilayers with only minor dipole component of membrane potential showed that addition of PAA produced similar response in the boundary potential (by 50mV) but negligible response in dipole potential of monolayer. These observations agree with our assumption that dipole potential is an important driving force for the insertion of polyacids into biological membranes.► Polyarylic acid forms small pores in lipid bilayers permeable for small solutes. ► Insertion of polyacrylic acid decreases the membrane dipole potential. ► Membranes composed of ether lipids are less responsive to polyacids.

Keywords: Abbreviations; PAA; polyacrylic acid; BAEE; N-benzoyl-; L; -arginine ethyl ester; DODPC; 1,2-di-O-octadecyl-; sn; -glycero-3-phosphocholine; DPPC; dipalmitoylphosphatidylcholine; EYPC; egg yolk lecithin; RH-421; 4-{4-; [; 4-(Dipentylamino)phenyl; ]; -1,3-butadienyl}-1-(4-sulfobutyl)pyridinium; TMA-OH; tetramethylammonium hydroxide; Tris; Tris-hydroxymethyl aminomethane basePolyacrylic acid; Phosphatidylcholine; Ether lipid; Lipid bilayer; Dipole potential; RH-421

Interaction of the full-length Bax protein with biomimetic mitochondrial liposomes: A small-angle neutron scattering and fluorescence study by Dmitri Satsoura; Kucerka Norbert Kučerka; Sanjeevan Shivakumar; Jeremy Pencer; Corrie Griffiths; Brian Leber; David W. Andrews; John Katsaras; Cécile Fradin (pp. 384-401).

In response to apoptotic stimuli, the pro-apoptotic protein Bax inserts in the outer mitochondrial membrane, resulting in the formation of pores and the release of several mitochondrial components, and sealing the cell's fate. To study the binding of Bax to membranes, we used an in vitro system consisting of 50nm diameter liposomes prepared with a lipid composition mimicking that of mitochondrial membranes in which recombinant purified full-length Bax was inserted via activation with purified tBid. We detected the association of the protein with the membrane using fluorescence fluctuation methods, and found that it could well be described by an equilibrium between soluble and membrane-bound Bax and that at a high protein-to-liposome ratio the binding seemed to saturate at about 15 Bax proteins per 50nm diameter liposome. We then obtained structural data for samples in this saturated binding regime using small-angle neutron scattering under different contrast matching conditions. Utilizing a simple model to fit the neutron data, we observed that a significant amount of the protein mass protrudes above the membrane, in contrast to the conjecture that all of the membrane-associated Bax states are umbrella-like. Upon protein binding, we also observed a thinning of the lipid bilayer accompanied by an increase in liposome radius, an effect reminiscent of the action of antimicrobial peptides on membranes.► Interaction of full-length Bax with biomimetic mitochondrial liposomes. ► Binding of Bax to the liposomes saturates at high protein-to-liposome ratio. ► For bound Bax, most of the protein mass is found protruding above the membrane. ► Interaction of Bax with liposomes causes membrane thinning.

Keywords: Abbreviations; wtBax; wild-type Bax; tBid; truncated Bid; SANS; small-angle neutron scattering; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositol; PS; phosphatidylserine; CL; cardiolipin; DOPS; dioleoyl phosphatidylserine; TOCL; tetraoleoyl cardiolipin; EGFP; enhanced green fluorescent protein; FIDA; fluorescence intensity distribution analysis; FCS; fluorescence correlation spectroscopy; SDD; sample-to-detector distance; FWHM; full-width-at-half-maximum; SLD; scattering length densityApoptosis; Bcl-2 family protein; Bax; FCS; FIDA; SANS

Adhesion and hemifusion of cytoplasmic myelin lipid membranes are highly dependent on the lipid composition by Xavier Banquy; Kai Kristiansen; Dong Woog Lee; Jacob N. Israelachvili (pp. 402-410).

We report the effects of calcium ions on the adhesion and hemifusion mechanisms of model supported myelin lipid bilayer membranes of differing lipid composition. As in our previous studies Min et al. [], the lipid compositions used mimic “healthy” and “diseased-like” (experimental autoimmune encephalomyelitis, EAE) membranes. Our results show that the interaction forces as a function of membrane separation distance are well described by a generic model that also (and in particular) includes the hydrophobic interaction arising from the hydrophobically exposed (interior) parts of the bilayers. The model is able to capture the mechanical instability that triggers the onset of the hemifusion event, and highlights the primary role of the hydrophobic interaction in membrane fusion. The effects of lipid composition on the fusion mechanism, and the adhesion forces between myelin lipid bilayers, can be summarized as follows: in calcium-free buffer, healthy membranes do not present any signs of adhesion or hemifusion, while diseased membranes hemifuse easily. Addition of 2mM calcium favors adhesion and hemifusion of the membranes independently of their composition, but the mechanisms involved in the two processes were different: healthy bilayers systematically presented stronger adhesion forces and lower energy barriers to fusion compared to diseased bilayers. These results are of particular relevance for understanding lesion development (demyelination, swelling, vacuolization and/or vesiculation) in myelin associated diseases such as multiple sclerosis and its relationship to lipid domain formation in myelin membranes.► We studied the effect of lipid composition on adhesion and fusion of model myelin lipid membranes. ► The interaction forces between membranes can be well described by a generic model that include the hydrophobic interaction. ► Presence of calcium strongly affects both adhesion and hemifusion of the membranes studied.

Keywords: Abbreviations; a; molecular area of the lipid molecules; a; ₀; unperturbed molecular area of the lipid molecules; A; surfaces contact area; A; _hem; hemifused area; C; _ES; ,; C; _HI; ,; C; _SI; constant pre-factor; D; _ES; ,; D; _HI; ,; D; _SI; characteristic decay length; D; _w; water gap thickness; D; _b; bilayer thickness; D; _ad; separation distance at which jump out occurs; D; separation distance; E; total molecular energy of the lipids in the bilayers; e; is the electronic charge; F; total interaction force; F; _ad; adhesion force between bilayers; F; _c; critical force at which breakthrough occurs; k; is the Boltzmann constant; R; radius of the surfaces; r; radius of the contact area; T; temperature; W; total interaction energy; z; ion valency; γ; _i; interfacial tension of the hydrocarbon–water interface; ψ; the surface potential of the membrane; σ; effective surface charge densityMultiple sclerosis; Hemi-fusion; Lipid membrane

Elastic, electrostatic and electrokinetic forces influencing membrane curvature by M. Tajparast; Glavinovic M.I. Glavinović (pp. 411-424).

Many cellular and intracellular processes critically depend on membrane shape, but the shape generating mechanisms are still to be fully understood. In this study we evaluate how electrostatic/electrokinetic forces contribute to membrane curvature. Membrane bilayer had finite thickness and was either elastically anisotropic or anisotropic overall, but isotropic per sections (heads and tails). The physics of the situation was evaluated using a coupled system of elastic and electrostatic/electrokinetic (Poisson–Nernst–Planck) equations. The fixed charges present only on the upper membrane surface lead to the accumulation of counter-ions and depletion of co-ions that decay spatially very rapidly (Debye length<1nm), as does the potential and electric field. Spatially uneven electric field and the permittivity mismatch also induce charges at the membrane–solution interface, which are not fixed but influence the electrostatics nevertheless. Membrane bends due to — Coulomb force (caused by fixed membrane charges in the electric field) and the dielectric force (due to the non-uniform electric field and the permittivity mismatch between the membrane and the solution). Both act as membrane surface forces, and both depend supra-linearly on the fixed charge density. Regardless of sign of the fixed charges, the membrane bends toward the charged (upper) surface owing to the action of the Coulomb force, but this is opposed by the smaller dielectric force. The spontaneous membrane curvature becomes very pronounced at high fixed charge densities, leading to very small spontaneous radii (<50nm). In conclusion the electrostatic/electrokinetic forces contribute significantly to the membrane curvature.► We evaluate how electrostatic/electrokinetic forces contribute to membrane curvature. ► The physics was evaluated using a coupled system of elastic equations and electrostatic/electrokinetic equations. ► Two forces bend the membrane — Coulomb force and the dielectric force. ► The membrane always bends toward the surface with fixed charges. ► The electrostatic/electrokinetic forces contribute significantly to the membrane curvature.

Keywords: Lipid bilayer; Poisson–Nernst–Planck; Maxwell stress; Coulomb force; Dielectric force; Spontaneous curvature

StarD7 behaves as a fusogenic protein in model and cell membrane bilayers by Sofía Angeletti; Julieta M. Sanchez; Larry W. Chamley; Susana Genti-Raimondi; María A. Perillo (pp. 425-433).

StarD7 is a surface active protein, structurally related with the START lipid transport family. So, the present work was aimed at elucidating a potential mechanism of action for StarD7 that could be related to its interaction with a lipid–membrane interface. We applied an assay based on the fluorescence de-quenching of BD-HPC-labeled DMPC–DMPS 4:1 mol/mol SUVs (donor liposomes) induced by the dilution with non-labeled DMPC–DMPS 4:1 mol/mol LUVs (acceptor liposomes). Recombinant StarD7 accelerated the dilution of BD-HPC in a concentration-dependent manner. This result could have been explained by either a bilayer fusion or monomeric transport of the labeled lipid between donor and acceptor liposomes. Further experiments (fluorescence energy transfer between DPH-HPC/BD-HPC, liposome size distribution analysis by dynamic light scattering, and the multinuclear giant cell formation induced by recombinant StarD7) strongly indicated that bilayer fusion was the mechanism responsible for the StarD7-induced lipid dilution. The efficiency of lipid dilution was dependent on StarD7 electrostatic interactions with the lipid–water interface, as shown by the pH- and salt-induced modulation. Moreover, this process was favored by phosphatidylethanolamine which is known to stabilize non-lamellar phases considered as intermediary in the fusion process. Altogether these findings allow postulate StarD7 as a fusogenic protein.► Finding of a biological activity for this protein which so far had no known function. ► Description of a StarD7 activity not demonstrated (not tested?) for structurally related proteins. ► Demonstration of StarD7 participation in trophoblast differentiation through membrane fusion. ► The biophysical perspective accounted for the complexity of fluorophore dilution experiments.

Keywords: Abbreviations; DPH-HPC; 2-(3-(diphenylhexatrienyl) propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; BODIPY-FL-C5-HPC (also BD-HPC); 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; DLS; dynamic light scattering; DMPC; dimiristoylphosphatidylcholine; DMPS; dimiristoylphosphatidylserine; DMPE; dimiristoylphosphatidylethanolamine; StarD7; StAR-related lipid transfer (START) domain containing 7; LUVs; large unilamellar vesicles; FRET; fluorescence energy transfer; MLVs; multilamellar vesicles; PC; phosphatidylcholine; PS; phosphatidylserine; PE; phosphatidylethanolamine; START; StAR-related lipid transfer; StAR; steroidogenic acute regulatory protein; SUVs; small unilamellar vesiclesStarD7; Bilayer fusion; Steady state fluorescence de-quenching; Dynamic light scattering; FRET; Trophoblast syncytialisation

Na-glutamine co-transporters B⁰AT1 in villus and SN2 in crypts are differentially altered in chronically inflamed rabbit intestine by Prosenjit Saha; Subha Arthur; Ramesh Kekuda; Uma Sundaram (pp. 434-442).

Glutamine is a major nutrient utilized by the intestinal epithelium and is primarily assimilated via Na-glutamine co-transport (NGcT) on the brush border membrane (BBM) of enterocytes. Recently we reported that B⁰AT1 (SLC6A19) mediates glutamine absorption in villus while SN2 (SLC38A5) does the same in crypt cells. However, how B⁰AT1 and SN2 are affected during intestinal inflammation is unknown. In the present study it was shown that during chronic enteritis NGcT was inhibited in villus cells, however, it was stimulated in crypt cells. Our studies also demonstrated that the mechanism of inhibition of NGcT during chronic enteritis was secondary to a reduction in the number of B⁰AT1 co-transporters in the villus cell BBM without a change in the affinity of the co-transporter. In contrast, stimulation of NGcT in crypt cells was secondary to an increase in the affinity of SN2 for glutamine without an alteration in the number of co-transporters. Thus, glutamine assimilation which occurs via distinct transporters in crypt and villus cells is altered in the chronically inflamed intestine.► During chronic intestinal inflammation NGcT was uniquely regulated. ► NGcT was inhibited in villus cells. ► NGcT inhibition was secondary to reduced B⁰AT1 co-transporters in villus cell BBM. ► NGcT was stimulated in crypt cells. ► NGcT stimulation was via increased affinity of SN2 for glutamine in crypt cell BBM.

Keywords: Abbreviations; NGcT; Na-glutamine co-transport; BBM; Brush Border MembraneInflammatory bowel disease; Chronic enteritis; Na-glutamine co-transport; B0AT1; SN2

Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na⁺ channels by John P. O'Reilly; Penny E. Shockett (pp. 443-447).

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na⁺ channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na⁺ current (I_Na). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in I_Na. Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4min), there is little effect on I_Na. However, with longer exposures (4–8min), there is a large decrease in I_Na, but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) — MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na⁺ channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.► ► Wild-type hNav1.5 reacts readily with MTSEA, reducing I_Na. ► The mutant hNav1.5-C373Y in the closed state does not react with MTSEA. ► When inactivated (fast or slow), hNav1.5-C373Y does react with MTSEA after 4min. ► The isoform hNav1.4 does not show the MTSEA effect. ► Molecular and/ or kinetic differences between the isoforms may explain the results.

Keywords: hNav1.5; hNav1.5-C373Y; hNav1.4; Methanethiosulfonate (MTSEA); Slow inactivation; Fast inactivation

Cellular uptake and biophysical properties of galactose and/or tryptophan containing cell-penetrating peptides by Lecorche Pascaline Lécorché; Astrid Walrant; Fabienne Burlina; Laurence Dutot; Sandrine Sagan; Jean-Maurice Mallet; Bernard Desbat; Gérard Chassaing; Isabel D. Alves; Solange Lavielle (pp. 448-457).

Glycosylated cell penetrating peptides (CPPs) have been conjugated to a peptide cargo and the efficiency of cargo delivery into wild type Chinese hamster ovary (CHO) and proteoglycan deficient CHO cells has been quantified by MALDI-TOF mass spectrometry and compared to tryptophan- or alanine containing CPPs. In parallel, the behavior of these CPPs in contact with model membranes has been characterized by different biophysical techniques: Differential Scanning and Isothermal Titration Calorimetries, Imaging Ellipsometry and Attenuated Total Reflectance IR spectroscopy. With these CPPs we have demonstrated that tryptophan residues play a key role in the insertion of a CPP and its conjugate into the membrane: galactosyl residues hampered the internalization when introduced in the middle of the amphipathic secondary structure of a CPP but not when added to the N-terminus, as long as the tryptophan residues were still present in the sequence. The insertion of these CPPs into membrane models was enthalpy driven and was related to the number of tryptophans in the sequence of these secondary amphipathic CPPs. Additionally, we have observed a certain propensity of the investigated CPP analogs to aggregate in contact with the lipid surface.Display Omitted► Internalization of a cargo by glycosylated CPPs was investigated by MALDI-TOF MS. ► Substitution of tryptophan(s) in the CPP decreased the internalization efficiency. ► The behavior of these CPPs was studied with model membranes. ► The insertion of CPPs is enthalpy driven and is related to the number of tryptophan. ► These CPPs have a certain propensity to aggregate in contact with the lipid surface.

Keywords: Abbreviations; ATR Spectroscopy; Attenuated Total Reflection Spectroscopy; CPP; cell-penetrating peptide; CHO; Chinese hamster ovary; DIPEA; diisopropylethylamine; DMF; dimethylformamide; DMPC; dimyristoylphosphatidylcholine; DMPG; dimyristoylphosphatidylglycine; DSC; differential scanning calorimetry; DSPG; distearylphosphatidylglycine; GAG; glycosaminoglycan; DMEM; Dulbecco's modified Eagle's medium; ITC; isothermal titration calorimetry; KLAK peptide; (KLAKLAK); ₂; LUVs; large unilamellar vesicles; MALDI-TOF; matrix-assisted laser desorption ionization time-of-flight; MLVs; multilamellar vesicles; MS; mass spectrometry; NMP; N; -methylpyrrolidone; PKCi; peptide inhibitor of protein kinase C; RP-HPLC; reverse-phase high-pressure chromatography; RT; room temperature; TFA; trifluoroacetic acid; TIS; triisopropylsilaneCell penetrating peptide; Internalization; Glycosylation; Microcalorimetry; Tryptophan; MALDI-TOF mass-spectrometry

Membrane microdomain components of Histoplasma capsulatum yeast forms, and their role in alveolar macrophage infectivity by Loriane Tagliari; Marcos S. Toledo; Tanil G. Lacerda; Erika Suzuki; Anita H. Straus; Helio K. Takahashi (pp. 458-466).

Analysis of membrane lipids of Histoplasma capsulatum showed that ~40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4°C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30kDa laminin-binding protein, and a 50kDa protein recognized by anti-α5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (mβCD). Removal of ergosterol by mβCD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. mβCD treatment did not displace/remove the 50kDa α5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with mβCD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30kDa laminin-binding protein; ergosterol and/or the 30kDa protein may be involved in macrophage infectivity.► H. capsulatum expresses ergosterol-dependent and -independent membrane microdomains. ► Pma1p and laminin binding protein are present in ergosterol dependent microdomains. ► Ergosterol independent rafts present integrin-like protein and glycosphingolipids. ► mβCD reduces by 45% H. capsulatum infectivity of alveolar macrophages. ► Ergosterol restores raft organization and infectivity of mβCD treated H. capsulatum.

Keywords: Abbreviations; BHI; brain heart infusion; BSA; bovine serum albumin; Cho; cholesterol; DRM; detergent-resistant membrane microdomain; ECM; extracellular matrix; Erg; ergosterol; FAMEs; fatty acid methyl esters; Fr.; fraction; GC–MS; gas chromatography coupled to mass spectrometry; GIPC; glycoinositol phosphorylceramide; GSLs; glycosphingolipids; HPTLC; high performance thin layer chromatography; HRP; horseradish peroxidase; mβCD; methyl-beta-cyclodextrin; MHC; monohexosylceramide; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositol; PS; phosphatidylserine; Pho; phospholipid; PVDF; polyvinylidene fluoride; TBST; Tris, NaCl, Tween-20 buffer; TNE; Tris–HCl, NaCl, EDTA buffer Histoplasma capsulatum; Membrane microdomain; Ergosterol; Macrophage infectivity; Glycosphingolipids; Methyl-beta-cyclodextrin

Actin-dependent clustering of insulin receptors in membrane microdomains by Peter W. Winter; Alan K. Van Orden; Deborah A. Roess; B. George Barisas (pp. 467-473).

Recent evidence suggests that, after binding insulin, insulin receptors (IR) interact with specialized, cholesterol-containing, membrane microdomains and components of the actin cytoskeleton. Using single particle tracking techniques, we examined how binding of insulin, depletion of membrane cholesterol and disruption of actin filaments affect the lateral diffusion of individual quantum dot-labeled native IR on live rat basophilic leukemia 2H3 cells. We also examined the effects of similar treatments on IR clustering and multivalent insulin binding on these cells using both photon counting histogram analysis and polarization-based fluorescence resonance energy homo-transfer imaging. Our analyses indicate that binding of insulin to IR on these cells is multivalent, involving at least two insulin molecules per IR as labeling concentrations approach 1μM. Insulin binding also reduces lateral diffusion of IR and the size of membrane compartments accessed by IR. For IR that have not bound insulin, lateral diffusion of IR and the size of membrane compartments accessed by IR increase after disrupting actin filaments or depleting membrane cholesterol. However, clustering of insulin-occupied IR is reduced only by disrupting actin filaments or by fixing cells with paraformaldehyde prior to exposure to insulin, but not by depleting membrane cholesterol. Thus, it appears that, although restriction of IR lateral diffusion on these cells is sensitive to both actin filament dynamics and membrane cholesterol content, clustering of insulin-occupied IR primarily involves an actin-dependent mechanism.► Membrane-localized insulin receptors can each bind at least two insulin molecules. ► Insulin restricts lateral diffusion of membrane-localized insulin receptors. ► Clustering of insulin receptors is reduced by disrupting actin filaments. ► Clustering of insulin receptors is not reduced by depleting membrane cholesterol.

Keywords: Abbreviations; IR; insulin receptor; RBL-2H3; 2H3 rat basophilic leukemia; FcεRI; Type I Fcε receptor; SPT; single particle tracking; PCH; photon counting histogram analysis; homoFRET; polarization-based fluorescence resonance energy homo-transfer; FITC; fluorescein isothiocyanate; MβCD; methyl-β-cyclodextrin); BSA; bovine serum albumin; FITC-insulin; human recombinant insulin N-terminally tagged with FITC; R6G; rhodamine 6G; MEM; modified essential medium; FBS; fetal bovine serum; N; average number of particles detected; ε; photon counts per molecule; F; out-of-focus emission ratio; s; total intensity; r; fluorescence anisotropy; %E; FRET efficiencies; Δr; change in anisotropyInsulin receptor; Photon counting histogram analysis; Single particle tracking; Polarization homo-transfer FRET; Membrane microdomain

Structure–activity relationship of sphingomyelin analogs with sphingomyelinase from Bacillus cereus by Christian Sergelius; Sanna Niinivehmas; Terhi Maula; Mayuko Kurita; Shou Yamaguchi; Tetsuya Yamamoto; Shigeo Katsumura; Pentikainen Olli T. Pentikäinen; J. Peter Slotte (pp. 474-480).

The aim of this study was to examine how structural properties of different sphingomyelin (SM) analogs affected their substrate properties with sphingomyelinase (SMase) from Bacillus cereus. Using molecular docking and dynamics simulations (for SMase–SM complex), we then attempted to explain the relationship between SM structure and enzyme activity. With both micellar and monolayer substrates, 3O-methylated SM was found not to be degraded by the SMase. 2N-methylated SM was a substrate, but was degraded at about half the rate of its 2NH–SM control. PhytoPSM was readily hydrolyzed by the enzyme. PSM lacking one methyl in the phosphocholine head group was a good substrate, but PSM lacking two or three methyls failed to act as substrates for SMase. Based on literature data, and our docking and MD simulations, we conclude that the 3O-methylated PSM fails to interact with Mg2⁺ and Glu53 in the active site, thus preventing hydrolysis. Methylation of 2NH was not crucial for binding to the active site, but appeared to interfere with an induced fit activation of the SMase via interaction with Asp156. An OH on carbon 4 in the long-chain base of phytoPSM appeared not to interfere with the 3OH interacting with Mg2⁺ and Glu53 in the active site, and thus did not interfere with catalysis. Removing two or three methyls from the PSM head group apparently increased the positive charge on the terminal N significantly, which most likely led to ionic interactions with Glu250 and Glu155 adjacent to the active site. This likely interaction could have misaligned the SM substrate and hindered proper catalysis.► We have examined how substrate structure affects sphingomyelinase activity. ► We have computationally docked the substrate into the active site. ► 2N-methylation of sphingomyelin interferes with induced fit stabilization. ► Head-group methyls are important for proper active site alignment. ► Phytosphingomyelin is a good substrate despite additional hydroxyl.

Keywords: Abbreviations; CPE; N; -palmitoyl-; d; -; erythro; -sphingosylphosphorylethanolamine; CPE-Me; ₁; N; -palmitoyl-; d; -; erythro; -sphingosylphosphorylethanol-; N; -monomethylamine; CPE-Me; ₂; N; -palmitoyl-; d; -; erythro; -sphingosylphosphorylethanol-; N; ,; N; -dimethylamine; CPE-Me; ₃; N; -palmitoyl-; d; -; erythro; -sphingosylphosphorylethanol-; N; ,; N; ,; N; -trimethylamine (i.e., PSM); MD; molecular dynamics; NMePSM; N; -palmitoyl-; d; -; erythro; -2NMe-sphingomyelin; OMePSM; N; -palmitoyl-; d; -; erythro; -3OMe-sphingomyelin; PSM; N; -palmitoyl-; d; -; erythro; -sphingomyelin; PC; phosphatidylcholine; PhytoPSM; N; -palmitoyl-; d; -; erythro; -phytosphingomyelin; SM; sphingomyelin; SMase; sphingomyelinase3O-methylated sphingomyelin; 2N-methylated sphingomyelin; Head group methyl analog; Phytosphingomyelin

Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane by S. De Oliveira; V. Vitorino de Almeida; A. Calado; Rosario H.S. Rosário; C. Saldanha (pp. 481-490).

Fibrinogen is a multifunctional plasma protein that plays a crucial role in several biological processes. Elevated fibrinogen induces erythrocyte hyperaggregation, suggesting an interaction between this protein and red blood cells (RBCs). Several studies support the concept that fibrinogen interacts with RBC membrane and this binding, due to specific and non-specific mechanisms, may be a trigger to RBC hyperaggregation in inflammation. The main goals of our work were to prove that human RBCs are able to specifically bind soluble fibrinogen, and identify membrane molecular targets that could be involved in this process. RBCs were first isolated from blood of healthy individuals and then separated in different age fractions by discontinuous Percoll gradients. After isolation RBC samples were incubated with human soluble fibrinogen and/or with a blocking antibody against CD47 followed by fluorescence confocal microscopy, flow cytometry acquisitions and zeta potential measurements. Our data show that soluble fibrinogen interacts with the human RBC membrane in an age-dependent manner, with younger RBCs interacting more with soluble fibrinogen than the older cells. Importantly, this interaction is abrogated in the presence of a specific antibody against CD47. Our results support a specific and age-dependent interaction of soluble fibrinogen with human RBC membrane; additionally we present CD47 as a putative mediator in this process. This interaction may contribute to RBC hyperaggregation in inflammation.► We studied soluble fibrinogen interaction with human erythrocyte membrane. ► Fibrinogen–erythrocyte interaction is integrin-like receptor independent. ► This interaction is erythrocyte age-dependent. ► CD47 is a mediator of the soluble fibrinogen binding to human erythrocyte membrane.

Keywords: RBCs; Soluble fibrinogen; Binding; RBC age; Molecular target; CD47

Changes in membrane lipid composition cause alterations in epithelial cell–cell adhesion structures in renal papillary collecting duct cells by Marquez María Gabriela Márquez; Nicolás Octavio Favale; Francisco Leocata Nieto; Lucila Gisele Pescio; Norma Sterin-Speziale (pp. 491-501).

In epithelial tissues, adherens junctions (AJ) mediate cell–cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell–cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell–cell adhesion structures in renal papillary collecting duct cells.► Cyclodextrin, neomycin and LiCl provoke membrane lipid redistribution. ► Cyclodextrin treatment promotes E-cadherin internalization. ► Neomycin promotes a non-favorable lipid domain for the adherens junction proteins. ► LiCl changes the relative proportion of membrane neutral and acidic phospholipids. ► Cyclodextrin, neomycin and LiCl provoke dissipation of adherens junctions.

Keywords: Adherens junctions; Detergent-resistant membrane; Renal papilla; Collecting duct cell

The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides by Mager Imre Mäger; Kent Langel; Taavi Lehto; Eiriksdottir Emelía Eiríksdóttir; Ülo Langel (pp. 502-511).

Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin–CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.► Endocytosis inhibitors affect strongly the cytosolic delivery kinetics of CPPs. ► Luciferin–CPP conjugates are delivered into cytosol mainly via macropinocytosis. ► Involvement of other routes depends on the particular conjugate and its concentration. ► Kinetic analysis reveals clearly the concentration dependent internalization routes.

Keywords: Abbreviations; CPP; Cell-penetrating peptide; siRNA; Short interfering ribonucleic acid; EGF; Epidermal growth factor; EGFR; Epidermal growth factor receptor; CME; Clathrin-mediated endocytosis; SPPS; Solid phase peptide synthesis; t; -Boc; tert; -Butyloxycarbonyl; HOBt; Hydroxyl-benzotriazole; DCC; N; ,; N; ′-Dicyclohexylcarbodiimide; DIEA; N; ,; N; ′-Diisopropylethylamine; DMF; N,N; ′-Dimethylformamide; Cpz; Chlorpromazine; CyD; Cytochalasin D; MP; Macropinocytosis; Nys; Nystatin; C/LR; Caveolae/lipid raft dependent endocytosis; CQ; Chloroquine; TBTU; 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate; LDH; Lactate dehydrogenase; AUC; Area-under-curve; RLU; Relative luminescence unitCell-penetrating peptide; Uptake kinetics; Uptake mechanism; Endocytosis; Bioluminescence

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes by Imola G. Zigoneanu; Yoo Jeong Yang; Alexander S. Krois; Md. Emdadul Haque; Gary J. Pielak (pp. 512-519).

α-Synuclein, an intrinsically-disordered protein associated with Parkinson's disease, interacts with mitochondria, but the details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles by using fluorescence anisotropy and¹⁹F nuclear magnetic resonance. Both proteins interact strongly with large unilamellar vesicles whose composition is similar to that of the inner mitochondrial membrane, which contains cardiolipin. However, the proteins have no affinity for vesicles mimicking the outer mitochondrial membrane, which lacks cardiolipin. The¹⁹F data show that the interaction involves α-synuclein's N-terminal region. These data indicate that the middle of the N-terminal region, which contains the KAKEGVVAAAE repeats, is involved in binding, probably via electrostatic interactions between the lysines and cardiolipin. We also found that the strength of α-synuclein binding depends on the nature of the cardiolipin acyl side chains. Eliminating one double bond increases affinity, while complete saturation dramatically decreases affinity. Increasing the temperature increases the binding of wild-type, but not the A30P variant. The data are interpreted in terms of the properties of the protein, cardiolipin demixing within the vesicles upon binding of α-synuclein, and packing density. The results advance our understanding of α-synuclein's interaction with mitochondrial membranes.►α-Synuclein binds the inner mitochondrial membrane, not the outer membrane. ►Head group charge and side-chain saturation are important. ►Quantitative, atomic-level interactions of α-synuclein with mitochondria.

Keywords: Fluorescence anisotropy; Fluorine NMR; Lipid vesicles; Mitochondria; Parkinson's disease; α-Synuclein

Saturation with cholesterol increases vertical order and smoothes the surface of the phosphatidylcholine bilayer: A molecular simulation study by Elżbieta Plesnar; Witold K. Subczynski; Marta Pasenkiewicz-Gierula (pp. 520-529).

Molecular dynamics (MD) simulations of a mono- cis-unsaturated 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer and a POPC bilayer containing 50mol% cholesterol (POPC–Chol50) were carried out for 200ns to compare the spatial organizations of the pure POPC bilayer and the POPC bilayer saturated with Chol. The results presented here indicate that saturation with Chol significantly narrows the distribution of vertical positions of the center-of-mass of POPC molecules and POPC atoms in the bilayer. In the POPC–Chol50 bilayer, the same moieties of the lipid molecules are better aligned at a given bilayer depth, forming the following clearly separated membrane regions: the polar headgroup, the rigid core consisting of steroid rings and upper fragments of the acyl chains, and the fluid hydrocarbon core consisting of Chol chains and the lower fragments of POPC chains. The membrane surface of the POPC–Chol50 bilayer is smooth. The results have biological significance because the POPC–Chol50 bilayer models the bulk phospholipid portion of the fiber-cell membrane in the eye lens. It is hypothesized that in the eye lens cholesterol-induced smoothing of the membrane surface decreases light-scattering and helps to maintain lens transparency.Display Omitted► POPC and POPC–Cholesterol (1:1 molar ratio) bilayers were MD simulated for 200ns each. ► Distributions of vertical positions of POPC and cholesterol atoms were calculated. ► Cholesterol significantly narrows the distribution of vertical positions of each of the lipid atoms. ► The surface of the POPC bilayer saturated with cholesterol is smoother than of the POPC bilayer without cholesterol.

Keywords: Abbreviations; CBD; cholesterol bilayer domain; PCD; phospholipid–cholesterol domain; PC; phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-phosphatidylcholine; Chol; cholesterol; MD; molecular dynamics; CM; center-of-mass; RP; roughness parameterFiber-cell membrane; Eye lens; Lens transparency; Chain order; Isomerization

Apolipoprotein A-1 (apoA-1) deposition in, and release from, the enterocyte brush border: A possible role in transintestinal cholesterol efflux (TICE)? by E. Michael Danielsen; Gert H. Hansen; Karina Rasmussen; Lise-Lotte Niels-Christiansen; Franz Frenzel (pp. 530-536).

Transintestinal cholesterol efflux (TICE) has been proposed to represent a non-hepatobiliary route of cholesterol secretion directly “from blood to gut” and to play a physiologically significant role in excretion of neutral sterols, but so far little is known about the proteins involved in the process. We have previously observed that apolipoprotein A-1 (apoA-1) synthesized by enterocytes of the small intestine is mainly secreted apically into the gut lumen during fasting where its assembly into chylomicrons and basolateral discharge is at a minimal level. In the present work we showed, both by immunomicroscopy and subcellular fractionation, that a fraction of the apically secreted apoA-1 in porcine small intestine was not released from the cell surface but instead deposited in the brush border. Cholesterol was detected in immunoisolated microvillar apoA-1, and it was partially associated with detergent resistant membranes (DRMs), indicative of localization in lipid raft microdomains. The apolipoprotein was not readily released from microvillar vesicles by high salt or by incubation with phosphatidylcholine-specific phospholipase C or trypsin, indicating a relatively firm attachment to the membrane bilayer. However, whole bile or taurocholate efficiently released apoA-1 at low concentrations that did not solubilize the transmembrane microvillar protein aminopeptidase N. Based on these findings and the well known role played by apoA-1 in extrahepatic cellular cholesterol removal and reverse cholesterol transport (RCT), we propose that brush border-deposited apoA-1 in the small intestine acts in TICE by mediating cholesterol efflux into the gut lumen.► Apolipoprotein A-1 is synthesized and deposited in the small intestinal brush border. ► Apolipoprotein A-1 is released from the brush border by bile and taurocholate. ► Apolipoprotein A-1 may participate in transintestinal cholesterol efflux.

Keywords: Cholesterol excretion; Apolipoprotein A-1; Small intestine; Enterocyte; Brush border; Transintestinal cholesterol efflux (TICE)

Probing molecular interactions of poly(styrene- co-maleic acid) with lipid matrix models to interpret the therapeutic potential of the co-polymer by Shubhadeep Banerjee; Tapan K. Pal; Sujoy K. Guha (pp. 537-550).

To understand and maximize the therapeutic potential of poly(styrene- co-maleic acid) (SMA), a synthetic, pharmacologically-active co-polymer, its effect on conformation, phase behavior and stability of lipid matrix models of cell membranes were investigated. The modes of interaction between SMA and lipid molecules were also studied. While, attenuated total reflection-Fourier-transform infrared (ATR-FTIR) and static³¹P nuclear magnetic resonance (NMR) experiments detected SMA-induced conformational changes in the headgroup region, differential scanning calorimetry (DSC) studies revealed thermotropic phase behavior changes of the membranes.¹H NMR results indicated weak immobilization of SMA within the bilayers. Molecular interpretation of the results indicated the role of hydrogen-bond formation and hydrophobic forces between SMA and zwitterionic phospholipid bilayers. The extent of membrane fluidization and generation of isotropic phases were affected by the surface charge of the liposomes, and hence suggested the role of electrostatic interactions between SMA and charged lipid headgroups. SMA was thus found to directly affect the structural integrity of model membranes.► SMA-lipid matrix models varying in composition and surface charge prepared. ► ATR-FTIR detected SMA-induced reorganization in packing of lipid molecules. ► DSC revealed thermotropic phase behavior change and fluidization of vesicles. ► NMR identified changes in polar regions and weak immobilization of SMA in bilayers. ► Presence of SMA affected structural integrity of model membranes.

Keywords: Poly(styrene-; co; -maleic acid); Liposome; ATR-FTIR; DSC; NMR

Functional interactions between voltage-gated Ca2⁺ channels and Rab3-interacting molecules (RIMs): New insights into stimulus–secretion coupling by María A. Gandini; Ricardo Felix (pp. 551-558).

Stimulus–secretion coupling is a complex set of intracellular reactions initiated by an external stimulus that result in the release of hormones and neurotransmitters. Under physiological conditions this signaling process takes a few milliseconds, and to minimize delays cells have developed a formidable integrated network, in which the relevant molecules are tightly packed on the nanometer scale. Active zones, the sites of release, are composed of several different proteins including voltage-gated Ca2⁺ (Ca_V) channels. It is well acknowledged that hormone and neurotransmitter release is initiated by the activation of these channels located close to docked vesicles, though the mechanisms that enrich channels at release sites are largely unknown. Interestingly, Rab3 binding proteins (RIMs), a diverse multidomain family of proteins that operate as effectors of the small G protein Rab3 involved in secretory vesicle trafficking, have recently identified as binding partners of Ca_V channels, placing both proteins in the center of an interaction network in the molecular anatomy of the active zones that influence different aspects of secretion. Here, we review recent evidences providing support for the notion that RIMs directly bind to the pore-forming and auxiliary β subunits of Ca_V channels and with RIM-binding protein, another interactor of the channels. Through these interactions, RIMs regulate the biophysical properties of the channels and their anchoring relative to active zones, significantly influencing hormone and neurotransmitter release.► RIM proteins operate as effectors of Rab3, a small G protein involved in secretory vesicle trafficking. ► Ca2⁺ entry through Ca_V channels leads to the exocytosis of secretory vesicles. ► Through multiple interactions, RIMs regulate Ca_V channel activity and anchoring to the sites of release. ► Ca_V channels and RIMs interaction contribute to determine different aspects of secretion.

Keywords: Ca; _V; channels; RIM; Exocytosis; Stimulus–secretion coupling; Hormone secretion; Neurotransmitter release

The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn's disease by Lorena Pochini; Mariafrancesca Scalise; Michele Galluccio; Giovambattista Pani; Katherine A. Siminovitch; Cesare Indiveri (pp. 559-565).

The organic cation transporter (OCTN1) plays key roles in transport of selected organic cations, but understanding of its biological functions remains limited by restricted knowledge of its substrate targets. Here we show capacity of human OCTN1-reconstituted proteoliposomes to mediate uptake and efflux of [³H]acetylcholine, the Km of transport being 1.0mM with V_max of 160nmol⋅mg−¹protein⋅min−¹. OCTN1-mediated transport of this neurotransmitter was time-dependent and was stimulated by intraliposomal ATP. The transporter operates as uniporter but translocates acetylcholine in both directions. [³H]acetylcholine uptake was competitively inhibited by tetraethylammonium, γ-butyrobetaine and acetylcarnitine, and was also inhibited by various polyamines. Decreasing intraliposomal ATP concentrations increased OCTN Km for acetylcholine, but V_max was unaffected. Evaluation of the acetylcholine transporter properties of a variant form of OCTN1, the Crohn's disease-associated 503F variant, revealed time course, Km and V_max for acetylcholine uptake to be comparable to that of wild-type OCTN1. Km for acetylcholine efflux was also comparable for both OCTN1 species, but V_max of OCTN1 503F-mediated acetylcholine efflux (1.9nmol⋅mg−¹protein⋅min−¹) was significantly lower than that of wild-type OCTN1 (14nmol⋅mg−¹protein⋅min−¹). These data identify a new transport role for OCTN1 and raise the possibility that its involvement in the non-neuronal acetylcholine system may be relevant to the pathogenesis of Crohn's disease.► hOCTN1 transporter reconstituted in liposomes catalyzes acetylcholine transport. ► The transporter in proteoliposome has the same orientation as in cell membrane. ► Transport occurs as uptake or efflux from proteoliposomes (cells). ► Efflux is physiologically related to the non-neuronal acetylcholine system. ► Acetylcholine efflux is defective in the Crohn's disease associated L503F mutant.

Keywords: Abbreviations; TEA; tetraethylammonium; PLP; pyridoxal 5-phosphate; ANTP; adenosine 5′-(β,γ-imido)triphosphate; MTSET; sodium (2-(trimethylammonium)ethyl methanethiosulfonate); p-OHMB; p-hydroxymercuribenzoate; NEM; N-ethylmaleimideOCTN1; Transporter; Reconstitution; Acetylcholine; Crohn's disease; Liposome

Model membrane studies for characterization of different antibiotic activities of lipopeptides from Pseudomonas by Katrin Reder-Christ; Yvonne Schmidt; Dorr Marius Dörr; Hans-Georg Sahl; Michaele Josten; Jos M. Raaijmakers; Harald Gross; Gerd Bendas (pp. 566-573).

Lipopeptides (LPs) are a structurally diverse class of amphipathic natural products that were in the past mainly known for their surfactant properties. However, the recent discovery of their antimicrobial and cytotoxic bioactivities have fueled and renewed the interest in this compound class. Propelled by the antimicrobial potential of this compound class, in this study a range of six underinvestigated LPs from Pseudomonads were examined with respect to their antibiotic activities towards bacteria. The assays revealed that only the glycosylated lipodipeptide SB-253514, produced by Pseudomonas strain SH-C52, showed significant antibacterial activity. Since the bioactivity of LPs is commonly attributed to membrane interactions, we analyzed the molecular interactions between the LPs and bacteria-like lipid model membranes in more detail via complementary biophysical approaches. Application of the quartz crystal microbalance (QCM) showed that all LPs possess a high binding affinity towards the model membranes. Despite their similar membrane affinity, monolayer studies displayed different tendencies of LPs to incorporate into the membrane. The degree of membrane incorporation could be correlated with specific structural features of the investigated LPs, such as distance between the peptidic macrocycle and the fatty acid, but did not fully reflect their respective antibacterial activity. Cyclic voltammetry (CV) experiments further demonstrated that SB-253514 showed no membrane permeabilization effects at inhibitory concentrations. Collectively, these results suggests that the antibacterial activity of SB-253514 cannot be explained by an unspecific detergent-like mechanism generally proposed for amphiphilic molecules but instead appears to occur via a defined structural target.► Underinvestigated Pseudomonas-lipopeptides were screened for antibiotic activity. ► Lipodipeptide SB-253514 possesses significant antibacterial activity. ► All CLPs showed high membrane binding affinity and insertion tendency. ► Membrane disintegration is not key for the antibiotic mode of action. ► The data imply a targeted mechanism for the antimicrobial activity of SB-253514.

Keywords: Lipopeptide; Cyclic voltammetry (CV); Langmuir–Blodgett (LB); Pseudomonas; Quartz crystal microbalance (QCM); SB-253514

Eicosapentaenoic acid plays a role in stabilizing dynamic membrane structure in the deep-sea piezophile Shewanella violacea: A study employing high-pressure time-resolved fluorescence anisotropy measurement by Keiko Usui; Toshiki Hiraki; Jun Kawamoto; Tatsuo Kurihara; Yuichi Nogi; Chiaki Kato; Fumiyoshi Abe (pp. 574-583).

Shewanella violacea DSS12 is a psychrophilic piezophile that optimally grows at 30MPa. It contains a substantial amount of eicosapentaenoic acid (EPA) in the membrane. Despite evidence linking increased fatty acid unsaturation and bacterial growth under high pressure, little is known of how the physicochemical properties of the membrane are modulated by unsaturated fatty acids in vivo. By means of the newly developed system performing time-resolved fluorescence anisotropy measurement under high pressure (HP-TRFAM), we demonstrate that the membrane of S. violacea is highly ordered at 0.1MPa and 10°C with the order parameter S of 0.9, and the rotational diffusion coefficient D_w of 5.4μs^–1 for 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene in the membrane. Deletion of pfaA encoding the omega-3 polyunsaturated fatty acid synthase caused disorder of the membrane and enhanced the rotational motion of acyl chains, in concert with a 2-fold increase in the palmitoleic acid level. While the wild-type membrane was unperturbed over a wide range of pressures with respect to relatively small effects of pressure on S and D_w, the Δ pfaA membrane was disturbed judging from the degree of increased S and decreased D_w. These results suggest that EPA prevents the membrane from becoming hyperfluid and maintains membrane stability against significant changes in pressure. Our results counter the generally accepted concept that greater fluidity is a membrane characteristic of microorganisms that inhabit cold, high-pressure environments. We suggest that retaining a certain level of membrane physical properties under high pressure is more important than conferring membrane fluidity alone.► The dynamic structure of the membrane of a deep-sea piezophile was investigated. ► The membrane was highly rigid with respect to a high order parameter. ► EPA deficiency resulted in membrane disorder and an increased palmitoleic acid level. ► EPA plays a role in preventing the membrane hyperfluid and maintaining the stability.

Keywords: Abbreviations; EPA; eicosapentaenoic acid; POPC; palmitoyl-oleylphosphatidylcholine; UFA; unsaturated fatty acid; MUFA; monounsaturated fatty acid; PUFA; polyunsaturated fatty acid; DPH; 1,6-diphenyl-1,3,5-hexatriene; TMA-DPH; 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene; TCSPC; time-correlated single-photon counting; r; _s; steady-state anisotropy; r; ₀; maximum anisotropy; r; _∞; limiting anisotropy; θ; rotational correlation time; S; order parameter; D; _w; rotational diffusion coefficient; τ; fluorescence lifetime; HP-TRFAM; high-pressure time-resolved fluorescence anisotropy measurement Shewanella violacea; High hydrostatic pressure; EPA; Time-resolved fluorescence anisotropy measurement; Membrane fluidity

Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination by Sang Ho Park; Fabio Casagrande; Mignon Chu; Klaus Maier; Hans Kiefer; Stanley J. Opella (pp. 584-591).

The human chemokine receptor CXCR1 is a G-protein coupled receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with¹³C and¹⁵N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.► It is possible to purify CXCR1 as homogeneous monomers. ► 7mg of pure CXCR1 is obtained from 1L culture. ► Isotopically labeled CXCR1 is stable for more than 6months.

Keywords: Abbreviations; DHPC; 1,2-dihexanoyl-sn-glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPC; dodecylphosphocholine; GST; glutathione S-transferase; HEPES; 4-(2-hydroxyethyl)-1 piperazineethane-sulfonic acid; HPC; n-hexadecylphosphocholine; MβCD; methyl-β-cyclodextrin; MAS; magic angle spinning; OS; oriented sample; SDS; sodium dodecyl sulfate; TCEP; tris-2-carboxyethyl-phosphineGPCR; CXCR1; Proteoliposome; Phospholipid bilayer; Solid-state NMR

Current and selectivity in a model sodium channel under physiological conditions: Dynamic Monte Carlo simulations by Csanyi Éva Csányi; Dezső Boda; Dirk Gillespie; Kristof Tamás Kristóf (pp. 592-600).

A reduced model of a sodium channel is analyzed using Dynamic Monte Carlo simulations. These include the first simulations of ionic current under approximately physiological ionic conditions through a model sodium channel and an analysis of how mutations of the sodium channel's DEKA selectivity filter motif transform the channel from being Na⁺ selective to being Ca2⁺ selective. Even though the model of the pore, amino acids, and permeant ions is simplified, the model reproduces the fundamental properties of a sodium channel (e.g., 10 to 1 Na⁺ over K⁺ selectivity, Ca2⁺ exclusion, and Ca2⁺ selectivity after several point mutations). In this model pore, ions move through the pore one at a time by simple diffusion and Na⁺ versus K⁺ selectivity is due to both the larger K⁺ not fitting well into the selectivity filter that contains amino acid terminal groups and K⁺ moving more slowly (compared to Na⁺) when it is in the selectivity filter.► Selectivity and permeation in model sodium channel studied with Dynamic Monte Carlo. ► Ion permeation is single ion moving by simple diffusion. ► Model reproduces Na⁺ vs. K⁺ selectivity, Ca2⁺ exclusion, DEKA to DEEE mutations. ► Na⁺ vs. K⁺ selectivity: K⁺ excluded from crowded pore and moves slower through pore. ► Point mutations DEKA→DEEE make calcium channel, reproducing experiments.

Keywords: Sodium channel; Modeling; Selectivity; Permeation

Regulation of plasma membrane Ca2⁺-ATPase activity by acetylated tubulin: Influence of the lipid environment by N.E. Monesterolo; M.R. Amaiden; A.N. Campetelli; V.S. Santander; C.A. Arce; Pie J. Pié; C.H. Casale (pp. 601-608).

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca2⁺-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca2⁺ transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.► Tubulin inhibits PMCA activity in plasma membrane through a reversible interaction. ► When the enzyme is reconstituted in the presence of acidic or neutral lipids and low concentrations of tubulin, PMCA is activated. ► PMCA is activated independently of surrounding lipid composition at low tubulin concentrations. ► PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. ► Regulation of PMCA activity by tubulin is dependent on lipid composition and tubulin concentration.

Keywords: Abbreviations; PMCA; plasma membrane Ca; 2; ⁺; ATPase; PC; L-α phosphatidylcholine type XVI-E from fresh egg yolk; BE; lipidic extract from bovine brain containing acidic lipids; PA; L-α-phosphatidic acid from egg yolk; DAG; diacylglycerol; C; ₁₂; E; ₁₀; polyoxyethylene-10-laurylether; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PLC; phospholipase C; PMSF; phenylmethyl-sulfonyl-fluoride; p-NPPC; para-nitro-phenyl-phosphatidyl choline; PMVs; plasma membrane vesiclesPlasma membrane Ca; 2; ⁺; -ATPase; Tubulin; Lipid composition; Phospholipase C

Structure, dynamics, and hydration of POPC/POPS bilayers suspended in NaCl, KCl, and CsCl solutions by Piotr Jurkiewicz; Lukasz Cwiklik; Vojtiskova Alžběta Vojtíšková; Pavel Jungwirth; Martin Hof (pp. 609-616).

Effects of alkali metal chlorides on the properties of mixed negatively charged lipid bilayers are experimentally measured and numerically simulated. Addition of 20mol% of negatively charged phosphatidylserine to zwitterionic phosphatidylcholine strengthens adsorption of monovalent cations revealing their specificity, in the following order: Cs⁺++. Time-resolved fluorescence solvent relaxation shows significant decrease both in mobility and hydration of the lipid carbonyls probed by Laurdan upon addition of the cations. The experimental findings are supported by molecular dynamics simulations, which show deep penetration of the cations down to the glycerol level of the lipid bilayer where they pair with oxygen atoms of carbonyl groups (with pairing with sn-2 carbonyl being about twice stronger than pairing with the sn-1 one). Moreover, the cations bridge neighboring lipids forming clusters of up to 4 lipid molecules, which decreases the area per lipid, thickens the membrane, causes rising of lipid headgroups, and hinders lipid dynamics. All these effects follow the same Hofmeister ordering as the cationic adsorption to the bilayer.► Fluorescence experiments and MD simulations report on specific ion-lipid interactions. ► Alkali metal chlorides dehydrate and compress the POPC/POPS (4:1, mol:mol) bilayer. ► No binding of the studied cations to the carboxylate group of POPS was observed. ► Cations penetrate toward the carbonyls and bridge neighboring lipid molecules. ► The observed changes follow the Hofmeister ordering: Na>K>Cs.

Keywords: Solvent relaxation; Time-dependent fluorescence shift; Molecular dynamics simulation; Specific ionic effect; Anionic/zwitterionic phospholipid bilayer; Model lipid membrane

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor by Tanxing Cui; David Mowrey; Vasyl Bondarenko; Tommy Tillman; Dejian Ma; Elizabeth Landrum; Jose Manuel Perez-Aguilar; Jing He; Wei Wang; Jeffery G. Saven; Roderic G. Eckenhoff; Pei Tang; Yan Xu (pp. 617-626).

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828–1833, 2004; Ma et al., PNAS, 105: 16537–42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T₂ dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.► We designed a water-soluble analog to the acetylcholine receptor α1 subunit. ► We determined the high-resolution structure of the water-soluble analog by NMR. ► The analog preserved structural features observed in homologous TM domains. ► Photoaffinity labeling identified a relevant anesthetic binding site. ► We affirm the water-solubilization method for high-resolution studies of TM proteins.

Keywords: Nicotinic acetylcholine receptor; Water-solubilization; Nuclear magnetic resonance (NMR) spectroscopy; Membrane protein; Computational protein redesign

Acyrthosiphon pisum AQP2: A multifunctional insect aquaglyceroporin by Ian S. Wallace; Ally J. Shakesby; Jin Ha Hwang; Won Gyu Choi; Martinkova Natália Martínková; Angela E. Douglas; Daniel M. Roberts (pp. 627-635).

Annotation of the recently sequenced genome of the pea aphid ( Acyrthosiphon pisum) identified a gene ApAQP2 (ACYPI009194, Gene ID: 100168499) with homology to the Major Intrinsic Protein/aquaporin superfamily of membrane channel proteins. Phylogenetic analysis suggests that ApAQP2 is a member of an insect-specific clade of this superfamily. Homology model structures of ApAQP2 showed a novel array of amino acids comprising the substrate selectivity-determining “aromatic/arginine” region of the putative transport pore. Subsequent characterization of the transport properties of ApAQP2 upon expression in Xenopus oocytes supports an unusual substrate selectivity profile. Water permeability analyses show that the ApAQP2 protein exhibits a robust mercury-insensitive aquaporin activity. However unlike the water-specific ApAQP1 protein, ApAQP2 forms a multifunctional transport channel that shows a wide permeability profile to a range of linear polyols, including the potentially biologically relevant substrates glycerol, mannitol and sorbitol. Gene expression analysis indicates that ApAQP2 is highly expressed in the insect bacteriocytes (cells bearing the symbiotic bacteria Buchnera) and the fat body. Overall the results demonstrate that ApAQP2 is a novel insect aquaglyceroporin which may be involved in water and polyol transport in support of the Buchnera symbiosis and aphid osmoregulation.► A novel aquaglyceroporin ApAQP2 has been identified in the pea aphid A. pisum. ► ApAQP2 is a member of an insect-specific clade of the AQP gene family. ► Members of this clade have unusual pore selectivity sequences. ► ApAQP2 transports a diverse set of substrates including water and multiple polyols. ► ApAQP2 is expressed predominantly in symbiotic bacteriocyte and fat body tissues.

Keywords: Aphid; Aquaporins; Buchnera aphidicola; Osmoregulation; Polyols; Symbiosis

Effects of the pore-forming agent nystatin on giant phospholipid vesicles by Luka Kristanc; Saša Svetina; Gomiscek Gregor Gomišček (pp. 636-644).

The effects of the polyene pore-forming agent nystatin were investigated on individual giant unilamellar phospholipid vesicles (GUVs), made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in different methanol–water solutions using phase-contrast optical microscopy. Three characteristic effects were detected in three different nystatin concentration ranges: vesicle shape changes (between 150 and 250μM); transient, nonspecific, tension pores (between 250 and 400μM); and vesicle ruptures (above 400μM). Both the appearance of the transient tension pores and the vesicle ruptures were explained as being a consequence of the formation of size-selective nystatin channels, whose membrane area density increases with the increasing nystatin concentrations. Our results also show that nystatin is able to form pores in the absence of sterols. In addition, study of the cross-interactions between nystatin and methanol revealed mutually antagonizing effects on the vesicle behavior for methanol volume fractions higher than 10%.► The response of individual, POPC sterol-free GUVs to nystatin was studied using phase-contrast microscopy. ► Three characteristic nystatin-related features are revealed: vesicle shape changes, transient tension pores and vesicle ruptures. ► The alteration of the area difference between lipid monolayers determines the vesicle shape changes. ► A two-stage model of the nystatin membrane permeabilization is explained by a single mechanism. ► The mutually antagonizing effects of nystatin and methanol at higher methanol volume fractions can be attributed to phospholipid interdigitation.

Keywords: Nystatin; Giant phospholipid vesicle; Transmembrane pore

Probing the helical tilt and dynamic properties of membrane-bound phospholamban in magnetically aligned bicelles using electron paramagnetic resonance spectroscopy by Harishchandra Ghimire; Shadi Abu-Baker; Indra D. Sahu; Andy Zhou; Daniel J. Mayo; Ryan T. Lee; Gary A. Lorigan (pp. 645-650).

Wild-type phospholamban (WT-PLB), a Ca2⁺-ATPase (SERCA) regulator in the sarcoplasmic reticulum membrane, was studied using TOAC nitroxide spin labeling, magnetically aligned bicelles, and electron paramagnetic resonance (EPR) spectroscopy to ascertain structural and dynamic information. Different structural domains of PLB (transmembrane segment: positions 42 and 45, loop region: position 20, and cytoplasmic domain: position 10) were probed with rigid TOAC spin labels to extract the transmembrane helical tilt and structural dynamic information, which is crucial for understanding the regulatory function of PLB in modulating Ca2⁺-ATPase activity. Aligned experiments indicate that the transmembrane domain of wild-type PLB has a helical tilt of 13°±4° in DMPC/DHPC bicelles. TOAC spin labels placed on the WT-PLB transmembrane domain showed highly restricted motion with more than 100ns rotational correlation time (τ_c); whereas the loop, and the cytoplasmic regions each consists of two distinct motional dynamics: one fast component in the sub-nanosecond scale and the other component is slower dynamics in the nanosecond range.► EPR spectroscopic methods developed for determining helical tilt of PLB peptide. ► Dynamic properties of rigid TOAC spin label attached to PLB measured. ► Bicelles are an excellent model membrane system.

Keywords: Abbreviations; PLB; Phospholamban (PLB); SERCA; sarco/endoplasmic reticulum Ca; 2; ⁺; -ATPase; DMPC; 1,2-Dimyristoyl-; sn; -Glycero-3-Phosphocholine; DHPC; 1,2-Dihexanoyl-; sn; -Glycero-3-Phosphocholine (DHPC); HPLC; High Performance Liquid Chromatography; MALDI-TOF; Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry; TOAC; 2,2,6,6-tetramethylpiperidine-1oxyl-4-amino-4-carboxylic acidPhospholamban (PLB); TOAC nitroxide spin label; Bicelles; Membrane proteins; Electron paramagnetic resonance spectroscopy

The iron transporter ferroportin can also function as a manganese exporter by Michael S. Madejczyk; Nazzareno Ballatori (pp. 651-657).

The present study examined the hypothesis that the iron exporter ferroportin (FPN1/SLC40A1) can also mediate cellular export of the essential trace element manganese, using Xenopus laevis oocytes expressing human FPN1. When compared to oocytes expressing only the divalent metal transporter-1 (DMT1/NRAMP2),⁵⁴Mn accumulation was lower in oocytes also expressing FPN1. FPN1-expressing oocytes exported more⁵⁴Mn than control oocytes (26.6±0.6% versus 7.1±0.5%, respectively, over 4h at pH 7.4 when preloaded with approximately 16μM⁵⁴Mn); however, there was no difference in⁵⁴Mn uptake between control and FPN1-expressing oocytes. FPN1-mediated Mn export was concentration dependent and could be partially cis-inhibited by 100μM Fe, Co, and Ni, but not by Rb. In addition, Mn export ability was significantly reduced when the extracellular pH was reduced from 7.4 to 5.5, and when Na⁺ was substituted with K⁺ in the incubation media. These results indicate that Mn is a substrate for FPN1, and that this export process is inhibited by a low extracellular pH and by incubation in a high K⁺ medium, indicating the involvement of transmembrane ion gradients in FPN1-mediated transport.► The ability of a known iron exporter, namely ferroportin, to transport manganese was examined in Xenopus laevis oocytes. ► Ferroportin had no affect on Mn uptake into the oocytes, but markedly enhanced Mn export. ► Mn export was concentration dependent, and inhibited by some divalent metals, low extracellular pH, and a high K⁺ medium. ► The results indicate that Mn is a substrate for ferroportin, and that this transporter may contribute to Mn homeostasis.

Keywords: Abbreviations; DMT1; divalent metal transporter-1/NRAMP2; FPN1; ferroportin/SLC40A1Ferroportin; FPN1; Manganese; Metal export; Divalent metal transporter; Xenopus laevis; oocytes

Arg 901 in the AE1 C-terminal tail is involved in conformational change but not in substrate binding by Shinya Takazaki; Yoshito Abe; Tomohiro Yamaguchi; Mikako Yagi; Tadashi Ueda; Dongchon Kang; Naotaka Hamasaki (pp. 658-665).

In our previous paper, we demonstrated that Arg 901 in the C-terminal tail of human AE1 (band 3, anion exchanger 1) had a functional role in conformational change during anion exchange. To further examine how Arg 901 is involved in conformational change, we expressed various Arg 901 mutants and alanine mutants of the C-terminal tail (from Leu 886 to Val 911) on the plasma membrane of Saccharomyces cerevisiae and evaluated the kinetic parameters of sulfate ion transport. As a result, Vmax decreased as the hydrophobicities of the 901st and peripheral hydrophilic residues increased, indicating that the hydrophobicity of the C-terminal residue is involved in the conformational change. We also found the alkali and protease resistance of the C-terminal region after Arg 901 modification with hydroxyphenylglyoxal (HPG) or phenylglyoxal (PG), a hydrophobic reagent. These results suggested that the increased hydrophobicity of the C-terminal region around Arg 901 leads to inefficient conformational change by the newly produced hydrophobic interaction.► Kinetic parameters of anion transport of C-terminal AE1 mutants were evaluated. ► Vmax decreased as the hydrophobicities of C-terminal residues increased. ► Arg 901 modification with HPG or PG increased alkali and protease resistance. ► Increase of C-terminal hydrophobicity leads to inefficient conformational change.

Keywords: Abbreviations; AE1; anion exchanger 1; C; ₁₂; E; ₈; octaethyleneglycol monododecyl ether; DEPC; diethylpyrocarbonate; DIDS; 4,4-diisothiocyanostilbene-2,2-disulfonic acid; GPA; glycophorin A; HPG; hydroxyphenylglyoxal; PG; phenylglyoxal; PMSF; phenylmethanesulfonyl fluoride; TCA; trichloroacetic acid; TM; transmembrane spanning portionBand 3 protein; Anion exchange; Membrane protein structure

Photo-activated phase separation in giant vesicles made from different lipid mixtures by Christopher K. Haluska; Mauricio S. Baptista; Adjaci U. Fernandes; André P. Schroder; Carlos M. Marques; Rosangela Itri (pp. 666-672).

Using giant unilamellar vesicles (GUVs) made from POPC, DPPC, cholesterol and a small amount of a porphyrin-based photosensitizer that we name PE-porph, we investigated the response of the lipid bilayer under visible light, focusing in the formation of domains during the lipid oxidation induced by singlet oxygen. This reactive species is generated by light excitation of PE-porf in the vicinity of the membrane, and thus promotes formation of hydroperoxides when unsaturated lipids and cholesterol are present. Using optical microscopy we determined the lipid compositions under which GUVs initially in the homogeneous phase displayed Lo–Ld phase separation following irradiation. Such an effect is attributed to the in situ formation of both hydroperoxized POPC and cholesterol. The boundary line separating homogeneous Lo phase and phase coexistence regions in the phase diagram is displaced vertically towards the higher cholesterol content in respect to ternary diagram of POPC:DPPC:cholesterol mixtures in the absence of oxidized species. Phase separated domains emerge from sub-micrometer initial sizes to evolve over hours into large Lo–Ld domains completely separated in the lipid membrane. This study provides not only a new tool to explore the kinetics of domain formation in mixtures of lipid membranes, but may also have implications in biological signaling of redox misbalance.► Membrane photosensitization promotes lipid peroxidation ► Lipid hydroperoxide formation induces lipid phase separation ► Lo–Ld domain formation may imply in biological signaling of redox misbalance

Keywords: Lipid raft; Lo-Ld phase separation; Giant unilamellar vesicle; Membrane photosensitization; Lipid hydroperoxidation; Porphyrin-based photosensitizer

Characterization of daptomycin oligomerization with perylene excimer fluorescence: Stoichiometric binding of phosphatidylglycerol triggers oligomer formation by Jawad K. Muraih; Jesse Harris; Scott D. Taylor; Michael Palmer (pp. 673-678).

Daptomycin is a lipopeptide antibiotic that binds to and depolarizes bacterial cell membranes. Its antibacterial activity requires calcium and correlates with the content of phosphatidylglycerol in the target membrane. Daptomycin has been shown to form oligomers on liposome membranes. We here use perylene excimer fluorescence to further characterize the membrane-associated oligomer. To this end, the N-terminal fatty acyl chain was replaced with perylene-butanoic acid. The perylene derivative retains one third of the antibacterial activity of native daptomycin. On liposomes containing phosphatidylcholine and phosphatidylglycerol, as well as on Bacillus subtilis cells, the perylene-labeled daptomycin forms excimers, which shows that the N-terminal acyl chains of neighboring oligomer subunits are in immediate contact with one another. In a lipid bicelle system, oligomer formation can be titrated with stoichiometric amounts of phosphatidylglycerol. Therefore, the interaction of daptomycin with a single molecule of phosphatidylglycerol is sufficient to trigger daptomycin oligomerization.► Daptomycin was labeled with perylene to produce excimer fluorescence. ► Excimer fluorescence on liposomes and bacterial cells prove oligomerization. ► Oligomerization is triggered by stoichiometric quantities of phosphatidylglycerol.

Keywords: Daptomycin; Lipopeptide; Perylene; Excimer fluorescence; Phosphatidylglycerol; Oligomerization

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b₅₆₁ enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells by Lucia Cenacchi; Manuela Busch; Philipp G. Schleidt; Muller Florian G. Müller; Tina V.M. Stumpp; Mantele Werner Mäntele; Paolo Trost; C. Roy D. Lancaster (pp. 679-688).

Cytochrome (cyt) b₅₆₁ proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome- b₅₆₁-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b₅₆₁ paralogs from Arabidopsis thaliana ( Acyt b₅₆₁-A, Acyt b₅₆₁-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acyt b₅₆₁-A resembles the best characterised member of the CYBASC family, the cytochrome b₅₆₁ from adrenomedullary chromaffin vesicles, and that Acyt b₅₆₁-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (E_M) values were found to be fully consistent with ascorbate oxidation activities and Fe3⁺-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b₅₆₁ from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E_M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E_M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe3⁺-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E_M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b₅₆₁ paralogs exist as homodimers.► Heterologous production, purification of functional cytochrome b₅₆₁-A and B-paralogs. ► Characterisation of a novel and atypical CYBASC protein from Arabidopsis thaliana. ► Determination of ascorbate oxidation and Fe3⁺-chelates reductase activities. ► Determination of the haem midpoint potential (E_m) values of the paralogs. ► Effects on E_m of inhibitor allow assignment of ascorbate-binding site close to haem b_L.

Keywords: Abbreviations; A; _x; absorption at a wavelength of x nm; BCA; bicinchoninic acid; BMGY; buffered glycerol-complex medium; BMMY; buffered methanol-complex medium; BSA; bovine serum albumin; CV; column volumes; CYBASC; cytochrome; b; ₅₆₁; ascorbate reducible family; cyt; b; ₅₆₁; cytochrome; b; ₅₆₁; Da; Dalton; ΔE; M,; b; _H; shift in E; M,; b; _H; ΔE; M,; b; _L; shift in E; M,; b; _L; DEPC; diethyl pyrocarbonate; DDΜ; n-dodecyl-β-D-maltoside; DMSO; Dimethyl sulfoxide; DTT; Dithiothreitol E; _M; , oxidation–reduction midpoint potential; E; _M7.2; E; _M; value at pH 7.2; E; M,; b; _H; E; _M; of the high-potential haem; E; M,; b; _L; E; _M; of the low-potential haem; ESI; Electrospray ionisation; FOS-12; Fos-choline 12, H; _10,; deca-histidine affinity tag; IEF; isoelectric focusing; IMAC; immobilised metal affinity chromatography; IPTG; Isopropyl β-D-1-thiogalactopyranoside; LB; Luria-Bertani; MDHA; mono-dehydroascorbate; MNNG; N-methyl-N′-nitro-N-nitrosoguanidine; MS; Mass spectrometry; MW; molecular weight; M9; Salts Minimal Media; NZYM; NZ amine; NaCl; bacto-yeast extract and magnesium sulphate; OD; _x; optical density at a wavelength of x nm; ORF; open reading frame; RCA; relative catalytic activity; S; _II; streptavidin affinity tag II; S. c.; Saccharomyces cerevisiae; SDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresis; TMPD; N,N,N′,N′,tetramethyl-p-phenylenediamine dihydrochloride; YNB; Yeast Nitrogen Bases; Zm; Zea mays Arabidopsis thaliana; Biochemical characterisation; Cytochrome; b; ₅₆₁; paralog; Heterologous production; Membrane protein

Listeria monocytogenes cell wall constituents exert a charge effect on electroporation threshold by Alex Golberg; Chris S. Rae; Boris Rubinsky (pp. 689-694).

Genetically engineered cells with mutations of relevance to electroporation, cell membrane permeabilization by electric pulses, can become a promising new tool for fundamental research on this important biotechnology. Listeria monocytogenes mutants lacking DltA or MprF and assayed for sensitivity to the cathelicidin like anti-microbial cationic peptide (mCRAMP), were developed to study the effect of cell wall charge on electroporation. Working in the irreversible electroporation regime (IRE), we found that application of a sequence of 50 pulses, each 50μs duration, 12.5kV/cm field, delivered at 2Hz led to 2.67±0.29 log reduction in wild-type L. monocytogenes, log 2.60±0.19 in the MprF-minus mutant, and log 1.33±0.13 in the DltA-minus mutant. The experimental observation that the DltA-minus mutant was highly susceptible to cationic mCRAMP and resistant to IRE suggests that the charge on the bacterial cell wall affects electroporation and shows that this approach may be promising for fundamental studies on electroporation.► Cells with modified surface charge for electroporation research. ► Cell surface charge impacts on electropermeabilization threshold. ► Cell surface charge impacts cell resistance to pulse electric fields.

Keywords: Irreversible electroporation; Membrane charge; Electroporation threshold; Listeria monocytogenes; Lipoteichoic acid

Calcium-dependent aggregation and fusion of phosphatidylcholine liposomes induced by complexes of flavonoids with divalent iron by Yury S. Tarahovsky; Elena A. Yagolnik; Eugeny N. Muzafarov; Bolatbek S. Abdrasilov; Yuri A. Kim (pp. 695-702).

It was found that complexes of the flavonoids quercetin, taxifolin, catechin and morin with divalent iron initiated an increase in light scattering in a suspension of unilamellar 100nm liposomes. The concentration of divalent iron in the suspension was 10μM. Liposomes were prepared from 1-palmitoyl-2-oleoylglycero-3-phoshpatidylcholine. The fluorescent resonance energy transfer (FRET) analysis of liposomes labeled with NBD-PE and lissamine rhodamine B dyes detected a slow lipid exchange in liposomes treated with flavonoid-iron complexes and calcium, while photon correlation spectroscopy and freeze-fracture electron microscopy revealed the aggregation and fusion of liposomes to yield gigantic vesicles. Such processes were not found in liposomes treated with phloretin because this flavonoid is unable to interact with iron. Rutin was also unable to initiate any marked changes because this water-soluble flavonoid cannot interact with the lipid bilayer. The experimental data and computer calculations of lipophilicity (cLogP) as well as the charge distribution on flavonoid-iron complexes indicate that the adhesion of liposomes is provided by an iron link between flavonoid molecules integrated in adjacent bilayers. It is supposed that calcium cations facilitate the aggregation and fusion of liposomes because they interact with the phosphate moieties of lipids.Display Omitted►We studied interaction of flavonoid-iron(II) complexes with liposomes. ►We found that flavonoid-iron(II) can initiate aggregation of liposomes. ►Calcium facilitates aggregation and fusion of liposomes treated by flavonoid-iron. ►We concluded that iron(II) produces links between bilayers contained flavonoids.

Keywords: Abbreviations; PC; phosphatidylcholine; POPC; 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine; DMSO; dimethyl sulfoxide; NBD-PE; [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)]; Rhodamine-PE; [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)]; PCS; Photon correlation spectroscopyFlavonoid; Liposome; Iron; Calcium; Membrane adhesion; Membrane fusion

Bioinformatic characterization of the 4-Toluene Sulfonate Uptake Permease (TSUP) family of transmembrane proteins by Maksim A. Shlykov; Wei Hao Zheng; Jonathan S. Chen; Milton H. Saier, Jr. (pp. 703-717).

The ubiquitous sequence diverse 4-Toluene Sulfonate Uptake Permease (TSUP) family contains few characterized members and is believed to catalyze the transport of several sulfur-based compounds. Prokaryotic members of the TSUP family outnumber the eukaryotic members substantially, and in prokaryotes, but not eukaryotes, extensive lateral gene transfer occurred during family evolution. Despite unequal representation, homologues from the three taxonomic domains of life share well-conserved motifs. We show that the prototypical eight TMS topology arose from an intragenic duplication of a four transmembrane segment (TMS) unit. Possibly, a two TMS α-helical hairpin structure was the precursor of the 4 TMS repeat unit. Genome context analyses confirmed the proposal of a sulfur-based compound transport role for many TSUP homologues, but functional outliers appear to be prevalent as well. Preliminary results suggest that the TSUP family is a member of a large novel superfamily that includes rhodopsins, integral membrane chaperone proteins, transmembrane electron flow carriers and several transporter families. All of these proteins probably arose via the same pathway: 2→4→8 TMSs followed by loss of a TMS either at the N- or C-terminus, depending on the family, to give the more frequent 7 TMS topology.► The 4-Toluene Sulfonate Uptake Permease (TSUP) family is ubiquitous in nature. ► Uptake of sulfur-containing compounds is mediated by members of the TSUP family. ► No bioinformatic characterization of this family of porters has yet been described. ► We here demonstrate that these proteins arose by intragenic duplication of 4 TMSs. ► Phylogeny, motifs, topologies and functional predictions are described herein.

Keywords: Transport protein; Secondary active transport; TSUP; Uptake/efflux; Evolution; Superfamily

Chaperoning α7 neuronal nicotinic acetylcholine receptors by Valles Ana S. Vallés; Francisco J. Barrantes (pp. 718-729).

The α7 subtype of nicotinic acetylcholine receptors (AChRs) is one of the most abundant members of the Cys-loop family of receptors present in the central nervous system. It participates in various physiological processes and has received much attention as a potential therapeutic target for a variety of pathologies. The importance of understanding the mechanisms controlling AChR assembly and cell-surface delivery lies in the fact that these two processes are key to determining the functional pool of receptors actively engaged in synaptic transmission. Here we review recent studies showing that RIC-3, a protein originally identified in the worm Caenorhabditis elegans, modulates the expression of α7 AChRs in a subtype-specific manner. Potentiation of AChR expression by post-transcriptional events is also critically assessed.► The α7 nicotinic acetylcholine receptor (AChR) is one of the most abundant AChR subtypes in the hippocampus. ► This receptor is a potential therapeutic target for important psychiatric diseases. ► RIC-3, a protein originally found in C. elegans, modulates the expression of α7 receptors. ► Modulation of receptor expression by RIC-3 is critically reviewed.

Keywords: Molecular chaperone; RIC-3; α7; Receptor trafficking

Thapsigargin decreases the Na⁺- Ca2⁺ exchanger mediated Ca2⁺ entry in pig coronary artery smooth muscle by Gauri Akolkar; Jyoti Pande; Sue E. Samson; Ashok K. Grover (pp. 730-737).

Na⁺- Ca2⁺ exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca2⁺ pool along with the SER Ca2⁺ pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca2⁺ depletion on NCX–SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na⁺-loaded and then placed in either a Na⁺-containing or in a Na⁺-substituted solution. Subsequently, the difference in Ca2⁺ entry between the two groups was examined and defined as the NCX mediated Ca2⁺ entry. The NCX mediated Ca2⁺ entry in the smooth muscle cells was monitored using two methods: Ca2⁺sensitive fluorescence dye Fluo-4 and radioactive Ca2⁺. Ca2⁺-entry was greater in the Na⁺-substituted cells than in the Na⁺-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca2⁺ entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na⁺-substituted solution with or without thapsigargin. SER Ca2⁺ depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca2⁺ entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca2⁺ entry may protect the cells against Ca2⁺-overload during ischemia–reperfusion when SERCA2 is known to be damaged.►Inhibiting SER Ca2⁺ pump with thapsigargin blocks Ca2⁺ entry through NCX. ►There is an increased proximity between the two proteins when SER Ca2⁺ is depleted. ►A mechanism for the NCX blockade is proposed based on organellar rearrangements. ►This blockade may protect cells from Ca2⁺ overload when SERCA is not functional.

Keywords: Abbreviations; [Ca; 2; ⁺; ]i; cytosolic Ca; 2; ⁺; EGTA; ethyleneglycol bis (2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; NCX; Na; ⁺; -Ca; 2; ⁺; -exchanger; PBS; phosphate buffered saline; NMG; N-methylglucamine; PM; plasma membrane; SER; sarco/endoplasmic reticulum; SERCA; SR Ca; 2; ⁺; pumpNCX; SERCA; Immunofluorescence; Confocal microscopy; Linkage; SOCE

Effects of an antimalarial quinazoline derivative on human erythrocytes and on cell membrane molecular models by Yareli Rojas-Aguirre; Hernandez-Luis Francisco Hernández-Luis; Mendoza-Martinez César Mendoza-Martínez; Carlos Patricio Sotomayor; Luis Felipe Aguilar; Fernando Villena; Ivan Castillo; Hernandez David J. Hernández; Mario Suwalsky (pp. 738-746).

Plasmodium, the parasite which causes malaria in humans multiplies in the liver and then infects circulating erythrocytes. Thus, the role of the erythrocyte cell membrane in antimalarial drug activity and resistance has key importance. The effects of the antiplasmodial N⁶-(4-methoxybenzyl)quinazoline-2,4,6-triamine (M4), and its inclusion complex (M4/HPβCD) with 2-hydroxypropyl-β-cyclodextrin (HPβCD) on human erythrocytes and on cell membrane molecular models are herein reported. This work evidences that M4/HPβCD interacts with red cells as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a 10μM concentration; b) in isolated unsealed human erythrocyte membranes (IUM) a concentration as low as 1μM induced sharp DPH fluorescence anisotropy decrease whereas increasing concentrations produced a monotonically decrease of DPH fluorescence lifetime at 37°C; c) X-ray diffraction studies showed that 200μM induced a complete structural perturbation of dimyristoylphosphatidylcholine (DMPC) bilayers whereas no significant effects were detected in dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of lipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively; d) fluorescence spectroscopy data showed that increasing concentrations of the complex interacted with the deep hydrophobic core of DMPC large unilamellar vesicles (LUV) at 18°C. All these experiments are consistent with the insertion of M4/HPβCD in the outer monolayer of the human erythrocyte membrane; thus, it can be considered a promising and novel antimalarial agent.SEM micrographs showing the morphological effects induced by the antiplasmodial quinazoline M4 complexed with the cyclodextrin HPβCD on human erythrocyte membrane.Display Omitted► Malaria parasites infect human erythrocytes. ► The antiplasmodial quinazoline derivative M4 was complexed with 2-hydroxypropyl-β-cyclodextrin. ► The complex induced in vitro morphological changes to human erythrocytes. ► This inclusion complex interacted with the outer monolayer of the human erythrocyte membrane. ► Cyclodextrin facilitated the interaction of M4 with the erythrocyte membrane.

Keywords: Abbreviations; SEM; scanning electron microscopy; IUM; isolated unsealed erythrocyte membrane; RBCS; red blood cell suspensions; LUV; large unilamellar vesicles; DMPC; dimyristoylphosphatidylcholine; DMPE; dimyristoylphosphatidylethanolamine; r; fluorescence anisotropy; GP; general polarization; DPH; 1,6-diphenyl-1,3,5-hexatriene; laurdan; 6-dodecanoyl-2-dimethylaminonaphtalene; PfDHFR; dihydrofolate reductase; M4; N; ⁶; -(4-methoxybenzyl)quinazoline-2,4,6-triamine; CDs; cyclodextrins; HPβCD; 2-hydroxypropyl-β-cyclodextrinMalaria; Quinazoline derivative; Cyclodextrin; Inclusion complex; Erythrocyte membrane; Drug–membrane interaction

Kinetics of demetallation of a zinc–salophen complex into liposomes by Carla Gasbarri; Guido Angelini; Antonella Fontana; Paolo De Maria; Gabriella Siani; Ilaria Giannicchi; Antonella Dalla Cort (pp. 747-752).

A Zn–salophen complex has been incorporated into POPC large unilamellar liposomes (LUV) obtained in phosphate buffer at pH 7.4. Fluorescence optical microscopy and anisotropy measurements show that the complex is located at the liposomal surface, close to the polar headgroups. The interaction of the POPC phosphate group with Zn2⁺ slowly leads to demetallation of the complex. The process follows first order kinetics and rate constants have been measured fluorimetrically in pure water and in buffered aqueous solution.The coordination of the phosphate group of monomeric POPC with salophen zinc also occurs in chloroform as detected by ESI-MS measurements.The effect of the Zn–salophen complex on the stability of POPC LUV has been evaluated at 25°C by measuring the rate of release of entrapped 5(6)-carboxyfluorescein (CF) in the presence and in the absence of Triton X-100 as the perturbing agent. It turns out that the inclusion of the complex significantly increases the stability of POPC LUV.Display Omitted► A Zn–salophen complex has been incorporated into POPC extruded liposomes. ► The phospholipid-Zn–salophen coordination slowly leads to complex demetallation. ► The process follows first order kinetics and can be followed fluorimetrically. ► Demetallation rate constants have been measured in water and in buffer solution. ► The incorporation of the complex enhances the stability of liposomes.

Keywords: Zinc-salophen; Liposomes; ESI-MS; Kinetics; Fluorescence anisotropy

Cholesterol dependence of Newcastle Disease Virus entry by Martin Juan José Martín; Javier Holguera; Sanchez-Felipe Lorena Sánchez-Felipe; Enrique Villar; Munoz-Barroso Isabel Muñoz-Barroso (pp. 753-761).

Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-β-cyclodextrin (MβCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MβCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4°C and then shifted to detergent-soluble fractions at 37°C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.► We examine the role of cellular cholesterol on NDV activities. ► Removal of cholesterol partially reduces viral binding, fusion and infectivity. ► This negative effect was reversible. ► The HN viral attachment protein partially localized to cell lipid rafts at 4°C. ► Cellular cholesterol may be required for optimal cell entry in NDV infection.

Keywords: Lipid raft; Cholesterol; Viral entry; Paramyxovirus; NDV

Structural and biophysical characterization of an antimicrobial peptide chimera comprised of lactoferricin and lactoferrampin by Evan F. Haney; Kamran Nazmi; Jan G.M. Bolscher; Hans J. Vogel (pp. 762-775).

Lactoferricin and lactoferrampin are two antimicrobial peptides found in the N-terminal lobe of bovine lactoferrin with broad spectrum antimicrobial activity against a range of Gram-positive and Gram-negative bacteria as well as Candida albicans. A heterodimer comprised of lactoferrampin joined to a fragment of lactoferricin was recently reported in which these two peptides were joined at their C-termini through the two amino groups of a single Lys residue (Bolscher et al., 2009, Biochimie 91(1):123–132). This hybrid peptide, termed LFchimera, has significantly higher antimicrobial activity compared to the individual peptides or an equimolar mixture of the two. In this work, the underlying mechanism behind the increased antibacterial activity of LFchimera was investigated. Differential scanning calorimetry studies demonstrated that all the peptides influenced the thermotropic phase behaviour of anionic phospholipid suspensions. Calcein leakage and vesicle fusion experiments with anionic liposomes revealed that LFchimera had enhanced membrane perturbing properties compared to the individual peptides. Peptide structures were evaluated using circular dichroism and NMR spectroscopy to gain insight into the structural features of LFchimera that contribute to the increased antimicrobial activity. The NMR solution structure, determined in a miscible co-solvent mixture of chloroform, methanol and water, revealed that the Lys linkage increased the helical content in LFchimera compared to the individual peptides, but it did not fix the relative orientations of lactoferricin and lactoferrampin with respect to each other. The structure of LFchimera provides insight into the conformation of this peptide in a membranous environment and improves our understanding of its antimicrobial mechanism of action.Display Omitted► Unique antimicrobial peptide comprised of lactoferricin and lactoferrampin. ► First NMR structure reported for a peptide heterodimer joined through a Lys linker. ► Enhanced membrane perturbation by the heterodimer compared to constituent peptides. ► Biophysical evidence correlates with the increased antimicrobial potency. ► Merits of a co-solvent mixture as a membrane mimetic in NMR studies is discussed.

Keywords: Abbreviations; COSY; Correlation spectroscopy; DPPG; 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol); DSC; Differential scanning calorimetry; ePE; egg derived phosphatidylethanolamine; ePG; egg derived phosphatidylglycerol; LFampB; Bovine lactoferrampin; LFchimera; Lys linked LFcin17–30 and LFampB heterodimer; LFcin; Lactoferricin; LUV; Large unilamellar vesicle; NMR; Nuclear magnetic resonance; NOESY; Nuclear Overhauser spectroscopy; PLE; E. coli; polar lipid extract; RMSD; Root mean squared deviation; SDS; Sodium dodecyl sulphate; TOCSY; Total correlation spectroscopyAntimicrobial peptide; Lactoferricin; Lactoferrampin; NMR solution structure; Peptide–membrane interactions

Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae by Jiwon Youm; Milton H. Saier Jr. (pp. 776-797).

The co-emergence of multidrug resistant pathogenic bacterial strains and the Human Immunodeficiency Virus pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life was identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth.►Starting point for experimental studies in dependencies for mycobacteria. ►Suggestions regarding transporters that function in intra- v. extracellular growth. ►Co-emergence of multidrug resistant bacterial strains as a public health threat.

Keywords: Tuberculosis; Leprosy; Transport protein; Intracellular versus extracellular life; Whole genome analyses; Comparative genomics

Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant by Iulia Blesneac; Stéphanie Ravaud; Céline Juillan-Binard; Laure-Anne Barret; Manuela Zoonens; Ange Polidori; Bruno Miroux; Bernard Pucci; Eva Pebay-Peyroula (pp. 798-805).

Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.► We expressed UCP1, a mitochondrial carrier, in a cell free system. ► The yield of pure protein is compatible with structural studies. ► Well-folded protein is expressed in the presence of fluorinated surfactants. ► The strategy is a new alternative for expressing all mitochondrial carriers.

Keywords: Abbreviations; UCP1; uncoupling protein 1; MCF; mitochondrial carrier family; AAC; ADP/ATP carrier; BAT; brown adipose tissues; cmc; critical micellar concentration; CD; circular dichroism; RTS; Rapid Translation SystemMembrane protein expression; Mitochondrial carriers; Uncoupling protein; Cell-free expression; Fluorinated surfactants; Circular dichroism

FtsZ polymers bound to lipid bilayers through ZipA form dynamic two dimensional networks by Pablo Mateos-Gil; Ileana Márquez; Lopez-Navajas Pilar López-Navajas; Jimenez Mercedes Jiménez; Miguel Vicente; Jesús Mingorance; Germán Rivas; Velez Marisela Vélez (pp. 806-813).

Bacteria divide by forming a contractile ring around their midcell region. FtsZ, a cytoskeletal soluble protein structurally related to tubulin, is the main component of this division machinery. It forms filaments that bundle at the inner side of the cytoplasmic membrane. These FtsZ bundles do not attach to bare lipid surfaces. In Escherichia coli they remain near the membrane surface by attaching to the membrane protein ZipA and FtsA. In order to study the structure and dynamics of the ZipA–FtsZ bundles formed on a lipid surface, we have oriented a soluble form of ZipA (sZipA), with its transmembrane domain substituted by a histidine tag, on supported lipid membranes. Atomic force microscopy has been used to visualize the polymers formed on top of this biomimetic surface. In the presence of GTP, when sZipA is present, FtsZ polymers restructure forming higher order structures. The lipid composition of the underlying membrane affects the aggregation kinetics and the shape of the structures formed. On the negatively charged E. coli lipid membranes, filaments condense from initially disperse material to form a network that is more dynamic and flexible than the one formed on phosphatidyl choline bilayers. These FtsZ–ZipA filament bundles are interconnected, retain their capacity to dynamically restructure, to fragment, to anneal and to condense laterally.► FtsZ attached to ZipA polymerizes differently on bilayers of different lipid compositions. ► FtsZ forms dynamic filament networks on E. coli lipid surfaces. ► Dynamic rearrangement of FtsZ filament aggregates on lipid surfaces could contribute to the contractile force.

Keywords: FtsZ; ZipA; Supported lipid membrane; Atomic force microscope

Reconstitution of integral membrane proteins into isotropic bicelles with improved sample stability and expanded lipid composition profile by Emma A. Morrison; Katherine A. Henzler-Wildman (pp. 814-820).

Reconstitution of integral membrane proteins into membrane mimetic environments suitable for biophysical and structural studies has long been a challenge. Isotropic bicelles promise the best of both worlds—keeping a membrane protein surrounded by a small patch of bilayer-forming lipids while remaining small enough to tumble isotropically and yield good solution NMR spectra. However, traditional methods for the reconstitution of membrane proteins into isotropic bicelles expose the proteins to potentially destabilizing environments. Reconstituting the protein into liposomes and then adding short-chain lipid to this mixture produces bicelle samples while minimizing protein exposure to unfavorable environments. The result is higher yield of protein reconstituted into bicelles and improved long-term stability, homogeneity, and sample-to-sample reproducibility. This suggests better preservation of protein structure during the reconstitution procedure and leads to decreased cost per sample, production of fewer samples, and reduction of the NMR time needed to collect a high quality spectrum. Furthermore, this approach enabled reconstitution of protein into isotropic bicelles with a wider range of lipid compositions. These results are demonstrated with the small multidrug resistance transporter EmrE, a protein known to be highly sensitive to its environment.Display Omitted► Reconstitution without requirement for organic solvent or specific detergent ► Increased stability and homogeneity of isotropic bicelle samples ► Reconstitution with expanded, more physiologically relevant lipid profile ► Significant improvements for applications in biophysical and structural studies

Keywords: Abbreviations; NMR; nuclear magnetic resonance; DM; decylmaltoside; DHPC; 1,2-dihexanoyl-sn-glycero-3-phosphocholine; DLPC; 1,2-dilauroyl-sn-glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-sn-glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG; 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); TPP; ⁺; tetraphenylphosphonium; ⁺; PS; phosphatidylserine; PE-DTPA; phosphatidylethanolamine-N-diethylenetriaminepentaacetic acid; IMP; integral membrane protein; DLS; dynamic light scattering; TLC; thin layer chromatographyIsotropic bicelles; Membrane mimetic; Membrane protein reconstitution; EmrE; NMR

Time-resolved EPR immersion depth studies of a transmembrane peptide incorporated into bicelles by Nisreen A. Nusair; Daniel J. Mayo; Tia D. Dorozenski; Thomas B. Cardon; Johnson J. Inbaraj; Ethan S. Karp; Justin P. Newstadt; Stuart M. Grosser; Gary A. Lorigan (pp. 821-828).

The reduction in EPR signal intensity of nitroxide spin-labels by ascorbic acid has been measured as a function of time to investigate the immersion depth of the spin-labeled M2δ AChR peptide incorporated into a bicelle system utilizing EPR spectroscopy. The corresponding decay curves of n-DSA (n=5, 7, 12, and 16) EPR signals have been used to (1) calibrate the depth of the bicelle membrane and (2) establish a calibration curve for measuring the depth of spin-labeled transmembrane peptides. The kinetic EPR data of CLS, n-DSA (n=5, 7, 12, and 16), and M2δ AChR peptide spin-labeled at Glu-1 and Ala-12 revealed excellent exponential and linear fits. For a model M2δ AChR peptide, the depth of immersion was calculated to be 5.8Å and 3Å for Glu-1, and 21.7Å and 19Å for Ala-12 in the gel-phase (298K) and L_α-phases (318K), respectively. The immersion depth values are consistent with the pitch of an α-helix and the structural model of M2δ AChR incorporated into the bicelle system is in a good agreement with previous studies. Therefore, this EPR time-resolved kinetic technique provides a new reliable method to determine the immersion depth of membrane-bound peptides, as well as, explore the structural characteristics of the M2δ AChR peptide.► EPR spectroscopic methods developed for measuring membrane depth. ► Kinetic decay curves reveal membrane depth. ► Bicelles are an excellent model membrane system.

Keywords: Abbreviations; L; _α; -phase; liquid–crystalline phase; M2δ; M2 segment of the δ subunit; AChR; nicotinic acetylcholine receptor; EPR spectroscopy; electron paramagnetic resonance spectroscopy; CLS; cholestane (3β-doxyl-5α-cholestane); n; -DSA (; n; =; 5; 7, 12, and 16), 5, 7, 12, and 16-doxylstearic acids; DMPC; 1, 2-Dimyristoyl-; sn; -glycerol-3-phosphocholine; DHPC; 1, 2-Dihexanoyl-; sn; -glycerol-3-phosphocholine; Δχ; magnetic susceptibility anisotropy tensor; [1]; amino acid residue 1; [12]; amino acid residue 12; SL; spin-labeled; [1] SL-M2δ AChR; M2δ AChR peptide subunit spin-labeled at amino acid residue 1; [12] SL-M2δ AChR; M2δ AChR peptide subunit spin-labeled at amino acid residue 12; NMR spectroscopy; nuclear magnetic resonance spectroscopy; FRET; fluorescence resonance energy transfer; MTSSL; 1-oxyl-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl-methanethiosulfonateImmersion depth; M2δ acetylcholine receptor; EPR spectroscopy; Bicelle; Transmembrane peptide; Kinetics; Ascorbic acid

Strain, stress and energy in lipid bilayer induced by electrostatic/electrokinetic forces by M. Tajparast; Glavinovic M.I. Glavinović (pp. 829-838).

Lipid bilayer was deformed by the electrostatic/electrokinetic forces induced by the fixed charges on the top monolayer–solution interface. The strains, stresses and energy were simulated using finite element method. The elastic moduli of the heads were four times greater than those of tails sections, but were individually isotropic. The physics of the situation was evaluated using a coupled system of linear elastic equations and electrostatic–electrokinetic (Poisson–Nernst–Planck) equations. The Coulomb force (due to fixed charges in the electric field), and the dielectric force (due to uneven electric field and the solution-membrane permittivity mismatch) bend the membrane, but unevenly. Whereas the bottom monolayer extends vertically (towards charged surface), the top monolayer compresses. In contrast the top monolayer extends horizontally, but the bottom monolayer compresses. The horizontal normal stress is higher in the heads than in the tails sections, but is similar in two monolayers, whereas the vertical normal stress is small. The horizontal normal stress is associated with horizontal normal strain, and vertical with both vertical and horizontal strain. Surprisingly, the shear stress (an indicator where the membrane will deform), is greater in the tails sections. Finally, the elastic energy (which is clearly greater in the heads sections) is dominated by its horizontal component and peaks in the middle of the membrane. The shear component dominates in the tails sections, and is minimal in the membrane center. Even spatially uniform external force thus leads to complex membrane deformation and generates complex profiles of stress and elastic energy.►Lipid bilayer was deformed by electrostatic forces due to membrane surface charges. ►The physics was evaluated using elastic–electrostatic–electrokinetic equations. ►The bottom monolayer extends vertically and compresses horizontally. ►Unexpectedly the shear stress is greater in tails sections. ►Uniform external force can produce complex membrane deformation and stress profiles.

Keywords: Lipid bilayer; Normal stress; Shear stress; Dielectric force; Maxwell stress; Poisson–Nernst–Planck

Reconstitution of water channel function and 2D-crystallization of human aquaporin 8 by Maria Agemark; Julia Kowal; Wanda Kukulski; Norden Kristina Nordén; Niklas Gustavsson; Urban Johanson; Andreas Engel; Per Kjellbom (pp. 839-850).

Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H₂O₂ in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent–lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3Å.► Human AQP8 forms a separate subfamily distinct from the other mammalian AQPs. ► Functional human AQP8 is overexpressed in P. pastoris at 2mg/mL per liter of culture. ► Optimal solubilization is achieved with the detergent decyl maltoside. ► Removal of affinity tag yields highly ordered crystal arrays diffracting to 3Å.

Keywords: Heterologous expression; AQP8; Aquaporin; 2D crystallization

Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating by Wuyang Wang; Paul Linsdell (pp. 851-860).

Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single “gate” within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)₂⁻ ions was reduced when ATP concentration was reduced from 1mM to 10μM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)₂⁻ was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.► Opening of the CFTR channel pore is controlled by ATP binding and hydrolysis. ► The conformational changes in the pore triggered by ATP are not known. ► We have used site-specific modification to identify changes in pore conformation. ► Pharmacological or mutagenic interference with ATP hydrolysis alters modification. ► Our results identify the location of a gate within the pore that is opened by ATP.

Keywords: Abbreviations; ABC; ATP-binding cassette; BHK; baby hamster kidney; CFTR; cystic fibrosis transmembrane conductance regulator; MTS; methanethiosulfonate; MTSES; [2-sulfonatoethyl] methanethiosulfonate; NBD; nucleotide binding domain; PKA; protein kinase A; PPi; pyrophosphate; TM; transmembrane α-helix; TMD; transmembrane domainABC protein; Channel gating; Channel pore; Chloride channel; Cystic fibrosis transmembrane conductance regulator; Nucleotide binding domain

Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy by Opacic Milena Opačić; Ben H. Hesp; Fabrizia Fusetti; Bauke W. Dijkstra; Jaap Broos (pp. 861-868).

The mannitol transporter EII^mtl from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EII^mtl is functional as a dimer and its membrane-embedded C domain, IIC^mtl, harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IIC^mtl. At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IIC^mtl. Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EII^mtl is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IIC^mtl structure are involved in the sugar translocation. The structure of EII^mtl, as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EII^mtl, the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.► 17 single Trp mutants of the mannitol transporter were labeled with 5-fluorotryptophan. ► The structure of the two protomers in this dimeric transporter are different. ► B domain phosphorylation induces long-range conformational changes in the C domain. ► Many parts of the C domain are involved in sugar translocation.

Keywords: Abbreviations; EII; ^mtl; the mannitol-specific transporting and phosphorylating enzyme from; E. coli; IIC; ^mtl; membrane-embedded C domain of EII; ^mtl; Trp; tryptophan; 5-FTrp; 5-fluorotryptophan; TOE; tryptophan octyl ester: mannitol,; d; -mannitol; FRET; Förster resonance energy transfer; FWHH; full width at the half height; ϕ; rotational correlation time; NATA; N-acetyl-tryptophanamide; PEG; Polyethylene glycolAnisotropy; Fluorotryptophan; Iodide quenching; Membrane protein; PTS; Time-resolved fluorescence spectroscopy

Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms by Kathryn E. Pavia; Sara A. Spinella; Donald E. Elmore (pp. 869-876).

The increase in multidrug resistant bacteria has sparked an interest in the development of novel antibiotics. Antimicrobial peptides that operate by crossing the cell membrane may also have the potential to deliver drugs to intracellular targets. Buforin 2 (BF2) is an antimicrobial peptide that shares sequence identity with a fragment of histone subunit H2A and whose bactericidal mechanism depends on membrane translocation and DNA binding. Previously, novel histone-derived antimicrobial peptides (HDAPs) were designed based on properties of BF2, and DesHDAP1 and DesHDAP3 showed significant antibacterial activity. In this study, their DNA binding, permeabilization, and translocation abilities were assessed independently and compared to antibacterial activity to determine whether they share a mechanism with BF2. To investigate the importance of proline in determining the peptides’ mechanisms of action, proline to alanine mutants of the novel peptides were generated. DesHDAP1, which shows significant similarities to BF2 in terms of secondary structure, translocates effectively across lipid vesicle and bacterial membranes, while the DesHDAP1 proline mutant shows reduced translocation abilities and antimicrobial potency. In contrast, both DesHDAP3 and its proline mutant translocate poorly, though the DesHDAP3 proline mutant is more potent. Our findings suggest that a proline hinge can promote membrane translocation in some peptides, but that the extent of its effect on permeabilization depends on the peptide's amphipathic properties. Our results also highlight the different antimicrobial mechanisms exhibited by histone-derived peptides and suggest that histones may serve as a source of novel antimicrobial peptides with varied properties.► Not all histone-derived antimicrobial peptides (HDAPs) use the same mechanism. ► DesHDAP1 shows proline-dependent translocation, analogous to buforin II. ► In DesHDAP3, proline to alanine mutation increases membrane lysis and potency. ► Proline promotes membrane translocation in some antimicrobial peptides. ► Amphipathicity, not α-helicity, determines lytic abilities of proline mutants.

Keywords: Antimicrobial peptide; Histone-derived peptide; Translocation; Membrane permeabilization; Proline hinge; Amphipathic

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: Implications for cell internalization by Ana M.S. Cardoso; Sara Trabulo; Ana L. Cardoso; Annely Lorents; Catarina M. Morais; Paula Gomes; Cláudia Nunes; Lucio Marlene Lúcio; Salette Reis; Kärt Padari; Margus Pooga; Maria C. Pedroso de Lima; Amália S. Jurado (pp. 877-888).

The present work aims to gain insights into the role of peptide–lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide–lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.► Cellular internalization of S4₁₃-PV peptide depends on membrane–lipid interactions. ► S4₁₃-PV and a scrambled analogue differently affect lipid physical behavior. ► S4₁₃-PV induces bilayer destabilization in lipid models and HeLa cell membranes. ► S4₁₃-PV uptake may be driven by lipid domain segregation and non-lamellar phases. ► Our data contribute to gain insights into peptide structure–activity relationships.

Keywords: Abbreviations; CPP; cell-penetrating peptide; DOPE; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPG; 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol); DPH-PA; 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid; DPPC; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPE; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DPPG; 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol); DSC; differential scanning calorimetry; LUV; large unilamellar vesicles; MLV; multilamellar vesicles; POPG; 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol); SAXS; small angle X-ray scattering; WAXS; wide angle X-ray scattering; 2-AS; 2-(9-anthroyloxy) stearic acid; 6-AS; 6-(9-anthroyloxy) stearic acid; 12-AS; 12-(9-anthroyloxy) stearic acid and 16-AP, 16-(9-anthroyloxy) palmitic acidCell-penetrating peptide; Membrane model; DSC; Fluorescence polarization; WAXS/SAXS; Electron microscopy

Transmembrane and extramembrane contributions to membrane protein thermal stability: Studies with the NaChBac sodium channel by Andrew M. Powl; Andrew J. Miles; B.A. Wallace (pp. 889-895).

The thermal stabilities of the extramembranous and transmembranous regions of the bacterial voltage-gated sodium channel NaChBac have been characterised using thermal-melt synchrotron radiation circular dichroism (SRCD) spectroscopy. A series of constructs, ranging from the full-length protein containing both the C-terminal cytoplasmic and the transmembranous domains, to proteins with decreasing amounts of the cytoplasmic domain, were examined in order to separately define the roles of these two types of domains in the stability and processes of unfolding of a membrane protein. The sensitivity of the SRCD measurements over a wide range of wavelengths and temperatures has meant that subtle but reproducible conformational changes could be detected with accuracy. The residues in the C-terminal extramembranous domain were highly susceptible to thermal denaturation, but for the most part the transmembrane residues were not thermally-labile and retained their helical character even at very elevated temperatures. The process of thermal unfolding involved an initial irreversible unfolding of the highly labile distal extramembranous C-terminal helical region, which was accompanied by a reversible unfolding of a small number of helical residues in the transmembrane domain. This was then followed by the irreversible unfolding of a limited number of additional transmembrane helical residues at greatly elevated temperatures. Hence this study has been able to determine the different contributions and roles of the transmembrane and extramembrane residues in the processes of thermal denaturation of this multipass integral membrane protein.► The NaChBac channel is very resistant to thermal denaturation. ► Differential thermal stability profiles of extramembrane and transmembrane residues. ► Membrane protein constructs with sequentially removed extramembranous residues. ► Synchrotron Radiation Circular Dichroism spectroscopy detection of subtle effects.

Keywords: Abbreviations; CD; circular dichroism; Cymal-5; 5-cyclohexyl-1-pentyl-β-D maltoside; EM; extramembrane; FL; full-length (native) NaChBac; NaChBac; voltage-gated sodium channel from; B. halodurans; SDS; sodium dodecyl sulfate; SRCD; synchrotron radiation circular dichroism; TM; transmembrane; VGSC; voltage-gated sodium channelProtein stability; Membrane protein folding/unfolding; Synchrotron radiation circular dichroism (SRCD) spectroscopy; Secondary structure; Extramembrane and transmembrane domains

Predicting the effect of steroids on membrane biophysical properties based on the molecular structure by Jorge J. Wenz (pp. 896-906).

The relationship between sterol structure and the resulting effects on membrane physical properties is still unclear, owing to the conflicting results found in the current literature. This study presents a multivariate analysis describing the physical properties of 83 steroid membranes. This first structure–activity analysis supports the generally accepted physical effects of sterols in lipid bilayers. The sterol chemical substituents and the sterol/phospholipid membrane physical properties were encoded by defining binary variables for the presence/absence of those chemical substituents in the polycyclic ring system and physical parameters obtained from phospholipid mixtures containing those sterols. Utilizing Principal Coordinates Analysis, the steroid population was grouped into five well-defined clusters according to their chemical structures. An examination of the membrane activity of each sterol structural cluster revealed that a hydroxyl group at C3 and an 8–10 carbon isoalkyl side-chain at C17 are mainly present in membrane active sterols having rigidifying, molecular ordering/condensing effects and/or a raft promoting ability. In contrast, sterol chemical structures containing a keto group at C3, a C4–C5-double bond, and polar groups or a short alkyl side-chain at C17 (3 to 7 atoms) are mostly found in sterols having opposite effects. Using combined multivariate approaches, it was concluded that the most important structural determinants influencing the physical properties of sterol-containing mixtures were the presence of an 8–10 carbon C17 isoalkyl side-chain, followed by a hydroxyl group at C3 and a C5–C6 double bond. Finally, a simple Logistic Regression model predicting the dependence of membrane activity on sterol chemical structure is proposed.Display Omitted► Structure and steroid activity on membranes was expressed by binary variables. ► Multivariate analysis provides insights on the structure-depending actions of steroids. ► An 8–10 carbon chain at C17 and a hydroxyl at C3 are the most relevant traits. ► Some chemical groups display opposite effects on steroid behavior. ► A structure-depending model predicts steroid activity on membranes.

Keywords: Abbreviations; PCoA; Principal Coordinate Analysis; LR; Logistic Regression; PC; principal coordinate; OR; odds ratio; CI; confidence intervalSterol structure; Membrane property; Multivariate analysis

Identification of residues involved in water versus glycerol selectivity in aquaporins by differential residue pair co-evolution by Xin Lin; Tian Hong; Yuguang Mu; Jaume Torres (pp. 907-914).

Aquaporins (AQPs) are members of the Major Intrinsic Protein (MIP) family that can transport water or glycerol, as well as other compounds. The rationale for substrate selectivity at the structural level is still incompletely understood. The information present in multiple sequence alignments (MSAs) can help identify both structural and functional features, especially the complex networks of interactions responsible for water or glycerol selectivity. Herein, we have used the method of Statistical Coupling Analysis (SCA) to identify co-evolving pairs of residues in two separate groups of sequences predicted to correspond to water or glycerol transporters. Differentially co-evolved pairs between the two groups were tested by their efficacy in correctly classifying a training set of MSAs, and binary classifiers were built with these pairs. Up to 50% of the residues found in hundreds of binary classifiers corresponded to only ten positions in the MSA of aquaporins. Most of these residues are close to the lining of the aquaporin pore and have been identified previously as important for selectivity. Therefore, this method can shed light on the residues that are important for substrate selectivity of aquaporins and other proteins. SCA requires a very large sequence dataset with relatively low homology amongst its members, and these requirements are met by aquaporins.► Aquaporins can be separated phylogenetically in water and glycerol transporters. ► SCA reveals residues important for substrate recognition in aquaporins. ► Most of these residues are close to the lumen of the pore. ► Most of these residues are in the extracellular half of the protein.

Keywords: Water channel; Aquaporin selectivity; Co-evolution analysis; Binary classifier; Water versus glycerol selectivity; Gating of aquaporins

Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane by Maja Anko; Janja Majhenc; Ksenija Kogej; Rannard Sillard; Ülo Langel; Gregor Anderluh; Matjaž Zorko (pp. 915-924).

The PepFect family of cell‐penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan‐10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.► Lipid–peptide interactions of cell-penetrating peptides (CPPs) TP10 and its modified analogs PF3 and PF6. ► Stearylation of TP10 improves insertion into neutral but not anionic lipid monolayers. ► Stearylation of TP10 decreases the membrane permeabilization activity of the modified CPPs. ► Modification of TP10 by trifluoromethylquinoline moieties has relatively little influence on lipid–CPP interaction.

Keywords: Abbreviations; CMC; critical micelle concentration; CPP; cell‐penetrating peptide; DCM; dichloromethane; DIEA; diisopropylethylamine; DLS; dynamic light scattering; DMF; dimethyl formamide; GUV; giant unilamellar vesicle; LUV; large unilamellar vesicle; MIP; maximum insertion pressure; MLV; multilamellar vesicle; PF; PepFect; POPC; 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine; POPG; 1palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glicerol); QN; N‐(2‐aminoethyl)‐N‐methyl‐N′‐[7(trifluoromethyl)‐quinolin‐4‐yl]ethane‐1,2‐diamine; Rh; hydrodynamic radius; RT‐HPLC; reversed‐phase high‐performance liquid chromatography; SPR; surface plasmon resonance; SUV; small unilamellar vesicle; TP10; transportan‐10Cell‐penetrating peptide; PepFect; Peptide‐membrane interaction; Pore; Stearylation

Sections

Personal tools

BBA - Biomembranes (v.1818, #3)

Sections

Personal tools

Local resources

BBA - Biomembranes (v.1818, #3)