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BBA - Biomembranes (v.1788, #2)

Editorial Board (pp. i).

Reviewer Acknowledgment (pp. iii-v).

Are specific nonannular cholesterol binding sites present in G-protein coupled receptors? by Yamuna Devi Paila; Shrish Tiwari; Amitabha Chattopadhyay ⁎ (pp. 295-302).

The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major drug targets in all clinical areas. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. Interestingly, recently reported crystal structures of GPCRs have shown structural evidence of cholesterol binding sites. Two possible mechanisms have been previously suggested by which membrane cholesterol could influence the structure and function of GPCRs (i) through a direct/specific interaction with GPCRs, which could induce a conformational change in the receptor, or (ii) through an indirect way by altering the membrane physical properties in which the receptor is embedded or due to a combination of both. We discuss here a novel mechanism by which membrane cholesterol could affect structure and function of GPCRs and propose that cholesterol binding sites in GPCRs could represent ‘nonannular’ binding sites. Interestingly, previous work from our laboratory has demonstrated that membrane cholesterol is required for the function of the serotonin_1A receptor, which could be due to specific interaction of the receptor with cholesterol. Based on these results, we envisage that there could be specific/nonannular cholesterol binding site(s) in the serotonin_1A receptor. We have analyzed putative cholesterol binding sites from protein databases in the serotonin_1A receptor, a representative GPCR, for which we have previously demonstrated specific requirement of membrane cholesterol for receptor function. Our analysis shows that cholesterol binding sites are inherent characteristic features of serotonin_1A receptors and are conserved over evolution. Progress in deciphering molecular details of the nature of GPCR-cholesterol interaction in the membrane would lead to better insight into our overall understanding of GPCR function in health and disease, thereby enhancing our ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.

Keywords: Abbreviations; 5-HT; _1A; receptor; 5-hydroxytryptamine; _1A; receptor; 7-DHC; 7-dehydrocholesterol; CCM; cholesterol consensus motif; CRAC; cholesterol recognition/interaction amino acid consensus; FRET; Fluorescence Resonance Energy Transfer; GPCR; G-protein coupled receptor; MbCD; methyl-b-cyclodextrin; SLOS; Smith–Lemli–Opitz Syndrome; SSD; sterol-sensing domainG-protein coupled receptor; Membrane cholesterol; Nonannular binding site; Serotonin; _1A; receptor; Specific cholesterol binding site

V H⁺-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes by Solange S. Samarão; Carlos E.S. Teodoro; Flavia E. Silva; Camila C. Ribeiro; Thais M. Granato; Natalia R. Bernardes; Cláudio A. Retamal; Arnoldo R. Façanha; Anna L. Okorokova-Façanha; Lev A. Okorokov ⁎ (pp. 303-313).

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin ( I₅₀=38.4 nM) than Golgi and vacuole pumps ( I₅₀=0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.

Keywords: V H; ⁺; -ATPase; H; ⁺; transport; Yeast; Endoplasmic reticulum; Golgi

Expression, purification and structural studies of a short antimicrobial peptide by Mateja Zorko; Boštjan Japelj; Iva Hafner-Bratkovič; Roman Jerala (pp. 314-323).

We have produced a small antimicrobial peptide PFWRIRIRR in bacteria utilizing production in the form of insoluble fusion protein with ketosteroid isomerase. The recombinant peptide was rapidly and efficiently isolated by acidic cleavage of the fusion protein based on the acid labile Asp–Pro bond at the N-terminus of the peptide. The peptide has antibacterial activity and neutralizes macrophage activation by LPS. The selectivity of the peptide against bacteria correlates with preferential binding to acidic phospholipid vesicles. Solution structure of the peptide in SDS and DPC micelles was determined by NMR. The peptide adopts a well-defined structure, comprising a short helical segment. Cationic and hydrophobic clusters are segregated along the molecular axis of the short helix, which is positioned perpendicular to the membrane plane. The position of the helix is shifted in two micellar types and more nonpolar surface is exposed in anionic micelles. Overall structure explains the advantageous role of the N-terminal proline residue, which forms an integral part of the hydrophobic cluster.

Keywords: Antimicrobial peptide; Solution structure; Recombinant peptide; NMR spectroscopy; Fluorescence; Lipid membrane

The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: Correlation with studies of DPH and Laurdan fluorescence by J. Sýkora; L. Bouřová; M. Hof; P. Svoboda (pp. 324-332).

The effect of non-ionic detergents on baclofen (GABA_B-R agonist)-stimulated G-protein activity was measured as a [³⁵S]GTPγS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic — a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABA_B-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58>Triton X-100>Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene ( r_DPH) incorporated into the hydrophobic PM interior. Decrease of r_DPH, in the order of Brij58>Triton X-100>Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time ϕ paralleled by an increase of diffusion wobbling constant D_w (analysis by time-resolved fluorescence according to “ wobble-in-cone” model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58>Triton X-100>Digitonin.

Keywords: Abbreviations; GABA; γ-aminobutyric acid, GABA; _B; -R, metabotropic receptor for GABA, GPCR, G-protein-coupled receptor; G-proteins; heterotrimeric guanine nucleotide-binding regulatory proteins; Gsα; G protein stimulating adenylyl cyclase activity; Gi/Goα; G-proteins inhibiting adenylyl cyclase activity in pertussis-toxin sensitive manner; Gq/G11α; G proteins stimulating phoshoplipase C in pertussis-toxin independent manner; [35S]GTPγS; guanosine-5′-[γ-35S] triphosphate; PBS; phosphate-buffered saline; PM; plasma membrane, PMSF, phenylmethylsulfonyl fluoride; PT; pertussis toxin; TBS; Tris-buffered saline; w.w.; wet weightDetergent; Fluorescence; Steady-state; Time-resolved; Diphenylhexatriene; DPH; Laurdan; Generalized polarization; Baclofen-stimulated G-protein activity; GABA; _B; -receptor; Rat brain; Plasma membrane

Interaction of an artificial antimicrobial peptide with lipid membranes by Lanlan Yu; Lin Guo; Jeak Ling Ding; Bow Ho; Si-shen Feng; Jonathan Popplewell; Marcus Swann; Thorsten Wohland ⁎ (pp. 333-344).

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab ( Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir–Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.

Keywords: Abbreviations; V4; cyclized CVKVQVKVGSGVKVQVKVC by an internal disulfide bond; TV4; VKVQVKVGSG; LPS; lipopolysaccharides; LB; Langmuir film balance; DPI; dual polarization interferometry; FCS; fluorescence correlation spectroscopy; REV; Rho 6G entrapping large unilamellar vesicle; RLV; Rho-PE labeled large unilamellar vesicle; GUV; giant unilamellar vesicle; POPC; 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine; POPG; 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)]; Rho-PE; 1,2–Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl) (Ammonium Salt); NBD-PE; 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (Ammonium Salt); Cap Biotinyl-DPPE; 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Cap Biotinyl) (Sodium Salt); MW; molecular weightAntimicrobial peptide; Fluorescence correlation spectroscopy; Dual polarization interference; Langmuir–Blodgett trough; Langmuir film balance; Lipid vesicle; Supported bilayer

Calorimetric and spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylglycerol bilayer membranes by Todd P.W. McMullen; Ruthven N.A.H. Lewis; Ronald N. McElhaney ⁎ (pp. 345-357).

We have examined the effects of cholesterol (Chol) on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylglycerols (PGs) by high-sensitivity differential scanning calorimetry and Fourier transform infrared and³¹P NMR spectroscopy. We find that the incorporation of increasing quantities of Chol alters the temperature and progressively reduces the enthalpy and cooperativity of the gel-to-liquid-crystalline phase transition of the host PG bilayer. With dimyristoyl-PG:Chol mixtures, cooperative chain-melting phase transitions are completely or almost completely abolished at Chol concentrations near 50 mol%, whereas with the dipalmitoyl- and distearoyl-PG:Chol mixtures, cooperative hydrocarbon chain-melting phase transitions are still discernable at Chol concentrations near 50 mol%. We are also unable to detect the presence of significant populations of separate domains of the anhydrous or monohydrate forms of Chol in our binary mixtures, in contrast to previous reports. We ascribe the previously reported large scale formation of Chol crystallites to the fractional crystallization of the Chol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. We further show that the direction and magnitude of the change in the phase transition temperature induced by Chol addition is dependent on the hydrocarbon chain length of the PG studied. This finding agrees with our previous results with phosphatidylcholine bilayers, where we found that Chol increases or decreases the phase transition temperature in a hydrophobic mismatch-dependent manner ( Biochemistry 1993, 32:516–522), but is in contrast to our previous results for phosphatidylethanolamine ( Biochim. Biophys. Acta 1999, 1416:119–234) and phosphatidylserine ( Biophys. J. 2000, 79:2056–2065) bilayers, where no such hydrophobic mismatch-dependent effects were observed. We also show that the addition of Chol facilitates the formation of the lamellar crystalline phase in PG bilayers, as it does in phosphatidylethanolamine and phosphatidylserine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of Chol. Moreover, the formation of the lamellar crystalline phase in PG bilayers at lower temperatures excludes Chol, resulting in an apparent Chol immiscibility in gel-state PG bilayers. We suggest that the magnitude of the effect of Chol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipids dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.

Keywords: Abbreviations; DMPC; dimyristoylphosphatidylcholine; DPPC; dipalmitoylphosphatidylcholine; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PS; phosphatidylserine; PG; phosphatidylglycerol; DMPG; dimyristoylphosphatidylglycerol; DPPG; dipalmitoylphosphatidylglycerol; DSPG; distearoylphosphatidylglycerol; DSC; differential scanning calorimetry; FTIR; Fourier transform infrared; ³¹; P; phosphorus-31; NMR; nuclear magnetic resonance; TLC; thin-layer chromatography; PPM; parts per million; L; _c; lamellar crystalline phase; L; _β; ′ and L; _β; lamellar gel phase with tilted or untilted hydrocarbon chains; P; _β; ′; rippled gel phase; L; _α; lamellar liquid-crystalline phaseCholesterol; Phosphatidylglycerol; Phospholipid bilayer; Model membrane; Hydrophobic mismatch; Differential scanning calorimetry; Fourier transform infrared spectroscopy; ³¹; P-nuclear magnetic resonance spectroscopy

Environmental tobacco smoke effects on lung surfactant film organization by Patrick C. Stenger; Coralie Alonso; Joseph A. Zasadzinski ⁎; Alan J. Waring; Chun-Ling Jung; Kent E. Pinkerton (pp. 358-370).

Adsorption of the clinical lung surfactants (LS) Curosurf or Survanta from aqueous suspension to the air–water interface progresses from multi-bilayer aggregates through multilayer films to a coexistence between multilayer and monolayer domains. Exposure to environmental tobacco smoke (ETS) alters this progression as shown by Langmuir isotherms, fluorescence microscopy and atomic force microscopy (AFM). After 12 h of LS exposure to ETS, AFM images of Langmuir–Blodgett deposited films show that ETS reduces the amount of material near the interface and alters how surfactant is removed from the interface during compression. For Curosurf, ETS prevents refining of the film composition during cycling; this leads to higher minimum surface tensions. ETS also changes the morphology of the Curosurf film by reducing the size of condensed phase domains from 8–12 μm to ∼2 μm, suggesting a decrease in the line tension between the domains. The minimum surface tension and morphology of the Survanta film are less impacted by ETS exposure, although the amount of material associated with the film is reduced in a similar way to Curosurf. Fluorescence and mass spectra of Survanta dispersions containing native bovine SP-B treated with ETS indicate the oxidative degradation of protein aromatic amino acid residue side chains. Native bovine SP-C isolated from ETS exposed Survanta had changes in molecular mass consistent with deacylation of the lipoprotein. Fourier Transform Infrared Spectroscopy (FTIR) characterization of the hydrophobic proteins from ETS treated Survanta dispersions show significant changes in the conformation of SP-B and SP-C that correlate with the altered surface activity and morphology of the lipid–protein film.

Keywords: Pulmonary surfactant; Inhibition; Inactivation; Second-hand smoke; Adsorption; Phospholipids

Spin labeling EPR studies of the properties of oxidized phospholipid-containing lipid vesicles by Francesco M. Megli; Luciana Russo; Elena Conte (pp. 371-379).

This study aims at characterizing the structure and some properties of phospholipid multi-lamellar vesicles (MLVs) containing the oxidized species γ-palmitoyl-β-(9-hydroperoxy-10,12-octadecanedienoyl)-lecithin (HPPLPC), γ-palmitoyl-β-(9-hydroxy-10,12-octadecanedienoyl)-lecithin (HOPLPC), γ-palmitoyl-β-glutaroyl-lecithin (GlPPC) and γ-palmitoyl-β-azelaoyl-lecithin (AzPPC). Sepharose 4B gel-chromatography was used to ensure and check that only MLVs are used in EPR measurements. Gel-solid to gel-liquid transition temperature (T_m), lateral phase separation, fluidity gradient and polarity profile were studied by use of EPR spectroscopy of enclosed n-doxylstearoyl lecithin spin labels. Contrarily to conjugate dienes and normal phospholipids, pure carboxyacyl species yielded aqueous suspensions showing gel-chromatography elution profile resembling that of lysolecithin micelles. Conjugate dienes/DPPC MLVs showed lateral phase separation at room temperature and T_m value lower than pure DPPC MLVs. Pure conjugate dienes MLVs resembled more PLPC MLVs and displayed free miscibility with PLPC in mixed MLVs. Pure HPPLPC MLV bilayer appeared to be slightly more rigid, while that of HOPLPC and the polarity profile of MLVs made of the pure conjugate dienes species were similar to those of normal PLPC. It is concluded that carboxyacyl lecithins in MLVs tend to disrupt vesicle structure, while conjugated dienes lecithins are more able to affect some physical properties of the bilayer, and that DPPC in MLVs enhances these effects while PLPC shows a better compatibility with the lipoperoxides.

Keywords: Abbreviations; DPPC; di-palmitoyl-lecithin; PLPC; γ-palmitoyl-β-linoleoyl-lecithin; EYPC; egg yolk lecithin; n-DSPPC; γ-palmitoyl-β-(n-doxylstearoyl)-lecithin HPPLPC, γ-palmitoyl-β-(9-hydroperoxy-10,12-octadecanedienoyl)-lecithin; HOPLPC; γ-palmitoyl-β-(9-hydroxy-10,12-octadecanedienoyl)-lecithin; CD-PC; conjugate dienes lecithins, the ensemble grouping HPPLPC and HOPLPC, full-chain phospholipoxides; GlPC; β-glutaroyl-lecithin; AzPC; β-azelaoyl-lecithin; CX-PC; β-carboxyacyl-lecithins, the ensemble grouping GlPC, AzPC and others, cleft-chain phospholipoxides; n-DSPPC; γ-palmitoyl-β-(n-doxylstearoyl)-lecithin (; n; =; 5, or 12, or 16); PUFA; poly unsaturated fatty acids; MLV; multilamellar vesicles; SUV; single unilamellar vesicles; ROS; reactive oxygen species; phospholipoxides; neologism standing for (per)oxidized phospholipidsLipoperoxidation; Spin labeling EPR; Membrane; Fluidity; Polarity; Transition temperature; Phase separation

Differential expression of BK channel isoforms and β-subunits in rat neuro-vascular tissues by Asser Nyander Poulsen; Helle Wulf; Anders Hay-Schmidt; Inger Jansen-Olesen; Jes Olesen; Dan A. Klaerke (pp. 380-389).

We investigated the expression of splice variants and β-subunits of the BK channel (big conductance Ca²⁺-activated K⁺ channel, Slo1, MaxiK, K_Ca1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An α-subunit splice variant X1+₂₄ was found expressed (RT-PCR) in nervous tissue only where also the SS4+₈₁ variant was dominating with little expression of the short form SS4₀. SS4+₈₁ was present in some cerebral vessels too. The SS2+₁₇₄ variant (STREX) was found in both blood vessels and in nervous tissue. In situ hybridization data supported the finding of SS4+₈₁ and SS2+₁₇₄ in vascular smooth muscle and trigeminal ganglion. β-subunits β2 and β4 showed high expression in brain and trigeminal ganglion and some in cerebral vessels while β1 showed highest expression in blood vessels. β3 was found only in testis and possibly brain. A novel splice variant X2+₉₂ was found, which generates a stop codon in the intracellular C-terminal part of the protein. This variant appears non-functional as a homomer but may modulate the function of other splice-variants when expressed in Xenopus oocytes. In conclusion a great number of splice variant and β-subunit combinations likely exist, being differentially expressed among nervous and vascular tissues.

Keywords: Isoform; Middle cerebral artery; Basilar artery; Migraine; Slo; BK; _Ca

Effects of temperature and pressure on the lateral organization of model membranes with functionally reconstituted multidrug transporter LmrA by Nagarajan Periasamy; Holger Teichert; Katrin Weise; Rudi F. Vogel; Roland Winter (pp. 390-401).

To contribute to the understanding of membrane protein function upon application of pressure, we investigated the influence of hydrostatic pressure on the conformational order and phase behavior of the multidrug transporter LmrA in biomembrane systems. To this end, the membrane protein was reconstituted into various lipid bilayer systems of different chain length, conformation, phase state and heterogeneity, including raft model mixtures as well as some natural lipid extracts. In the first step, we determined the temperature stability of the protein itself and verified its reconstitution into the lipid bilayer systems using CD spectroscopic and AFM measurements, respectively. Then, to yield information on the temperature and pressure dependent conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined, which report on the conformation and phase state of the lipid bilayer system. The temperature-dependent measurements were carried out in the temperature range 5–70 °C, and the pressure dependent measurements were performed in the range 1–200 MPa. The data show that the effect of the LmrA reconstitution on the conformation and phase state of the lipid matrix depends on the fluidity and hydrophobic matching conditions of the lipid system. The effect is most pronounced for fluid DMPC and DMPC with low cholesterol levels, but minor for longer-chain fluid phospholipids such as DOPC and model raft mixtures such as DOPC/DPPC/cholesterol. The latter have the additional advantage of using lipid sorting to avoid substantial hydrophobic mismatch. Notably, the most drastic effect was observed for the neutral/glycolipid natural lipid mixture. In this case, the impact of LmrA incorporation on the increase of the conformational order of the lipid membrane was most pronounced. As a consequence, the membrane reaches a mechanical stability which makes it very insensitive to application of pressures as high as 200 MPa. The results are correlated with the functional properties of LmrA in these various lipid environments and upon application of high hydrostatic pressure and are discussed in the context of other work on pressure effects on membrane protein systems.

Keywords: Abbreviations; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphatidylcholine; Chol; cholesterol; SM; sphingomyelin; PC; phosphatidylcholine; Laurdan; 6-dodecanoyl-2-dimethylamino-napthalene; GP; generalized polarization; LAB; lactic acid bacteria; MDR; multidrug resistance transporters; DDM; dodecyl maltosideLmrA; High hydrostatic pressure; Fluorescence spectroscopy; Phospholipid; Lipid phase transition; Raft mixture

The high turnover Drosophila multidrug resistance-associated protein shares the biochemical features of its human orthologues by Flóra Szeri ⁎; Attila Iliás; Viola Pomozi; Steven Robinow; Éva Bakos; András Váradi (pp. 402-409).

DMRP, an ABC transporter encoded by the dMRP/CG6214 gene, is the Drosophila melanogaster orthologue of the “long” human multidrug resistance-associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6, and MRP7/ABCC10). In order to provide a detailed biochemical characterisation we expressed DMRP in Sf9 insect cell membranes. We demonstrated DMRP as a functional orthologue of its human counterparts capable of transporting several human MRP substrates like β-estradiol 17-β-d-glucuronide, leukotriene C₄, calcein, fluo3 and carboxydichlorofluorescein. Unexpectedly, we found DMRP to exhibit an extremely high turnover rate for the substrate transport as compared to its human orthologues. Furthermore, DMRP showed remarkably high basal ATPase activity (68–75 nmol P_i/mg membrane protein/min), which could be further stimulated by probenecid and the glutathione conjugate of N-ethylmaleimide. Surprisingly, this high level basal ATPase activity was inhibited by the transported substrates. We discussed this phenomenon in the light of a potential endogenous substrate (or activator) present in the Sf9 membrane.

Keywords: Abbreviations; ABC; ATP-binding cassette; BB; benzbromarone; cAMP; 3′–5′-cyclic adenosine mono-phosphate; CDCF; carboxydichlorofluorescein; CFTR; cystic fibrosis transmembrane conductance regulator; DMRP; Drosophila; multidrug resistance-associated protein; E; ₂; 17βDG; β-estradiol 17-β-; d; -glucuronide; GSH; reduced glutathione; IM; indomethacin; LTC; ₄; leukotriene C; ₄; MRP; multidrug resistance-associated protein; NBD; nucleotide binding domain; NEM-GS; N-ethylmaleimide glutathione conjugate; PB; probenecid; SUR; sulfonylurea receptor; Sf9; Spodoptera frugiperda; ovarian cells; TMD; transmembrane domain; V; _i; orthovanadateABC-transporter; Transport activity; Substrate stimulated-substrate inhibited ATPase activity; Substrate-specificity

Binding–gating coupling in a nondesensitizing α7 nicotinic receptor by José Antonio Bernal; José Mulet; Mar Castillo; Manuel Criado; Salvador Sala; Francisco Sala ⁎ (pp. 410-416).

The highly conserved αLys145 has been suggested to play an important role in the early steps of activation of the nicotinic acetylcholine receptor (nAChR) by acetylcholine. Both macroscopic and single channel currents were recorded in the slowly desensitizing mutants L248T- and K145A- L248T-α7 receptors expressed in Xenopus oocytes. On ACh-evoked currents, substitution of Lys145 by alanine showed the same effects that in wild type receptors: moderately decreased gating function and a more-than-expected loss of ACh potency, thus validating the experimental model. Single channel analysis quantitatively agreed with macroscopic data and revealed that impaired gating function in the double mutant α7K145A/L248T is the consequence of a slower opening rate, β. Several nicotinic agonists were also studied, showing important features. Particularly, dimethylphenylpiperazinium (DMPP), acting as an antagonist in α7K145A, became a full agonist in α7K145A/L248T. Single channel analysis of DMPP-evoked currents showed effects of Lys145 removal similar to those observed with ACh. Data suggest that α7Lys145 facilitates the early steps of channel activation. Moreover, the slowly desensitizing mutant α7L248T could be an interesting tool for the study of channel activation in α7 receptors. Nevertheless, its extensively altered pharmacology precludes the simple extrapolation of pharmacological data obtained in singly mutated α7 receptors.

Keywords: Acetylcholine receptor; Cys-loop receptor; Channel gating; Mutant; Single channel

Interaction of preS domains of hepatitis B virus with phospholipid vesicles by Elena Núñez; Belén Yélamos; Carmen Delgado; Julián Gómez-Gutiérrez; Darrell L. Peterson; Francisco Gavilanes ⁎ (pp. 417-424).

The role of preS domains of the hepatitis B virus (HBV) envelope proteins in the first steps of viral infection has been restricted to their implication in virus attachment to a putative hepatocyte receptor. In order to explore a fusion activity in these regions, we used recombinant preS domains to characterize their interaction with liposomes. Binding experiments carried out with NBD-labeled proteins indicated that preS were able to interact in a monomeric way with acidic phospholipid vesicles, being the partition coefficient similar to that described for peptides which can insert deeply into bilayers. Fluorescence depolarization of DPH-labeled vesicles confirmed the specificity for negative charged phospholipids. Upon interaction the proteins induced aggregation, lipid mixing and release of internal contents of acidic vesicles at both acid and neutral pH in a concentration-dependent manner. Taken together, all these data indicate that preS domains are able to insert into the hydrophobic core of the bilayer. Moreover, the insertion resulted in a protein conformational change which increased the helical content. Therefore all these results suggest that, besides their participation in the recognition of a cellular receptor, the preS domains could be involved in the fusion mechanism of HBV with the plasma membrane of target cells.

Keywords: HBV; preS domain; Fusion; Phospholipid membrane

Fluorescent sterols monitor cell penetrating peptide Pep-1 mediated uptake and intracellular targeting of cargo protein in living cells by Anca D. Petrescu; Aude Vespa; Huan Huang; Avery L. McIntosh; Friedhelm Schroeder; Ann B. Kier (pp. 425-441).

Although cell-penetrating peptides (CPP) facilitate endocytic uptake of proteins, little is known regarding the extent to which CPPs facilitate protein cargo exit from endocytic vesicles for targeting to other intracellular sites. Since the plasma membrane and less so intracellular membranes contain cholesterol, the fluorescent sterol analogues dansyl-cholestanol (DChol) and dehydroergosterol (DHE) were used to monitor the uptake and intracellular distribution of fluorescent-tagged acyl coenzyme A binding protein (ACBP) into COS-7 cells and rat hepatoma cells. Confocal microscopy colocalized DChol and Texas Red-ACBP (TR-ACBP) with markers for the major endocytosis pathways, especially fluorescent-labeled cholera toxin (marker of ganglioside GM1 in plasma membrane lipid rafts) and dextran (macropinocytosis marker), but less so with transferrin (clathrin-mediated endocytosis marker). These findings were confirmed by multiphoton laser scanning microscopy colocalization of TR-ACBP with DHE (naturally-fluorescent sterol) and by double immunofluorescence labeling of native endogenous ACBP. Serum greatly and Pep-1 further 2.4-fold facilitated uptake of TR-ACBP, but neither altered the relative proportion of TR-ACBP colocalized with membranes/organelles (nearly 80%) vs cytoplasm and/or nucleoplasm (20%). Interestingly, Pep-1 selectively increased TR-ACBP associated with mitochondria while concomitantly decreasing that in endoplasmic reticulum. In summary, fluorescent sterols (DChol, DHE) were useful markers for comparing the distributions of both transported and endogenous proteins. Pep-1 modestly enhanced the translocation and altered the intracellular targeting of exogenous-delivered (TR-ACBP) in living cells.

Keywords: Abbreviations; ACBP; acyl coenzyme A binding protein; DChol; dansyl-cholestanol; DHE; dehydroergosterol; LUV; large unilamellar vesicles; CPP; cell penetrating peptide; CD; circular dichroism; LSCM; laser scanning confocal microscopy; MPLSM; multiphoton laser scanning microscopyAcyl-CoA binding protein; Confocal microscopy; Dansyl-cholestanol; Dehydroergosterol; Endocytosis; Macropinocytosis; Multiphoton excitation

Apolipoprotein E peptide-modified colloidal carriers: The design determines the mechanism of uptake in vascular endothelial cells by Eik Leupold ⁎; Heike Nikolenko; Margitta Dathe (pp. 442-449).

Supramolecular structures, particularly micelles and liposomes equipped with uptake-mediating address compounds, have attracted much attention as pharmaceutical formulations. Their development requires an understanding of the mechanism by which the carrier systems interact with and translocate into the target cells. We developed an apolipoprotein E-derived peptide, called A2, that efficiently translocates across cell membranes. Upon coupling of two palmitoyl chains (P2), the highly cationic sequence acquires detergent-like properties such as a strong tendency to self-associate and the ability to integrate into lipid bilayers. Confocal laser scanning microscopy and fluorescence activated cell sorting were used to compare the internalization of the fluorescence-labeled monomeric A2 with the uptake of the colloidal P2A2 micelles and P2A2-tagged liposomes into endothelial cells of blood vessels. Specific inhibitors of endocytosis were used to identify the underlying mechanisms. b.End3 and BAEC cells as example of endothelial cells of small capillaries and large vascular vessels, respectively, were examined. The uptake of monomeric A2 was characterized by poor cellular selectivity. A2 was efficiently internalized into both cell lines via at least two different mechanisms. Besides an endocytotic uptake route, a second passive pathway exists, that leads to a rapid distribution of A2 within the cytoplasm. Also liposomes tagged with P2A2 were non-selectively internalized into both b.End3 and BAEC cells. Their nonselective uptake was mediated by clathrin- and caveolin-independent endocytosis. In contrast, micellar P2A2 entered b.End3 cells via clathrin-mediated endocytosis, while no uptake of P2A2 into BAEC cells was observed. In conclusion, the specific clathrin-mediated uptake mode of P2A2 micelles might provide the basis for a blood brain barrier-specific targeting.

Keywords: Peptide micelles; Liposome; Endocytosis; Inhibitor; Cell-penetrating peptide

Cytoskeleton reorganization in influenza hemagglutinin-initiated syncytium formation by Jean-Philippe Richard; Eugenia Leikina; Leonid V. Chernomordik (pp. 450-457).

Little is known about the mechanisms of cell–cell fusion in development and diseases and, especially, about fusion stages downstream of an opening of nascent fusion pore(s). Earlier works on different cell–cell fusion reactions have indicated that cytoskeleton plays important role in syncytium formation. However, due to complexity of these reactions and multifaceted contributions of cytoskeleton in cell physiology, it has remained unclear whether cytoskeleton directly drives fusion pore expansion or affects preceding fusion stages. Here we explore cellular reorganization associated with fusion pore expansion in syncytium formation using relatively simple experimental system. Fusion between murine embryonic fibroblasts NIH3T3-based cells is initiated on demand by well-characterized fusogen influenza virus hemagglutinin. We uncouple early fusion stages dependent on protein fusogens from subsequent fusion pore expansion stage and establish that the transition from local fusion to syncytium requires metabolic activity of living cells. Effective syncytium formation for cells with disorganized actin and microtubule cytoskeleton argues against hypothesis that cytoskeleton drives fusion expansion.

Keywords: Cytoskeleton; Membrane fusion; Syncytium; Influenza hemagglutinin

Contribution of electrostatic and structural properties of Kv4.3 S4 arginine residues to the regulation of channel gating by Matthew R. Skerritt; Donald L. Campbell (pp. 458-469).

Previous work has demonstrated that replacing individual arginine (R) residues in the S4 domain of Kv4.3 with alanine (A) not only altered activation and deactivation processes, but also those of closed-state inactivation (CSI) and recovery. R → A mutants eliminated individual positive charge while substantially reducing side chain volume and hydrophilic character. Their novel effects on gating may thus have been the result of electrostatic and/or structural perturbations. To address this issue, and to gain further insights into the roles that S4 plays in the regulation of Kv4.3 gating transitions, we comparatively analyzed arginine to glutamine (R → Q) mutations at positions 290, 293, and 296. This maneuver maintained positive charge elimination of the R → A mutants, while partially restoring native side chain volume and hydrophilic properties. R → A and R → Q mutant pairs produced similar effects on the forward gating process of activation. In contrast, significant differences between the two substitutions were discovered on deactivation, CSI, and recovery, with the R → Q mutants partially restoring wild type characteristics. Our results argue that modification of individual S4 residue properties may result in altered localized interactions within unique microenvironments encountered during forward and reverse gating transitions. As such, predominant effects appear on the reverse gating transitions of deactivation and recovery. These results are consistent with the proposal that arginine residues in S4 are involved in regulating Kv4.3 CSI and recovery.

Keywords: Kv4.3; S4 domain; Gating; Closed-state inactivation; Recovery

Loss of membrane cholesterol influences lysosomal permeability to potassium ions and protons by Dong Deng; Nan Jiang; Shu-Jing Hao; Hui Sun; Guo-jiang Zhang ⁎ (pp. 470-476).

Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K⁺ and H⁺, and the organelle stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-β-cyclodextrin (MβCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K⁺/H⁺ exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MβCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K⁺ and H⁺. Loss of this membrane sterol is critical for the organelle acidification and stability.

Keywords: Abbreviations; CCCP; carbonyl cyanide m-chlorophenylhydrazone; CMA; chaperone-mediated autophapy; DPH; 1,6-diphenyl-1,3,5-hexatriene; FITC-Dextran; fluorescein isothiocyanate-dextran; LAMP-2A; lysosome associated membrane protein type 2A; MβCD; Methyl-β-cyclodextrin; oxonol VI; Bis(3-propyl-5-oxoisoxazol-4-yl) pentamethine-oxonol; SCP-2; Sterol carrier protein-2Lysosome; Cholesterol; Ion permeability; Membrane fluidity; Methyl-β-cyclodextrin (MβCD)

The structure of detergent-resistant membrane vesicles from rat brain cells by Xi Chen; M. Jayne Lawrence; David J. Barlow; Roger J. Morris; Richard K. Heenan; Peter J. Quinn ⁎ (pp. 477-483).

The size and the bilayer thickness of detergent-resistant membranes isolated from rat brain neuronal membranes using Triton X-100 or Brij 96 in buffers with or without the cations, K⁺/Mg²⁺ at a temperature of either 4 °C or 37 °C were determined by dynamic light scattering and small-angle neutron scattering. Regardless of the precise conditions used, isolated membrane preparations consisted of vesicles of ∼100 to 200 nm diameter as determined by dynamic light scattering methods, equating to an area of the lipid based membrane microdomain size of 200 to 400 nm diameter. By means of small angle neutron scattering it was established that the average thickness of the bilayers of the complete population of detergent-resistant membranes was similar to that of the parental membrane at between 4.6 and 5.0 nm. Detergent-resistant membranes prepared using buffers containing K⁺/Mg²⁺ uniquely formed unilamellar vesicles while membranes prepared in the absence of K⁺/Mg²⁺ formed a mixture of uni- and oligolamellar structures indicating that the arrangement of the membrane differs from that observed in the presence of cations. Furthermore, the detergent-resistant membranes prepared at 37 °C were slightly thicker than those prepared at 4 °C, consistent with the presence of a greater proportion of lipids with longer, more saturated fatty acid chains associated with the Lo (liquid-ordered) phase. It was concluded that the preparation of detergent-resistant membranes at 37 °C using buffer containing cations abundant in the cytoplasm might more accurately reflect the composition of lipid rafts present in the plasma membrane under physiological conditions.

Keywords: Lipid raft; Detergent resistant membrane; Membrane microdomain; Detergent solubilization; Small--angle neutron scattering; Dynamic light scattering

Probing molecular level interaction of oseltamivir with H5N1-NA and model membranes by molecular docking, multinuclear NMR and DSC methods by Charlotte D'Souza; Meena Kanyalkar; Mamata Joshi; Evans Coutinho; Sudha Srivastava ⁎ (pp. 484-494).

Structure-based drug design has led to the introduction of three drugs — oseltamivir (GS-4104), zanamivir (GG-167) and peramivir (RWJ-270201) which target the enzyme neuraminidase, for treatment of influenza infections. Using comparative docking studies we propose that more potent molecules against neuraminidase can be obtained by appending extra positively charged substituents at the C5 position of the oseltamivir skeleton. This provides an additional interaction with the enzyme and may overcome the problem of resistance encountered with these drugs. To get an insight into the transport and absorption of oseltamivir — the ethyl ester prodrug (GS-4104) as well as its mechanism of action, we have carried out¹H,¹³C,³¹P NMR, DSC and TEM studies on GS-4104 with model membranes prepared from DMPC/DPPC/POPC. These studies reveal that interactions between GS-4104 and the membrane are both electrostatic (involving H-bonding) and hydrophobic (involving the hydrophobic chain and cyclohexene ring of GS-4104) in nature. The prodrug is seen to increase the fluidity as well as stabilize the bilayer phase of the membrane. This property may be responsible for preventing viral entry into the cells by preventing fusion of the virus outer coat with the cell membrane.

Keywords: Neuraminidase; Oseltamivir; Model membrane; Docking; NMR; Differential scanning calorimeter

Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone by Andrey S. Klymchenko ⁎; Sule Oncul; Pascal Didier; Emmanuel Schaub; Luis Bagatolli; Guy Duportail; Yves Mély (pp. 495-499).

We characterized the recently introduced environment-sensitive fluorescent membrane probe based on 3-hydroxyflavone, F2N12S, in model lipid membranes displaying liquid disordered (Ld) phase, liquid ordered (Lo) phase, or their coexistence. Steady-state fluorescence studies in large unilamellar vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast, the absence of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen.

Keywords: Abbreviations; Chol; cholesterol; DMSO; dimethylsulfoxide; DOPC; dioleoylphosphatidylcholine; ESIPT; excited state intramolecular proton transfer; GUV; giant unilamellar vesicle; LUV; large unilamellar vesicle; Ld; liquid disordered phase; Lo; liquid ordered phase; SM; sphingomyelinLiquid disordered phase; Liquid ordered phase; Lipid rafts; Giant unilamellar vesicle; Fluorescent probe; Fluorescence imaging

K⁺ binding in the G-loop and water cavity facilitates Ba²⁺ movement in the Kir2.1 channel by Hsueh-Kai Chang; Laurence J. Marton; Kuo Kan Liang; Ru-Chi Shieh ⁎ (pp. 500-506).

K⁺ are selectively coordinated in the selectivity filter and concerted K⁺ and water movements in this region ensure high conduction rates in K⁺ channels. In channels with long pores many K⁺ binding sites are located intracellular to the selectivity filter (inner vestibule), but their contribution to permeation has not been well studied. We investigated this phenomenon by slowing the ion permeation process via blocking inwardly rectifying Kir2.1 channels with Ba²⁺ in the selectivity filter and observing the effect of K⁺ in the inner vestibule on Ba²⁺ exit. The dose–response effect of the intracellular K⁺ concentration ([K⁺]_i) on Ba²⁺ exit was recorded with and without intracellular polyamines, which compete with K⁺ for binding sites. Ba²⁺ exit was facilitated by the cooperative binding of at least three K⁺. Site-directed mutagenesis studies suggest that K⁺ interacting with Ba²⁺ bound in the selectivity filter were located in the region between selectivity filter and cytoplasmic pore, i.e. the water cavity and G-loop. One of the K⁺ binding sites was located at residue D172 and another was possibly at M301. This study provides functional evidence for the three K⁺ binding sites in the inner vestibule previously identified by crystal structure study.

Keywords: Permeation; Selectivity filter; Channel block; Polyamines

The role of membrane cholesterol in determining bile acid cytotoxicity and cytoprotection of ursodeoxycholic acid by Yong Zhou; Rand Doyen; Lenard M. Lichtenberger (pp. 507-513).

In cholestatic liver diseases, the ability of hydrophobic bile acids to damage membranes of hepatocytes/ductal cells contributes to their cytotoxicity. However, ursodeoxycholic acid (UDC), a hydrophilic bile acid, is used to treat cholestasis because it protects membranes. It has been well established that bile acids associate with and solubilize free cholesterol (CHOL) contained within the lumen of the gallbladder because of their structural similarities. However, there is a lack of understanding of how membrane CHOL, which is a well-established membrane stabilizing agent, is involved in cytotoxicity of hydrophobic bile acids and the cytoprotective effect of UDC. We utilized phospholipid liposomes to examine the ability of membrane CHOL to influence toxicity of individual bile acids, such as UDC and the highly toxic sodium deoxycholate (SDC), as well as the cytoprotective mechanism of UDC against SDC-induced cytotoxicity by measuring membrane permeation and intramembrane dipole potential. The kinetics of bile acid solubilization of phosphatidylcholine liposomes containing various levels of CHOL was also characterized. It was found that the presence of CHOL in membranes significantly reduced the ability of bile acids to damage synthetic membranes. UDC effectively prevented damaging effects of SDC on synthetic membranes only in the presence of membrane CHOL, while UDC enhances the damaging effects of SDC in the absence of CHOL. This further demonstrates that the cytoprotective effects of UDC depend upon the level of CHOL in the lipid membrane. Thus, changes in cell membrane composition, such as CHOL content, potentially influence the efficacy of UDC as the primary drug used to treat cholestasis.

Keywords: Ursodeoxycholic acid cytoprotection; Deoxycholic acid cytotoxicity; Cholesterol; Membrane permeability; Membrane dipole potential; Turbidity

Effects of arginine density on the membrane-bound structure of a cationic antimicrobial peptide from solid-state NMR by Ming Tang; Alan J. Waring; Mei Hong ⁎ (pp. 514-521).

Solid-state NMR spectroscopy is used to determine the membrane-bound topological structure of a cationic β-hairpin antimicrobial peptide in which the number of Arg residues has been halved. The parent peptide, PG-1, was previously found to form transmembrane β-barrels in anionic membranes where the Arg residues complex with the lipid phosphate groups to cause toroidal pore defects in the membrane. In comparison, the charge-attenuated and less active mutant studied here forms β-sheets that lie on the surface of the zwitterionic membrane and only partially insert into the anionic membrane. The mutant also exhibits much looser contact with the lipid headgroups. These results indicate that transmembrane insertion and tight Arg-phosphate association are two important elements for strong antimicrobial activities of this class of peptides. Comparison with other β-hairpin antimicrobial peptides studied so far further suggests a relative potency scale for the various mechanisms of action for the β-sheet family of antimicrobial peptides. The transmembrane insertion-toroidal pore mechanism is the most potent in disrupting the lipid bilayer, followed by the large-amplitude in-plane motional mechanism. The carpet model, where peptides aggregate on the membrane surface to cause lateral expansion and eventual micellization of the membrane, is a weaker mechanism of action.

Keywords: Antimicrobial peptide; Depth of insertion; Oligomeric structure; Arginine; Solid-state NMR; β-hairpin

Sensory rhodopsin II/transducer complex formation in detergent and in lipid bilayers studied with FRET by J. Kriegsmann; M. Brehs; J.P. Klare; M. Engelhard; J. Fitter ⁎ (pp. 522-531).

The photophobic receptor from Natronomonas pharaonis (NpSRII) forms a photo-signalling complex with its cognate transducer (NpHtrII). In order to elucidate the complex formation in more detail, we have studied the intermolecular binding of both constituents (NpSRII and NpHtrII₁₅₇; truncated at residue 157) in detergent buffers, and in lipid bilayers using FRET. The data for hetero-dimer formation of NpSRII/NpHtrII in detergent agrees well with K_D values (∼200 nM) described in the literature. In lipid bilayers, the binding affinity between proteins in the NpSRII/NpHtrII complex is at least one order of magnitude stronger. In detergent the strength of binding is similar for both homo-dimers (NpSRII/NpSRII and NpHtrII/NpHtrII) but significantly weaker ( K_D∼16 μM) when compared to the hetero-dimer. The intermolecular binding is again considerably stronger in lipid bilayers; however, it is not as strong as that observed for the hetero-dimer. At a molar transducer/lipid ratio of 1:2000, which is still well above physiological concentrations, only 40% homo-dimers are formed. Apparently, in cell membranes the formation of the assumed functionally active oligomeric 2:2 complex depends on the full-length transducer including the helical cytoplasmic part, which is thought to tighten the transducer–dimer association.

Keywords: Abbreviations; FRET; Förster resonance energy transfer; NpSRII; Natronomonas pharaonis; sensory rhodopsin II; NpHtrII; ₁₅₇; Natronomonas pharaonis; transducer of sensory rhodopsin II truncated at residue 157, DDM, β-dodecyl-D-maltoside; POPC; palmitoyl oleoyl phosphatidyl choline; LUV; large lamellar vesicle; QY; fluorescence quantum yield; ITC; isothermal titration calorimetry; EPR; electron paramagnetic resonanceFluorescence spectroscopy; Lipid vesicle; Photo signalling; Sensory rhodopsin; Membrane protein interaction; Dissociation constant

Potential-dependent permeabilization of plasma membrane by the peptide BTM-P1 derived from the Cry11Bb1 protoxin by Mauricio Arias; Sergio Orduz; Victor V. Lemeshko (pp. 532-537).

The peptide BTM-P1, which is derived from the amino acid sequence of the Cry11Bb1 protoxin, is able to permeabilize mitochondrial membranes and reveals antimicrobial activity. In this work we demonstrated that the permeabilizing activity of BTM-P1 for the plasma membrane of rat red blood cells increased in a dose-dependent manner for the concentration range of 1–4 μg/ml. Using osmotic protectants, the radius of pores formed at 4 μg/ml BTM-P1 was determined as 0.8 nm for 5 min hemolysis data, 0.7 nm for 5 min decrease in light dispersion of the cell suspension and 0.5 nm for the light dispersion slope measurements. The permeabilizing activity of 1 μg/ml peptide was increased by valinomycin-induced plasma membrane potential, especially under moderately hypotonic conditions. These results might explain the antimicrobial activity of BTM-P1 and support the hypothesis of potential-dependent and pro-apoptotic character of toxicity of naturally proteolysed Cry11Bb1 protoxin for epithelial cells of mosquito larvae midgut.

Keywords: Plasma membrane; Pore size; Cry11Bb protoxin; Polycation peptide; Hemolysis; Light dispersion

A peptide derived from the putative transmembrane domain in the tail region of E. coli toxin hemolysin E assembles in phospholipid membrane and exhibits lytic activity to human red blood cells: Plausible implications in the toxic activity of the protein by Sharada Prasad Yadav; Aqeel Ahmad; Brijesh Kumar Pandey; Dharamsheela Singh; Neeta Asthana; Richa Verma; Raj Kamal Tripathi; Jimut Kanti Ghosh ⁎ (pp. 538-550).

Hemolysin E (HlyE), a pore-forming protein-toxin and a potential virulence factor of Escherichia coli, exhibits cytotoxic activity to mammalian cells. However, very little is known about how the different individual segments contribute in the toxic activity of the protein. Toward this end, the role of a 33-residue segment comprising the amino acid region 88 to 120, which contains the putative transmembrane domain in the tail region of HlyE has been addressed in the toxic activity of the protein-toxin by characterizing the related wild type and mutant peptides and the whole protein. Along with the 33-residue wild type peptide, H-88, two mutants of the same size were synthesized; in one mutant a conserved valine at 89^th position was replaced by aspartic acid and in the other both glycine and valine at the 88^th and 89^th positions were substituted by aspartic acid residues. These mutations were also incorporated in the whole toxin HlyE. Results showed that only H-88 but not its mutants permeabilized both lipid vesicles and human red blood cells (hRBCs). Interestingly, while H-88 exhibited a moderate lytic activity to human red blood cells, the mutants were not active. Drastic reduction in the depolarization of hRBCs and hemolytic activity of the whole toxin HlyE was also observed as a result of the same double and single amino acid substitution in it. The results indicate an important role of the amino acid segment 88–120, containing the putative transmembrane domain of the tail region of the toxin in the toxic activity of hemolysin E.

Keywords: Abbreviations; HlyE; hemolysin E; TFE; Trifluoroethanol; NBD-fluoride; 4-fluoro-7-nitrobenz-2-oxa-1, 3-diazole; Rho; Tetramethylrhodamine; PC; Egg phosphatidylcholine; PG; Egg phosphatidylglycerol; Chol; Cholesterol; a.a.; amino acid; hRBC; human red blood cellPore-forming toxin; Escherichia coli; hemolysin E; Toxicity of hemolysin E; Putative transmembrane segment; Hemolysin E mutant; Lipid–peptide interaction; Protein-dissection

Stimulation of EAAC1 in C6 glioma cells by store-operated calcium influx by Andrew Murphy; Alice Vines; Gethin J. McBean ⁎ (pp. 551-558).

This study investigated how modulation of intracellular calcium alters the functional activity of the EAAC1 glutamate transporter in C6 glioma cells. Pre-incubation of C6 glioma cells with the endoplasmic reticulum Ca²⁺ ATP pump inhibitor, thapsigargin (10 μM) produced a time-dependent increase in the V_max ford-[³H]aspartate transport that reached a maximum at 15 min (143% of control; P<0.001) that was accompanied by increased plasma membrane expression of EAAC1 and was blocked by inhibition of protein kinase C. Pre-incubation of C6 glioma cells with phorbol myristate-3-acetate (100 nM for 20 min) also caused a significant increase in the V_max of sodium-dependentd-[³H]aspartate transport (190% of control; P<0.01). In contrast, in the absence of extracellular calcium, thapsigargin caused a significant inhibition ind-[³H]aspartate transport that was not mediated by protein kinase C. Blockade of store-operated calcium channels with 2-aminoethoxydiphenyl borate (50 μM) or SKF 96365 (10 μM) caused a net inhibition ofd-[³H]aspartate uptake. Co-incubation of C6 glioma cells with both thapsigargin and 2-aminoethoxydiphenyl borate (but not SKF 96365) prevented the increase ind-[³H]aspartate transport that was observed in the presence of thapsigargin alone. Furthermore, 2-aminoethoxydiphenyl borate, but not SKF 96365, reduced the increase in intracellular calcium that occurred following pre-incubation of the cells with thapsigargin. It is concluded that, in C6 glioma cells, stimulation of EAAC1-mediated glutamate transport by thapsigargin is dependent on entry of calcium via the NSCC-1 subtype of store operated calcium channel and is mediated by protein kinase C. In contrast, in the absence of store operated calcium entry, thapsigargin inhibits transport.

Keywords: Abbreviations; 2-APB; 2-aminoethoxy diphenylborate; CaMKII; calcium-calmodulin dependent kinase II; EAAC1; excitatory amino acid carrier 1; ER; endoplasmic reticulum; NSCC; non-selective cation channel; PI3K; phosphatidylinositol-3-kinase; PMA; phorbol myristate-3-acetate; SOCC; store-operated calcium channelGlutamate; EAAC1; Calcium; Thapsigargin; C6 glioma

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling? by Eleri Hughes; Jonathan C. Clayton; David A. Middleton ⁎ (pp. 559-566).

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB_1–23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB_1–23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.

Keywords: Abbreviations; SERCA; sarco(endo)plasmic reticulum Ca; ²⁺; -ATPase; PLB; phospholamban; NMR; nuclear magnetic resonance; ITC; isothermal titration calorimetry; EPR; electron paramagnetic resonance; SR; sarcoplasmic reticulum; AAA-PLB; a full-length, null-cysteine variant of PLB mutated as C36A C41A C46A; PLB; _1–23; the N-terminal cytoplasmic residues; _1–23; of phospholamban; DOPC; l; -α-dioleoylphosphatidylcholine; DOPE; l; -α-dioleoylphosphatidylethanolamine; DOPS; l; -α-dioleoylphosphatidylserine; DOPG; l; -α-dioleoylphosphatidylglycerol; DMPC; l; -α-dimyristoylphosphatidylcholine; MAS; magic angle spinning; PI; phosphatidylinositol; Δ; ν; _1/2; peak width at half heightPhospholamban; Sarco(endo)plasmic calcium ATPase; Nuclear magnetic resonance; Isothermal titration calorimetry

A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells by Yutetsu Kuruma; Pasquale Stano; Takuya Ueda; Pier Luigi Luisi ⁎ (pp. 567-574).

Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127–8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity – inside liposomes – of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within — which corresponds to the autopoietic growth mechanism.

Keywords: Cell-free translation system; Liposome; Membrane protein; Phospholipid biosynthesis; Self-reproduction; Minimal cell

Chemical modification of Bacillus thuringiensis Cry1Aa toxin single-cysteine mutants reveals the importance of domain I structural elements in the mechanism of pore formation by Frédéric Girard; Vincent Vachon; Geneviève Lebel; Gabrielle Préfontaine; Jean-Louis Schwartz; Luke Masson; Raynald Laprade (pp. 575-580).

Bacillus thuringiensis Cry toxins form pores in the apical membrane of insect larval midgut cells. To investigate their mechanism of membrane insertion, mutants in which cysteine replaced individual amino acids located within the pore-forming domain of Cry1Aa were chemically modified with sulfhydryl-specific reagents. The thiol group of cysteine was highly susceptible to oxidation and its reactivity was significantly increased when the toxins were purified under reducing conditions. Addition of a biotin group to the cysteine had little effect on the ability of the toxins to permeabilize Manduca sexta brush border membrane vesicles except for a slight reduction in activity for S252C and a large increase in activity for Y153C. The activity of Y153C was also significantly increased after modification by reagents that added an aromatic or a charged group to the cysteine. When permeability assays were performed in the presence of streptavidin, a large biotin-binding protein, the pore-forming activity of several mutants, including Y153C, where the altered residue is located within the hairpin comprising helices α4 and α5, or in adjacent loops, was significantly reduced. These results support the umbrella model of toxin insertion.

Keywords: Insecticidal toxin; Site-directed sulfhydryl modification; Membrane insertion; Pore formation; Bacillus thuringiensis; Manduca sexta

Domain complementation studies reveal residues critical for the activity of the mannitol permease from Escherichia coli by Erwin P.P. Vos; Ramon ter Horst; Bert Poolman; Jaap Broos ⁎ (pp. 581-586).

This paper presents domain complementation studies in the mannitol transporter, EII^mtl, from Escherichia coli. EII^mtl is responsible for the transport and concomitant phosphorylation of mannitol over the cytoplasmic membrane. By using tryptophan-less EII^mtl as a basis, each of the four phenylalanines located in the cytoplasmic loop between putative transmembrane helices II and III in the membrane-embedded C domain were replaced by tryptophan, yielding the mutants W97, W114, W126, and W133. Except for W97, these single-tryptophan mutants exhibited a high, wild-type-like, binding affinity for mannitol. Of the four mutants, only W114 showed a high mannitol phosphorylation activity. EII^mtl is functional as a dimer and the effect of these mutations on the oligomeric activity was investigated via heterodimer formation (C/C domain complementation studies). The low phosphorylation activities of W126 and W133 could be increased 7–28 fold by forming heterodimers with either the C domain of W97 (IIC^mtlW97) or the inactive EII^mtl mutant G196D. W126 and W133, on the other hand, did not complement each other. This study points towards a role of positions 97, 126 and 133 in the oligomeric activation of EII^mtl. The involvement of specific residue positions in the oligomeric functioning of a sugar-translocating EII protein has not been presented before.

Keywords: Abbreviations; dPEG; decylpoly-(ethyleneglycol)300; PEP; phosphoenolpyruvate; Phe; phenylalanine; Trp; tryptophan; K; _d; dissociation constant; PTS; phosphoenolpyruvate-dependent group-translocation system; EI; Enzyme I from the PTS system of; E. coli; HPr; histidine-containing protein of; E. coliPTS; Enzyme II; Heterodimer; Oligomeric activation

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BBA - Biomembranes (v.1788, #2)