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BBA - Biomembranes (v.1778, #2)

Editorial Board (pp. ii).

Studies on anticancer activities of antimicrobial peptides by David W. Hoskin; Ayyalusamy Ramamoorthy (pp. 357-375).

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.

Keywords: Antimicrobial peptide; Anticancer peptide; Membrane; Lipid bilayer; Drug

Structure-based interpretation of the mutagenesis database for the nucleotide binding domains of P-glycoprotein by J. Lawson; M.L. O'Mara; I.D Kerr (pp. 376-391).

P-glycoprotein (P-gp) is the most intensively studied eukaryotic ATP binding cassette (ABC) transporter, due to its involvement in the multidrug resistance phenotype of a number of cancers. In common with most ABC transporters, P-gp is comprised of two transmembrane domains (TMDs) and two nucleotide binding domains (NBD), the latter coupling ATP hydrolysis with substrate transport (efflux in the case of P-gp). Biochemical investigations over the past twenty years have attempted to unlock mechanistic aspects of P-glycoprotein through scanning and site-directed mutagenesis of both the TMDs and the NBDs. Contemporaneously, crystallographers have elucidated the atomic structure of numerous ABC transporter NBDs, as well as the intact structure (i.e. NBDs and TMDs) of a distantly related ABC-exporter Sav1866. Significantly, the structure of P-gp remains unknown, and only low resolution electron microscopy data exists. Within the current manuscript we employ crystallographic data for homologous proteins, and a molecular model for P-gp, to perform a structural interpretation of the existing “mutagenesis database” for P-gp NBDs. Consequently, this will enable testable predictions to be made that will result in further in-roads into our understanding of this clinically important drug pump.

Keywords: ABC transporter; P-glycoprotein; Mutagenesis; Nucleotide binding domain; Mechanism; Catalytic cycle; Modeling

A trimeric building block model for Cry toxins in vitro ion channel formation by Jaume Torres; Xin Lin; Panadda Boonserm (pp. 392-397).

The crystal (Cry) insecticidal toxins, or δ-endotoxins, are lethal to a wide variety of insect larvae, and are therefore very important in insect control. Toxicity has been explained by formation of transmembrane oligomeric pores or ion channels and, more recently, by the ability of the monomeric toxin to subvert cellular signaling pathways. The structure, topology, and precise role of the putative pore in toxicity are not known. However, in vitro biophysical studies suggest that helices α4 and α5 in domain I insert into the lipid bilayer as an α-helical hairpin. Mutagenesis studies have assigned an important role to α5 in maintaining oligomerization, and to α4 in channel formation. To detect the possible homo-oligomerizing tendencies of these two helices, we have used the evolutionary conservation data contained in sixteen Cry homologs in order to filter non-native interactions found during a global conformational search. No conserved homo-oligomer was found for α4, but a right handed trimeric α5 model was present in the simulations of all Cry sequences. We propose a model for Cry toxin oligomerization based on sequence analysis and available mutagenesis data.

Keywords: Cry toxin; Bacillus thuringiensis; Pore; Biopesticide; Molecular dynamics; Membrane

Impaired routing of wild type FXYD2 after oligomerisation with FXYD2-G41R might explain the dominant nature of renal hypomagnesemia by Edinio R. Cairo; Thomas Friedrich; Herman G.P. Swarts; Nine V. Knoers; René J.M. Bindels; Leo A. Monnens; Peter H.G.M. Willems; Jan Joep H.H.M. De Pont; Jan B. Koenderink (pp. 398-404).

Autosomal dominant renal hypomagnesemia, associated with hypocalciurea, has been linked to a G to A mutation at nucleotide position 121 in the FXYD2 gene, resulting in the substitution of Gly with Arg at residue 41 of the protein. FXYD2, also called the Na,K-ATPase γ-subunit, binds to Na,K-ATPase and influences its cation affinities. In this paper, we provide evidence for the molecular mechanism underlying the dominant character of the disorder. Co-immunoprecipitation experiments using tagged FXYD2 proteins demonstrated that wild type FXYD2 proteins oligomerise. Moreover, FXYD2-G41R also shows oligomerisation with itself and with the wild type protein. In the case of FXYD2-G41R, however, formation of homo-oligomers was prevented by addition of DTT or introduction of the C52A mutation. Finally, we demonstrated that artificial glycosylation of the wild type FXYD2 is reduced when co-expressed with FXYD2-G41R. These data indicate that binding of FXYD2-G41R to wild type FXYD2 subunit might abrogate the routing of wild type FXYD2 to the plasma membrane thus causing the dominant nature of this mutation.

Keywords: Na,K-ATPase; FXYD2; Hypomagnesemia

Electrogenic steps of the SR Ca-ATPase enzymatic cycle and the effect of curcumin by Gianluca Bartolommei; Francesco Tadini-Buoninsegni; Maria Rosa Moncelli; Rolando Guidelli (pp. 405-413).

Sarcoplasmic reticulum (SR) vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps in the presence of calcium ions. The resulting capacitive current transients are compared with those calculated on the basis of the enzymatic cycle of the calcium pump. This comparison provides information on the kinetics of the E₂–E₁ conformational change and on its pH dependence. The alteration in the current transients following ATP concentration jumps in the presence of curcumin is examined. In particular, curcumin decreases the rate of slippage of the Ca-ATPase, and at concentrations above 10 μM reduces calcium transport by this pump.

Keywords: SERCA; Electrogenic transport; Curcumin; Solid supported membrane

Effect of lysophosphatidylcholine on the surface hydration of phospholipid vesicles by Marilene Alves; Barney L. Bales; Miroslav Peric (pp. 414-422).

The interfacial properties of the negatively charged dimyristoyl-phosphatidylglycerol (DMPG) and the zwitterionic dimyristoyl-phosphatidylcholine (DMPC) vesicles mixed with the fusion inhibitor lysomyristoylphosphatidylcholine (LMPC) are investigated by electron paramagnetic resonance (EPR). At 35 °C, addition of 20 mol% of LMPC to the DMPG vesicles increases the effective concentration of water in the interfacial layer of DMPG vesicles from 19.3 M to 27.7 M, whereas in the case of mixed DMPC-LMPC vesicle the effective water concentration in the interfacial layer of DMPC vesicles only changes from 15.1 M to 18.4 M. The hydrogen bonding structure in both mixed DMPG-LMPC and mixed DMPC-LMPC vesicles becomes stronger with an increasing fraction of LMPC in the vesicles. The average area per phospholipid decreases in mixed DMPC-LMPC vesicles, while it increases in mixed DMPG-LMPC vesicles as the proportion of LMPC in the vesicle increases. The inhibitory nature of LMPC in both vesicle and biological fusion comes from the increase in surface hydration, as well as from the dynamic cone shape of LMPC in the phospholipid bilayer.

Keywords: Electron Paramagnetic Resonance (EPR) spectroscopy; Surface Hydration; Lysomyristoylphosphatidylcholine (LMPC); Dimyristoyl-phosphatidylglycerol (DMPG); Dimyristoyl-phosphatidylcholine (DMPC); Spin probes

MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion by Yuma Yamada; Hidetaka Akita; Hiroyuki Kamiya; Kentaro Kogure; Takenori Yamamoto; Yasuo Shinohara; Kikuji Yamashita; Hideo Kobayashi; Hiroshi Kikuchi; Hideyoshi Harashima (pp. 423-432).

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.

Keywords: Non-viral vector; Mitochondria; Mitochondrial drug delivery; MITO-Porter; Membrane fusion; Octaarginine

A comparative study of mechanisms of surfactant inhibition by Lasantha Gunasekara; W. Michael Schoel; Samuel Schürch; Matthias W. Amrein (pp. 433-444).

Pulmonary surfactant spreads to the hydrated air–lung interface and reduces the surface tension to a very small value. This function fails in acute respiratory distress syndrome (ARDS) and the surface tension stays high. Dysfunction has been attributed to competition for the air–lung interface between plasma proteins and surfactant or, alternatively, to ARDS-specific alterations of the molecular profile of surfactant. Here, we compared the two mechanisms in vitro, to assess their potential role in causing respiratory distress. Albumin and fibrinogen exposure at or above blood level concentrations served as the models for testing competitive adsorption. An elevated level of cholesterol was chosen as a known adverse change in the molecular profile of surfactant in ARDS. Bovine lipid extract surfactant (BLES) was spread from a small bolus of a concentrated suspension (27 mg/ml) to the air–water interface in a captive bubble surfactometer (CBS) and the bubble volume was cyclically reduced and increased to assess surface activity of the spread material. Concentrations of inhibitors and the concentration and spreading method of pulmonary surfactant were chosen in an attempt to reproduce the exposure of surfactant to inhibitors in the lung. Under these conditions, neither serum albumin nor fibrinogen was persistently inhibitory and normal near-zero minimum surface tension values were obtained after a small number of cycles. In contrast, inhibition by an increased level of cholesterol persisted even after extensive cycling. These results suggest that in ARDS, competitive adsorption may not sufficiently explain high surface tension, and that disruption of the surfactant film needs to be given causal consideration.

Keywords: Captive bubble surfactometer (CBS); Pulmonary surfactant; Fibrinogen; Serum albumin; Cholesterol; Polyethylene glycol (PEG); Bovine lipid extract surfactant (BLES)

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation by Jana Beranová; Małgorzata Jemioła-Rzemińska; Dana Elhottová; Kazimierz Strzałka; Ivo Konopásek (pp. 445-453).

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 °C) and low (20 °C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T_m (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 °C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T_m was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 °C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T_m by 10.5 °C. In mineral media at 20 °C the corresponding changes of T_m were almost negligible. After a temperature shift from 40 to 20 °C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.

Keywords: Abbreviations; CM; complex medium with glucose; MMGlu; mineral medium with glucose; MMGlyc; mineral medium with glycerol; DPH; 1,6-diphenyl-1,3,5-hexatriene; FA; fatty acid; i-; iso-branched fatty acid; a-; anteiso-branched fatty acid; n-; non-branched fatty acid; r; _ss; steady state anisotropy of DPH fluorescence; T; _m; gel to liquid-crystalline phase transition temperature; T; _c; temperature of cultivationBacillus; Fatty acid synthesis regulation; Cold shock; DPH; DSC

The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function by Runying Yang; Robert Scavetta; Xiu-bao Chang (pp. 454-465).

Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the β-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 °C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the β-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport.

Keywords: Abbreviations; BHK; baby hamster kidney; Sf21; Spodoptera frugiperda 21; ABC; ATP binding cassette; MRP1; multidrug resistance protein 1; P-gp; P-glycoprotein; CFTR; cystic fibrosis trans-membrane conductance regulator; BSA; bovine serum albumin; NBD; nucleotide binding domain; LTC4; leukotriene C4; 8-N; ₃; ATP; 8-azidoadenosine 5′-triphosphate; PBS; phosphate-buffered saline; EGTA; ethylene glycol-bis(β-aminoethyl ether); N,N,N,N; -tetraacetic acid; SDS; sodium dodecyl sulfateMRP1; Protein folding; Hydrogen-bond formation; Complex-glycosylated mature protein; ATP binding/hydrolysis; ATP-dependent solute transport

Docosahexaenoic acid alters the size and distribution of cell surface microdomains by Robert S. Chapkin; Naisyin Wang; Yang-Yi Fan; Joanne R. Lupton; Ian A. Prior (pp. 466-471).

We recently generated nutritional data suggesting that chemoprotective dietary n-3 polyunsaturated fatty acids (n-3 PUFA) are capable of displacing acylated proteins from lipid raft microdomains in vivo [D.W. Ma, J. Seo, L.A. Davidson, E.S. Callaway, Y.Y. Fan, J.R. Lupton, R.S. Chapkin, n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon, FASEB J. 18 (2004) 1040–1042; Y.Y. Fan, L.H. Ly, R. Barhoumi, D.N. McMurray, R.S. Chapkin, Dietary docosahexaenoic acid suppresses T cell protein kinase Cθ lipid raft recruitment and IL-2 recruitment, J. Immunol. 173 (2004) 6151–6160]. A primary source of very long chain n-3 PUFA in the diet is derived from fish enriched with docosahexaenoic acid (DHA, 22:6n-3). In this study, we sought to determine the effect of DHA on cell surface microdomain organization in situ. Using immuno-gold electron microscopy of plasma membrane sheets coupled with spatial point analysis of validated microdomain markers, morphologically featureless microdomains were visualized in HeLa cells at high resolution. Clustering of probes within cholesterol-dependent (GFP-tH) versus cholesterol-independent (GFP-tK) nanoclusters was differentially sensitive to n-3 PUFA treatment of cells. Univariate K-function analysis of GFP-tH (5 nm gold) revealed a significant increase in clustering ( p<0.05) by pre-treatment with DHA and linoleic acid (LA, 18:2^Δ9,12) compared to control fatty acids; whereas LA significantly ( p<0.05) reduced GFP-tK clustering. These novel data suggest that the plasma membrane organization of inner leaflets is fundamentally altered by PUFA-enrichment. We speculate that our findings may help define a new paradigm to better understand the complexity of n-3 PUFA modulation of signaling networks.

Keywords: Dynamic domain; Nanocluster; Omega-3 fatty acid; Microdomain

Insight into the location and dynamics of the annexin A2 N-terminal domain during Ca²⁺-induced membrane bridging by Jesus Ayala-Sanmartin; Mallik Zibouche; Françoise Illien; Michel Vincent; Jacques Gallay (pp. 472-482).

Annexin A2 (AnxA2) is a Ca²⁺- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca²⁺ binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth of data exists on the structure and dynamics of the core, but little is known about the N-terminal domain especially in the Ca²⁺-induced membrane-bridging process. To investigate this protein region in the monomeric AnxA2 and in the heterotetramer (AnxA2-p11)₂, the reactive Cys8 residue was specifically labelled with the fluorescent probe acrylodan and the interactions with membranes were studied by steady-state and time-resolved fluorescence. In membrane junctions formed by the (AnxA2-p11)₂ heterotetramer, the flexibility of the N-terminal domain increased as compared to the protein in solution. In “homotypic” membrane junctions formed by monomeric AnxA2, acrylodan moved to a more hydrophobic environment than in the protein in solution and the flexibility of the N-terminal domain also increased. In these junctions, this domain is probably not in close contact with the membrane surface, as suggested by the weak quenching of acrylodan observed with doxyl-PCs, but pairs of N-termini likely interact, as revealed by the excimer-forming probe pyrene-maleimide bound to Cys8. We present a model of monomeric AnxA2 N-terminal domain organization in “homotypic” bridged membranes in the presence of Ca²⁺.

Keywords: Abbreviations; Acrylodan; 6-acryloyl-2-dimethylaminonaphthalene; LUV; large unilamellar vesicles; MEM; maximum entropy method; n; -doxyl PC; 1-palmitoyl-2-stearoyl(; n; -doxyl)-; sn; -glycerophosphatidylcholine (; n; =; 5, 7 or 12); p11; S100A10 protein; Anx; annexin; AnxA2; ^acryl; annexin A2 labelled with acrylodan on Cys8; AnxA2; ^pyr; annexin A2 labelled with pyrene on Cys8; (AnxA2-p11); ₂; heterotetramer AnxA2-p11; Pyrene-maleimide; N; -(1-Pyrene)maleimide; PC; egg; l; -α-glycerophosphatidylcholine; PS; brain; l; -α-glycerophosphatidyl-; l; -serine; PE; egg; l; -α-phosphatidyl-; l; -ethanolamine; pCa; −; log [Ca; ²⁺; ]; L/P; lipid/protein molar ratioAnnexin; N-terminal domain; Acrylodan; Pyrene; Membrane aggregation; Time-resolved fluorescence

Monitoring lysosomal fusion in electrofused hybridoma cells by Mateja Gabrijel; Marko Kreft; Robert Zorec (pp. 483-490).

Dendritic and tumor cells are fused to produce hybridoma cells, which are considered to be used as cellular vaccines to treat cancer. Previous strategies for hybridoma cell production were based on the quantification of the electrofusion yield by labeling the cytoplasm of both parental cell types. However, a better physiological strategy would be to label subcellular structures related directly to the antigen presentation process. Therefore, we here electrofused the same amount of CHO cells stained with red and green fluorescent dextrans and have monitored the yield of hybridoma cell formation by measuring the fusion of red and green late endocytic organelles that are involved in antigen presentation. By using confocal microscopy, the level of fused, fluorescently labelled late endocytic compartments in a single hybridoma cell was determined. The results demonstrate that organellar fusion occurs in hybridomas, which is time- and temperature-dependent. This approach therefore provides a new method for the hybridoma cell vaccine evaluation, which is based on the intracellular physiological mechanism of antigen presentation.

Keywords: Lysosomal fusion; Quantitative 3D colocalization; Confocal microscopy; Hybridoma cells; Cancer

Effects of fluorescent probe NBD-PC on the structure, dynamics and phase transition of DPPC. A molecular dynamics and differential scanning calorimetry study by Luís M.S. Loura; Fábio Fernandes; A.C. Fernandes; J.P. Prates Ramalho (pp. 491-501).

We present a combined theoretical (molecular dynamics, MD) and experimental (differential scanning calorimetry, DSC) study of the effect of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) acyl chain-labeled fluorescent phospholipid analogs (C6-NBD-PC and C12-NBD-PC) on 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) bilayers. DSC measurements reveal that <1 mol% of NBD-PC causes elimination of the pre-transition and a large loss of cooperativity of the main transition of DPPC. Labeling with C6-NBD-PC or C12-NBD-PC shifts the main transition temperature to lower or higher values, respectively. Following our recent report on the location and dynamics of these probes ( BBA 1768 (2007) 467–478) in fluid phase DPPC, we present a detailed analysis of 100-ns MD simulations of systems containing either C6-NBD-PC or C12-NBD-PC, focused on their influence on several properties of the host bilayer. Whereas most monitored parameters are not severely affected for 1.6 mol% of probe, for the higher concentration studied (6.2 mol%) important differences are evident. In agreement with published reports, we observed that the average area per phospholipid molecule increases, whereas DPPC acyl chain order parameters decrease. Moreover, we predict that incorporation of NBD-PC should increase the electrostatic potential across the bilayer and, especially for C12-NBD-PC, slow lateral diffusion of DPPC molecules and rotational mobility of DPPC acyl chains.

Keywords: Fluorescence probe; NBD-labeled lipid; Molecular simulation; Membrane model system; Membrane perturbation

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: Lipid specificity and effect of the temperature by Danny Lassiseraye; Lesley Courtemanche; Annick Bergeron; Puttaswamy Manjunath; Michel Lafleur (pp. 502-513).

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 °C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 °C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.

Keywords: Isothermal calorimetry; Membrane; BSP protein; PDC-109; Lipid specificity; Lipid–protein interaction

Dehydration-inducible changes in expression of two aquaporins in the sleeping chironomid, Polypedilum vanderplanki by Takahiro Kikawada; Ayako Saito; Yasushi Kanamori; Mika Fujita; Katarzyna Śnigórska; Masahiko Watanabe; Takashi Okuda (pp. 514-520).

Aquaporin, AQP, is a channel protein that allows water to permeate across cell membranes. Larvae of the sleeping chironomid, Polypedilum vanderplanki, can withstand complete dehydration by entering anhydrobiosis, a state of suspended animation; however, the mechanism by which water flows out of the larval body during dehydration is still unclear. We isolated two cDNAs ( PvAqp1 and PvAqp2) encoding water-selective aquaporins from the chironomid. When expressed in Xenopus oocytes, PvAQP1 and PvAQP2 facilitated permeation of water but not glycerol. Northern blots and in situ hybridization showed that expression of PvAqp1 was dehydration-inducible and ubiquitous whereas that of PvAqp2 was dehydration-repressive and fat body-specific. These data suggest distinct roles for these aquaporins in P. vanderplanki, i.e., PvAqp2 controls water homeostasis of fat body during normal conditions and PvAqp1 is involved in the removal of water during induction of anhydrobiosis.

Keywords: Aquaporin; Anhydrobiosis; Polypedilum vanderplanki; Water transport; Desiccation-resistance; Desiccation-avoidance; Desiccation-tolerance; Dehydration-inducible gene; Dehydration-repressible gene

Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating by María José De Rosa; Jeremías Corradi; Cecilia Bouzat (pp. 521-529).

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of α and non-α subunits, or homo-pentameric, composed of α7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between α1 and ɛ or α7 subunits. The replacement of M3 in α1 by ɛM3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse ɛ chimeric subunit. The duration of the open state decreases with the increase in the number of α1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each α1M3 segment decreases the energy barrier of the closing process by ∼0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of α1 sequence by α7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.

Keywords: Nicotinic receptor; Single-channel; Patch-clamp; Transmembrane domains

The anti-tumor alkylphospholipid perifosine is internalized by an ATP-dependent translocase activity across the plasma membrane of human KB carcinoma cells by Francisco Muñoz-Martínez; Cristina Torres; Santiago Castanys; Francisco Gamarro (pp. 530-540).

Perifosine is a promising anticancer alkylphospholipid (ALP) that induces apoptosis in tumor cells. Here we report evidences against a role of endocytosis in perifosine uptake by human KB carcinoma cells. We have generated a KB cell line resistant to perifosine (KB PER^R clone10), which shows cross-resistance to the ALPs miltefosine and edelfosine, a marked impairment in the uptake of¹⁴C-perifosine at both 37 °C and 4 °C, and no signs for active efflux of the drug. KB PER^R clone10 cells show a similar rate of raft-dependent endocytosis with respect to the parental cells, and silencing of both clathrin and dynamin in the latter causes only minor changes in the rate of perifosine uptake. Perifosine uptake is a temperature- and ATP-dependent, N-ethylmaleimide- and orthovanadate-sensitive process in parental cells. Accumulation of¹⁴C-perifosine and the fluorescent phospholipid analogue 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl]-phosphatidylethanolamine (NBD-PE) is inhibited by perifosine in a concentration-dependent manner in parental cells. Moreover, NBD-PE accumulation is slower in PER^R clone10 cells and correlated with phosphatidylserine exposure in their plasma membrane surface. Together, all these data suggest a role of plasma membrane translocation by a putative phospholipid translocase, rather than endocytosis, as the true mechanism for ALPs uptake in KB carcinoma cells.

Keywords: Alkylphospholipids; Perifosine uptake; Tumor cells; Endocytosis; Aminophospholipid translocase

Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes by A.A. Pashkovskaya; V.E. Maizlish; G.P. Shaposhnikov; E.A. Kotova; Y.N. Antonenko (pp. 541-548).

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum–phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.

Keywords: Abbreviations; BLM; bilayer lipid membrane; gA; gramicidin A; DPhPC; diphytanoylphosphatidylcholine; DPhPG; diphytanoylphosphatidylglycerol; DGEPC; dipalmitoyl-glycero-ethylphosphocholine; AlPcS; ₄; aluminum tetrasulfophthalocyanine; AlPcN; ₄; hydroxoaluminum (III) tetra(tributylammoniomethyl)phthalocyanine chloride; FCS; fluorescence correlation spectroscopy; IFC; intramembrane field compensation method; Δ; φ; _b; difference of boundary potentials; CTAB; cetyltrimethylammonium bromideSensitized photoinactivation; Gramicidin A; Sulfonated phthalocyanine; Bilayer lipid membrane

Responding double-porous lipid membrane: Lyotropic phases in a polymer scaffold by Christoffer Åberg; Cécile Pairin; Fátima O. Costa-Balogh; Emma Sparr (pp. 549-558).

The large osmotic gradient over the outermost layer of human skin implies major structural changes along the gradient, which in turn affects transport. In particular, the possibility of phase changes introduces a non-linear element to the transport behaviour. We present a novel model membrane system to be used for studying these transport mechanisms, where we use a hydrophobic porous polymer membrane as a scaffold for lipid lyotropic phases. The polymer membrane provides mechanical robustness, but also prevents defects of the lipid lyotropic phases, and it can induce an orientation of anisotropic phases. We study the location, structure and phase behaviour of the confined phases. It is shown that this model membrane system allow for accurate measurements of transport through lipid membranes in the presence of different osmotic gradients. A theoretical description is evaluated and shows that this phenomenon can be understood in terms of the proposed mechanism of phase changes. The novel double-porous lipid membrane constitutes a mechanically robust system for studies in aligned systems, which is generally very difficult to achieve. This could have large implications for studies of transport processes in, e.g. skin and other biomembrane model systems.

Keywords: Diffusive transport; Permeability; Osmotic gradient; Phase transformation; Stratum corneum; Responding membrane

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BBA - Biomembranes (v.1778, #2)

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BBA - Biomembranes (v.1778, #2)