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BBA - Biomembranes (v.1758, #11)

Editorial Board (pp. ii).

Transfection of oral cancer cells mediated by transferrin-associated lipoplexes: Mechanisms of cell death induced by herpes simplex virus thymidine kinase/ganciclovir therapy by Sílvia S. Neves; Ana B. Sarmento-Ribeiro; Sérgio P. Simões; Maria C. Pedroso de Lima (pp. 1703-1712).

The Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene/ganciclovir (GCV) approach has been used for the treatment of a variety of cancers. The purpose of the present study was to evaluate the cytotoxic effect of ganciclovir in oral squamous cancer cells, previously transfected with HSV-tk gene delivered by transferrin-associated complexes (Tf-lipoplexes), as well as to investigate the mechanisms involved in the bystander effect and in the process of cell death. The delivery of HSV-tk gene to the oral cancer cells, HSC-3 and SCC-7, mediated by Tf-lipoplexes followed by ganciclovir treatment resulted in essentially 100% cytotoxicity, the observed toxic effect being dependent both on GCV dose and incubation time. Cell death was shown to occur mainly by an apoptotic process. Different experimental approaches demonstrated that the observed cytotoxicity was mainly due to diffusion of the toxic agent into neighbouring, non-transfected cells, via gap junctions. Preliminary in vivo studies in a murine model for oral squamous cell carcinoma have shown a significant inhibition of tumor growth upon injection of Tf-lipoplexes carrying HSV-tk followed by intraperitonal injection of GCV, as compared to controls.

Keywords: HSV-tk/GCV suicide gene therapy; Oral carcinoma; Transferrin-associated lipoplexes; Bystander effect; Apoptosis

WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes by M. Coraiola; P. Lo Cantore; S. Lazzaroni; A. Evidente; N.S. Iacobellis; M. Dalla Serra (pp. 1713-1722).

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (>27 μM). This effect was not detected for tolaasin I at the concentrations tested (<28 μM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.

Keywords: Lipodepsipeptide; Pseudomonas reactans; Pseudomonas tolaasii; Liposome; Transmembrane pore; Secondary structure

Role of membrane curvature in mechanoelectrical transduction: Ion carriers nonactin and valinomycin sense changes in integral bending energy by V.Gh. Shlyonsky; V.S. Markin; I. Andreeva; S.E. Pedersen; S.A. Simon; D.J. Benos; I.I. Ismailov (pp. 1723-1731).

We describe the phenomenon of mechanoelectrical transduction in macroscopic lipid bilayer membranes modified by two cation-selective ionophores, valinomycin and nonactin. We found that bulging these membranes, while maintaining the membrane tension constant, produced a marked supralinear increase in specific carrier-mediated conductance. Analyses of the mechanisms involved in mechanoelectrical transduction induced by the imposition of a hydrostatic pressure gradient or by an amphipathic compound chlorpromazine reveal similar changes in the charge carrier motility and carrier reaction rates at the interface(s). Furthermore, the relative change in membrane conductance was independent of membrane diameter, but was directly proportional to the square of membrane curvature, thus relating the observed phenomena to the bilayer bending energy. Extrapolated to biological membranes, these findings indicate that ion transport in cells can be influenced simply by changing shape of the membrane, without a change in membrane tension.

Keywords: Mechanosensitivity; Lipid bilayers; Liposomes; Vesicles; Ion carriers; Curvature

Membrane interaction and activity of the glycolipid transfer protein by Gun West; Matts Nylund; J. Peter Slotte; Peter Mattjus (pp. 1732-1742).

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.

Keywords: Abbreviations; GLTP; glycolipid transfer protein; DiO-C; ₁₆; dihexadecyloxacarbocyanine perchlorate; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; GalCer; galactosylceramide; AV-GalCer; N-[(11; E; )-12-(9-anthryl)-11-dodecenoyl]-1-O-β-galactosylsphingosine; NATA; N-acetyltryptophanamideFluorescence; Site-directed mutagenesis; GLTP; Membrane; Binding

Mechanism of the inhibitory effect of zwitterionic drugs (levofloxacin and grepafloxacin) on carnitine transporter (OCTN2) in Caco-2 cells by Takeshi Hirano; Satoru Yasuda; Yuki Osaka; Masaki Kobayashi; Shirou Itagaki; Ken Iseki (pp. 1743-1750).

l-Carnitine plays an important role in lipid metabolism by facilitating the transport of long-chain fatty acids across the mitochondrial inner membrane followed by fatty acid beta-oxidation. It is known thatl-carnitine exists as a zwitterion and that member of the OCTN family play an important role in its transport. The aims of this study were to characterizel-carnitine transport in the intestine by using Caco-2 cells and to elucidate the effects of levofloxacin (LVFX) and grepafloxacin (GPFX), which are zwitterionic drugs, onl-carnitine uptake. Kinetic analysis showed that the half-saturation Na⁺ concentration, Hill coefficient and K_m value ofl-carnitine uptake in Caco-2 cells were 10.3±4.5 mM, 1.09 and 8.0±1.0 μM, respectively, suggesting that OCTN2 mainly transportsl-carnitine. LVFX and GPFX have two p K_a values and the existence ratio of their zwitterionic forms is higher under a neutral condition than under an acidic condition. Experiments on the inhibitory effect of LVFX and GPFX onl-carnitine uptake showed that LVFX and GPFX inhibitedl-carnitine uptake more strongly at pH 7.4 than at pH 5.5. It was concluded that the zwitterionic form of drugs plays an important role in inhibition of OCTN2 function.

Keywords: l; -carnitine; Caco-2; Levofloxacin; Grepafloxacin; OCTN2

Molecular study of the diffusional process of DMSO in double lipid bilayers by Sukit Leekumjorn; Amadeu K. Sum (pp. 1751-1758).

As a way to quantify the diffusion process of molecular compounds through biological membranes, we investigated in this study the dynamics of DMSO through an 1,2-Dipalmitoyl- sn-Glycero-3-Phosphocholine (DPPC) bilayer system. To properly account for the diffusion of DMSO due to a concentration gradient, a double DPPC bilayer was setup for our simulations. In such configuration, the aqueous phases can be explicitly associated with the extra and intracellular domains of the membrane, which is seldom the case in studies of single lipid bilayer due to the periodicity imposed by the simulations. DMSO molecules were initially contained in one of the aqueous phases (extracellular region) at a concentration of 5 wt.%. Molecular dynamics simulation was performed in this system for 95 ns at 350 K and 1 bar. The simulations showed that although many DMSO molecules penetrated the lipid bilayer, only about 10% of them crossed the bilayer to reach the other aqueous phase corresponding to the intracellular region of the membrane. The simulation time considered was insufficient to reach equilibrium of the DMSO concentration between the aqueous phases. However, the simulations provided sufficient information to estimate parameters to apply Fick's Law to model the diffusion process of the system. Using this model, we predicted that for the time considered in our simulation, the concentration of DMSO in the intracellular domain should have been about half of the actual value obtained. The model also predicted that equilibrium of the DMSO concentration in the system would be reached after about 2000 ns, approximately 20 times longer than the performed simulation.

Keywords: Molecular dynamic; Double bilayer; Diffusion; Concentration gradient; DMSO; DPPC

Myristoylation of human LanC-like Protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin by Christine Landlinger; Ulrich Salzer; Rainer Prohaska (pp. 1759-1767).

Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-β-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.

Keywords: Abbreviations; BSA; bovine serum albumin; DOPC; 1,2-Dioleoyl-; sn; -glycero-3-phosphocholine; DMEM; Dulbecco's modified Eagle's medium; GFP; green fluorescent protein; GST; glutathione-S-transferase; HMA; 2-hydroxy myristic acid; LANCL2; lanthionine synthetase component C-like 2; LANCL2-GFP del.; N-terminal truncation of C-terminally GFP-tagged LANCL2; LANCL2-GFP mutG1A; C-terminally GFP-tagged LANCL2 with glycine-1 mutated to alanine; mβCD; methyl-β-cyclodextrin; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS; phosphate-buffered saline; P-gp; P-glycoprotein; PI; phosphatidylinositol; PIP; phosphatidylinositol phosphate(s); PMSF; phenylmethylsulfonyl fluoride; RT; room temperature; TASP; Testis-specific Adriamycin Sensitivity Protein; TBS; Tris-buffered saline; TNET; Tris–NaCl–EDTA–Triton X-100Myristoylation; Phosphatidylinositol phosphate; Adriamycin; Actin cytoskeleton; Cell attachment

Self-association of isolated large cytoplasmic domain of plasma membrane H⁺-ATPase from Saccharomyces cerevisiae: Role of the phosphorylation domain in a general dimeric model for P-ATPases by W.I. Almeida; O.B. Martins; P.C. Carvalho-Alves (pp. 1768-1776).

Large cytoplasmic domain (LCD) plasma membrane H⁺-ATPase from S. cerevisiae was expressed as two fusion polypeptides in E. coli: a DNA sequence coding for Leu353–Ileu674 (LCDh), comprising both nucleotide (N) and phosphorylation (P) domains, and a DNA sequence coding for Leu353–Thr543 (LCDΔh, lacking the C-terminus of P domain), were inserted in expression vectors pDEST-17, yielding the respective recombinant plasmids. Overexpressed fusion polypeptides were solubilized with 6 M urea and purified on affinity columns, and urea was removed by dialysis. Their predicted secondary structure contents were confirmed by CD spectra. In addition, both recombinant polypeptides exhibited high-affinity 2′,3′- O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP) binding ( K_d=1.9 μM and 2.9 μM for LCDh and LCDΔh, respectively), suggesting that they have native-like folding. The gel filtration profile (HPLC) of purified LCDh showed two main peaks, with molecular weights of 95 kDa and 39 kDa, compatible with dimeric and monomeric forms, respectively. However, a single elution peak was observed for purified LCDΔh, with an estimated molecular weight of 29 kDa, as expected for a monomer. Together, these data suggest that LCDh exist in monomer–dimer equilibrium, and that the C-terminus of P domain is necessary for self-association. We propose that such association is due to interaction between vicinal P domains, which may be of functional relevance for H⁺-ATPase in native membranes. We discuss a general dimeric model for P-ATPases with interacting P domains, based on published crystallography and cryo-electron microscopy evidence.

Keywords: Abbreviations; ATP; adenosine triphosphate; ATPase; adenosine-triphosphatase; bp; base pairs; CD; circular dichroism; DTT; dithiothreitol; HPLC; high performance liquid chromatography; LCD; Large cytoplasmic domain; PCR; polymerase chain reaction; PMSF; phenylmethylsulfonyl fluoride; SERCA; sarcoplasmic reticulum Ca; ²⁺; -ATPase; SDS; sodium dodecyl sulfate; PAGE; polyacrylamide gel electrophoresis; TNP-ATP; 2′,3′-; O; -(2,4,6-trinitrophenyl)adenosine-5′-triphosphate; MOPS; 3-; N; -morpholinopropanesulfonic acidP-type; ATPase; Oligomerization; Yeast; Protein structure; Self-association

Binding assays of inhibitors towards selected V-ATPase domains by F. Fernandes; L.M.S. Loura; A. Fedorov; N. Dixon; T.P. Kee; M. Prieto; M.A. Hemminga (pp. 1777-1786).

The macrolide antibiotic bafilomycin and the related synthetic compound SB 242784 are potent inhibitors of the vacuolar H⁺-ATPases (V-ATPase). It is currently believed that the site of action of these inhibitors is located on the membrane bound c-subunits of V-ATPases. To address the identification of the critical inhibitors binding domain, their specific binding to a synthetic peptide corresponding to the putative 4th transmembrane segment of the c-subunit was investigated using fluorescence resonance energy transfer (FRET), and for this purpose a specific formalism was derived. Another peptide of the corresponding domain of the c′ isoform, was checked for binding of bafilomycin, since it is not clear if V-ATPase inhibition can also be achieved by interaction of the inhibitor with the c′-subunit. It was concluded that bafilomycin binds to the selected peptides, whereas SB 242784 was unable to interact, and in addition for bafilomycin, its interaction with the peptides either corresponding to the c- or the c′-subunit isoforms is identical. Since the observed interactions are however much weaker as compared to the very efficient binding of both bafilomycin and SB 242784 to the whole protein, it can be concluded that assembly of all V-ATPase transmembrane segments is required for an efficient interaction.

Keywords: V-ATPase; Bafilomycin A; ₁; Fluorescence; FRET

Characterization and in vitro evaluation of spherulites as sequestering vesicles with potential application in drug detoxification by Anand Babu Dhanikula; Michel Lafleur; Jean-Christophe Leroux (pp. 1787-1796).

The aim of the present investigation was to prepare and characterize lecithin spherulites as parenteral drug sequestering agents with potential application in the treatment of drug overdose and chemical poisoning. The spherulites (∼200 nm) obtained by controlled hydration and shearing of lipid–alcohol mixtures, revealed unexpected differences in the physical properties of the bilayer when compared to liposomes. Differential scanning calorimetry, 31-phosphorus nuclear magnetic resonance, and pH-sensitive pyranine steady-state fluorescence studies indicated that although spherulites retained the typical bilayer conformation, the arrangement of the phospholipid molecules was perturbed relative to native liposome bilayer. The loosened packing of the phospholipids in bilayers was strongly supported by the relative ease with which spherulites lost the established pH-gradient. This permeability problem was overcome via incorporation of cholesterol in the bilayer. Subsequently, albumin/buffer components were encapsulated in these spherulites and the drug sequestration potential for detoxification application was examined. Citrate pH-gradient spherulites accumulated 75% of external haloperidol while those loaded with ∼20% (w/w) albumin were able to take up 45% of haloperidol and 91–95% of taxanes (docetaxel and paclitaxel). In cytotoxicity studies, the competitive internalization of docetaxel by albumin-loaded spherulites resulted in an increase of the IC₅₀ value for the free drug. Thus, the spherulite technology could be a versatile approach for actively sequestering toxins in the blood and for reducing the adverse effects by altering the pharmacokinetics and biodistribution of overdosed drugs.

Keywords: Spherulite; Detoxification; Uptake; Haloperidol; Taxanes

In-plane miscibility and mixed bilayer microstructure in mixtures of catanionic glycolipids and zwitterionic phospholipids by C.V. Teixeira; M. Blanzat; J. Koetz; I. Rico-Lattes; G. Brezesinski (pp. 1797-1808).

SAXS/WAXS studies were performed in combination with freeze fracture electron microscopy using mixtures of a new Gemini catanionic surfactant (Gem16-12, formed by two sugar groups bound by a hydrocarbon spacer with 12 carbons and two 16-carbon chains) and the zwitterionic phospholipid 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) to establish the phase diagram. Gem16-12 in water forms bilayers with the same amount of hydration water as DPPC. A frozen interdigitated phase with a low hydration number is observed below room temperature. The kinetics of the formation of this crystalline phase is very slow. Above the chain melting temperature, multilayered vesicles are formed. Mixing with DPPC produces mixed bilayers above the corresponding chain melting temperature. At room temperature, partially lamellar aggregates with local nematic order are observed. Splitting of infinite lamellae into discs is linked to immiscibility in frozen state. The ordering process is always accompanied by dehydration of the system. As a consequence, an unusual order–disorder phase transition upon cooling is observed.

Keywords: SAXS; Bilayer; Gemini surfactant; Ordering process; Anti-HIV; Miscibility

Signaling pathways involving the sodium pump stimulate NO production in endothelial cells by Alexander Eva; Ulrike Kirch; Georgios Scheiner-Bobis (pp. 1809-1814).

The cardiac steroid ouabain, a known inhibitor of the sodium pump (Na⁺,K⁺-ATPase), has been shown to release endothelin from endothelial cells when used at concentrations below those that inhibit the pump. The present study addresses the question of which signaling pathways are activated by ouabain in endothelial cells. Our findings indicate that ouabain, applied at low concentrations to human umbilical cord endothelial cells (HUAECs), induces a reaction cascade that leads to translocation of endothelial nitric oxide synthase (eNOS) and to activation of phosphatidylinositol 3-kinase (PI3K). These events are followed by phosphorylation of Akt (also known as protein kinase B, or PKB) and activation of eNOS by phosphorylation. This signaling pathway, which results in increased nitric oxide (NO) production in HUAECs, is inhibited by the PI3K-specific inhibitor LY294002. Activation of the reaction cascade is not due to endothelin-1 (ET-1) binding to the ET-1 receptor B (ET_B), since application of the ET_B-specific antagonist BQ-788 did not have any effect on Akt or eNOS phosphorylation. The results shown here indicate that ouabain binding to the sodium pump results in the activation of the proliferation and survival pathways involving PI3K, Akt activation, stimulation of eNOS, and production of NO in HUAECs. Together with results from previous publications, the current investigation implies that the sodium pump is involved in vascular tone regulation.

Keywords: Na; ⁺; ,K; ⁺; -ATPase; Ouabain; Signaling cascade; Akt; eNOs; PI3K

Assembly of a transmembrane b-Type cytochrome is mainly driven by transmembrane helix interactions by Thomas Volkmer; Christian Becker; Alexander Prodöhl; Carmen Finger; Dirk Schneider (pp. 1815-1822).

Folding, assembly and stability of α-helical membrane proteins is still not very well understood. Several of these membrane proteins contain cofactors, which are essential for their function and which can be involved in protein assembly and/or stabilization. The effect of heme binding on the assembly and stability of the transmembrane b-type cytochrome b₅₅₉^′ was studied by fluorescence resonance energy transfer. Cytochrome b₅₅₉^′ consists of two monomers of a 44 amino acid long polypeptide, which contains one transmembrane domain. The synthesis of two variants of the b₅₅₉^′ monomer, each carrying a specific fluorescent dye, allowed monitoring helix–helix interactions in micelles by resonance energy transfer. The measurements demonstrate that the transmembrane peptides dimerize in detergent in the absence and presence of the heme cofactor. Cofactor binding only marginally enhances dimerization and, apparently, the redox state of the heme group has no effect on dimerization.

Keywords: Abbreviations; DDM; n; -dodecyl-β-; d; -maltoside; FRET; Fluorescence Resonance Energy Transfer; MalE; maltose binding protein; SDS; sodium dodecylsulfateAssembly; Cytochrome; Heme; Membrane protein folding; Two-stage model

FTIR analysis of the interaction of arbutin with dimyristoyl phosphatidylcholine in anhydrous and hydrated states by M.A. Frías; S.B. Díaz; N.M. Ale; A. Ben Altabef; E.A. Disalvo (pp. 1823-1829).

In this paper, the interaction of arbutin with dimyristoylphosphatidylcholine bilayers was studied by FTIR spectrometry. The results show that arbutin interacts in different extents with the phosphate and carbonyl groups of membranes in the gel state, the liquid crystalline state or subjected to osmotic stress. The effect, in the presence of water, on the antisymmetric stretching of the phosphate groups is qualitatively similar to that found with other molecules composed by a glucose moiety such as trehalose and sucrose. However, significant differences were found between these compounds and arbutin in the carbonyl region. Arbutin displaces the PO₂⁻ antisymmetric stretching to lower frequencies in lipids dispersed in water. This indicates strong hydrogen bonding. In contrast, in the solid state, this frequency increases. The effect on the carbonyl groups varies depending on the hydration state of the bilayer, which is achieved by changing the phase state of the bilayer or by osmotic stress. The hydrocarbon region is not affected by arbutin in the excess of water. However, symmetric and antisymmetric stretching of CH₂ and CH₃ are strongly affected in the dry state.

Keywords: Arbutin; Dimyristoylphosphatidylcholine; Fourier Transform infrared spectroscopy; Carbonyl group; Phosphate group; Hydration

Coexistence of two domains in intercellular lipid matrix of stratum corneum by Ichiro Hatta; Noboru Ohta; Katsuaki Inoue; Naoto Yagi (pp. 1830-1836).

The outermost layer of the skin, the stratum corneum (SC), is composed of corneocytes and an intercellular lipid matrix. The matrix acts as both the main barrier and also as the pathway of water, drugs, etc. across the SC. In the mammalian SC, the longitudinal arrangement of the lipid molecules, consisting of long and short lamellar structures with repeat distances of about 13 nm and 6 nm, respectively, has been observed by small-angle X-ray diffraction. In the lateral arrangement of the lipid molecules, hexagonal and orthorhombic hydrocarbon-chain packing has been observed by wide-angle X-ray diffraction. From the systematic study of the temperature dependence of simultaneous small- and wide-angle X-ray diffraction patterns, we demonstrate that the intercellular lipid matrix forms two domains, which consist at room temperature of a long lamellar structure with hexagonal hydrocarbon-chain packing and a short lamellar structure with orthorhombic hydrocarbon-chain packing.

Keywords: Abbreviations; DSC; differential scanning calorimetry; SC; stratum corneum; SAXD; small-angle X-ray diffraction; WAXD; wide-angle X-ray diffractionHexagonal structure; Hydrocarbon chain; Long lamellar; Orthorhombic structure; SAXD; Short lamellar; Skin; WAXD

Modulation of Kir4.2 rectification properties and pHi-sensitive run-down by association with Kir5.1 by Hung D. Lam; Anne-Marie Lemay; M. Martha Briggs; Marco Yung; Ceredwyn E. Hill (pp. 1837-1845).

Inwardly rectifying K⁺ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2–Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2–Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to E_K were essentially time independent for Kir4.2 indicating rapid block by Mg²⁺. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.

Keywords: Inward rectifier; Channel kinetic; Mg; ²⁺; block and unblock; pH sensitivity; Heteromeric channel

Formation of transmembrane ionic channels of primary amphipathic cell-penetrating peptides. Consequences on the mechanism of cell penetration by Sébastien Deshayes; Thomas Plénat; Pierre Charnet; Gilles Divita; Gérard Molle; Frédéric Heitz (pp. 1846-1851).

The ability of three primary amphipathic Cell-Penetrating Peptides (CPPs) CH₃-CO-GALFLGFLGAAGSTMGAWSQPKKKRKV-NH-CH₂-CH₂-SH, CH₃-CO-GALFLAFLAAALS LMGLWSQPKKKRKV-NH-CH₂-CH₂-SH, and CH₃-CO-KETWWETWWTEWSQPKKKRKV-NH-CH₂-CH₂-SH called Pβ, Pα and Pep-1, respectively, to promote pore formation is examined both in Xenopus oocytes and artificial planar lipid bilayers. A good correlation between pore formation and their structural properties, especially their conformational versatility, was established. This work shows that the cell-penetrating peptides Pβ and Pep-1 are able to induce formation of transmembrane pores in artificial bilayers and that these pores are most likely at the basis of their ability to facilitate intracellular delivery of therapeutics. In addition, their behaviour provides some information concerning the positioning of the peptides with respect to the membrane and confirms the role of the membrane potential in the translocation process.

Keywords: Cell-penetrating peptides; Conformation; Membrane uptake; Ionic channel; Amphipathic peptides

Study of calix[4]resorcinarene–dopamine complexation in mixed phospholipid monolayers formed at the air–water interface by P. Vitovi�?; D.P. Nikolelis; T. Hianik (pp. 1852-1861).

We have studied the physical properties of monolayers formed by calix[4]resorcinarene and in mixtures with dipalmitoyl phosphatidylcholine (DPPC) in various molar ratios formed at the air–water interface and at presence of dopamine in water subphase by means of measurements of surface pressure and dipole potential. We showed that both calix[4]resorcinarene as well as its mixture with DPPC form stable monolayers at the water subphase. The presence of dopamine resulted in an increase of the mean molecular area and in a decrease of the compressibility modulus of the monolayers. For mixed monolayers at higher content of calix[4]resorcinarene (>0.2 molar fraction) a deviation from ideal miscibility took place especially for monolayers in a solid state. This can be connected with formation of aggregates of calix[4] resorcinarene. Lowest miscibility and weakest interaction of dopamine with a monolayer was observed for calix[4]resorcinarene molar fraction of 0.33 in the monolayer.

Keywords: Calix[4]resorcinarene; Monolayer; Surface pressure; Dipole potential; Dopamine; Aggregates

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BBA - Biomembranes (v.1758, #11)