Check out our New Publishers' Select for Free Articles

BBA - Biomembranes (v.1758, #10)

Editorial Board (pp. ii).

To see or not to see: Lateral organization of biological membranes and fluorescence microscopy by Luis A. Bagatolli / (pp. 1541-1556).

In the last few years several experimental strategies based on epi-, confocal and two photon excitation fluorescence microscopy techniques have been employed to study the lateral structure of membranes using giant vesicles as model systems. This review article discusses the methodological aspects of the aforementioned experimental approaches, particularly stressing the information obtained by the use of UV excited fluorescent probes using two-photon excitation fluorescence microscopy. Additionally, the advantages of utilizing visual information, to correlate the lateral structure of compositionally simple membranes with complex situations, i.e., biological membranes, will be addressed.

Keywords: LAURDAN; Cholesterol; Two photon excitation fluorescence microscopy; Giant vesicle; Lipid phase coexistence; Raft

Extra domains in secondary transport carriers and channel proteins by Ravi D. Barabote; Dorjee G. Tamang; Shannon N. Abeywardena; Neda S. Fallah; Jeffrey Yu Chung Fu; Jeffrey K. Lio; Pegah Mirhosseini; Ronnie Pezeshk; Sheila Podell; Marnae L. Salampessy; Mark D. Thever; Milton H. Saier Jr. (pp. 1557-1579).

“Extra�? domains in members of the families of secondary transport carrier and channel proteins provide secondary functions that expand, amplify or restrict the functional nature of these proteins. Domains in secondary carriers include TrkA and SPX domains in DASS family members, DedA domains in TRAP-T family members (both of the IT superfamily), Kazal-2 and PDZ domains in OAT family members (of the MF superfamily), USP, IIA^Fru and TrkA domains in ABT family members (of the APC superfamily), ricin domains in OST family members, and TrkA domains in AAE family members. Some transporters contain highly hydrophilic domains consisting of multiple repeat units that can also be found in proteins of dissimilar function. Similarly, transmembrane α-helical channel-forming proteins contain unique, conserved, hydrophilic domains, most of which are not found in carriers. In some cases the functions of these domains are known. They may be ligand binding domains, phosphorylation domains, signal transduction domains, protein/protein interaction domains or complex carbohydrate-binding domains. These domains mediate regulation, subunit interactions, or subcellular targeting. Phylogenetic analyses show that while some of these domains are restricted to closely related proteins derived from specific organismal types, others are nearly ubiquitous within a particular family of transporters and occur in a tremendous diversity of organisms. The former probably became associated with the transporters late in the evolutionary process; the latter probably became associated with the carriers much earlier. These domains can be located at either end of the transporter or in a central region, depending on the domain and transporter family. These studies provide useful information about the evolution of extra domains in channels and secondary carriers and provide novel clues concerning function.

Keywords: Transport; Secondary carriers; Evolution; Protein domains; DedA; TrkA; SPX; Kazal-2; PDZ; USP; IIA; ^Fru

Different modes in antibiotic action of tritrpticin analogs, cathelicidin-derived Trp-rich and Pro/Arg-rich peptides by Sung-Tae Yang; Song Yub Shin; Kyung-Soo Hahm; Jae Il Kim (pp. 1580-1586).

The cathelicidin-derived antimicrobial tritrpticin could be classified as either Trp-rich or Pro/Arg-rich peptide. We recently found that the sequence modification of tritrpticin focused on Trp and Pro residues led to considerable change in structure and antimicrobial potency and selectivity, but their mechanisms of microbial killing action were still unclear. Here, to better understand the bactericidal mechanisms of tritrpticin and its two analogs, TPA and TWF, we studied their effect on the viability of Gram-positive S. aureus and Gram-negative E. coli in relation to their membrane depolarization. Although TWF more effectively inhibited growth of S. aureus and E. coli than TPA, only a 30 min exposure to TPA was sufficient to kill both bacteria and TWF required a lag period of about 3–6 h for bactericidal activity. Their different bactericidal kinetics was associated with membrane permeabilization, i.e., TWF showed negligible ability to depolarize the cytoplasmic membrane potential of target cell membrane, whereas we observed significant membrane depolarization for TPA. In addition, while TPA caused rapid and large dye leakage from negatively charged model vesicles, TWF showed very little membrane-disrupting activity. Interestingly, we have looked for a synergism among the three peptides against E. coli, supporting that they are working with different modes of action. Collectively, our results suggest that TPA disrupts the ion gradients across the membrane, causing depolarization and a loss of microbial viability. By contrast, TWF more likely translocates across the cytoplasmic membrane without depolarization and then acts against one or more intracellular targets. Tritrpticin exhibits intermediate properties and appears to act via membrane depolarization coupled to secondary intracellular targeting.

Keywords: Abbreviations; PP-II; polyproline type II; CD; circular dichroism; MIC; minimal inhibitory concentration; FIC; fractional inhibitory concentration; CFU; colony forming units; diSC; ₃; (5); 3,3′-dipropylthiacarbocyanine; LPS; lipopolysaccharide; POPC; 1-palmitoyl-2-oleoyl-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-phosphatidylglycerol; LUVs; large unilamellar vesicles; SDS; sodium dodecyl sulfateCathelicidin; Tritrpticin; Membrane depolarization; Intracellular target; Synergy

Verapamil, a Ca²⁺ channel inhibitor acts as a local anesthetic and induces the sigma E dependent extra-cytoplasmic stress response in E. coli by C.L. Andersen; I.B. Holland; A. Jacq (pp. 1587-1595).

Verapamil is used clinically as a Ca²⁺ channel inhibitor for the treatment of various disorders such as angina, hypertension and cardiac arrhythmia. Here we study the effect of verapamil on the bacterium Escherichia coli. The drug was shown to inhibit cell division at growth sub inhibitory concentrations, independently of the SOS response. We show verapamil is a membrane active drug, with similar effects to dibucaine, a local anesthetic. Thus, both verapamil and dibucaine abolish the proton motive force and decrease the intracellular ATP concentration. This is accompanied by induction of degP expression, as a result of the activation of the RpoE (SigmaE) extra-cytoplasmic stress response, and activation of the psp operon. Such effects of verapamil, as a membrane active compound, could explain its general toxicity in eukaryotic cells.

Keywords: E. coli; Verapamil; Dibucaine; DegP; Membrane potential; PspA

Solvent-dependent structure of two tryptophan-rich antimicrobial peptides and their analogs studied by FTIR and CD spectroscopy by Valery V. Andrushchenko; Hans J. Vogel; Elmar J. Prenner (pp. 1596-1608).

Structural changes for a series of antimicrobial peptides in various solvents were investigated by a combined approach of FTIR and CD spectroscopy. The well-characterized and potent antimicrobial peptides indolicidin and tritrpticin were studied along with several analogs of tritrpticin, including Tritrp1 (amidated analog of tritrpticin), Tritrp2 (analog of Tritrp1 with Arg→Lys substitutions), Tritrp3 (analog of Tritrp1 with Pro→Ala substitutions) and Tritrp4 (analog of Tritrp1 with Trp→Tyr substitutions). All peptides were studied in aqueous buffer, ethanol and in the presence of dodecylphosphocholine (DPC) micelles. It was shown that tritrpticin and its analogs preferentially adopt turn structures in all solvents studied. The turn structures formed by the tritrpticin analogs bound to DPC micelles are more compact and more conformationally restricted compared to indolicidin. While several peptides showed a slight propensity for an α-helical conformation in ethanol, this trend was only strong for Tritrp3, which also adopted a largely α-helical structure with DPC micelles. Tritrp3 also demonstrated along with Tritrp1 the highest ability to interact with DPC micelles, while Tritrp2 and Tritrp4 showed the weakest interaction.

Keywords: Abbreviations; Indo; indolicidin; Trp; tritrpticin; FTIR; Fourier transform infrared spectroscopy; ATR; attenuated total reflectance; CD; circular dichroism; TFE; trifluoroethanol; DMSO; dimethyl sulphoxide; DPC; dodecylphosphocholine; TFA; trifluoroacetateAntimicrobial peptide; CD spectroscopy; FTIR spectroscopy; Indolicidin; Tritrpticin

Effect of humidity on the stability of lung surfactant films adsorbed at air–water interfaces by Yi Y. Zuo; Edgar Acosta; Zdenka Policova; Peter N. Cox; Michael L. Hair; A. Wilhelm Neumann (pp. 1609-1620).

The effect of humidity on the film stability of Bovine Lipid Extract Surfactant (BLES) is studied using the captive bubble method. It is found that adsorbed BLES films show distinctly different stability patterns at two extreme relative humidities (RHs), i.e., bubbles formed by ambient air and by air prehumidified to 100% RH at 37 °C. The differences are illustrated by the ability to maintain low surface tensions at various compression ratios, the behavior of bubble clicks, and film compressibility. These results suggest that 100% RH at 37 °C tends to destabilize the BLES films. In turn, the experimental results indicate that the rapidly adsorbed BLES film on a captive bubble presents a barrier to water transport that retards full humidification of the bubble when ambient air is used for bubble formation. These findings necessitate careful evaluation and maintenance of environmental humidity for all in vitro assessment of lung surfactants. It is also found that the stability of adsorbed bovine natural lung surfactant (NLS) films is not as sensitive as BLES films to high humidity. This may indicate a physiological function of SP-A and/or cholesterol, which are absent in BLES, in maintaining the extraordinary film stability in vivo.

Keywords: Lung surfactant; Film stability; Humidity; Surface tension; Captive bubble; Axisymmetric drop shape analysis (ADSA)

Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-B_Δc). Structure and interaction with lipid interfaces by Alicia G. Serrano; Elisa J. Cabré; José M. Oviedo; Antonio Cruz; Beatriz González; Alicia Palacios; Pilar Estrada; Jesús Pérez-Gil (pp. 1621-1632).

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid–protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-B_ΔC, a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-B_ΔC has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-B_ΔC indicated that the protein is properly folded, showing around 32% α-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-B_ΔC that were indicative of lipid–protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air–liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-B_ΔC in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.

Keywords: Abbreviations; CD; circular dichroism; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; IPTG; Isopropyl-β-; d; -thiogalactopyranoside; MLV; Multilamellar vesicles; PBS; phosphate buffer saline; PMSF; phenylmethanesulphonyl fluoride; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-; rac; -glycerol; SUV; Small unilamellar vesicles; SAPLIP; Saposin-like protein; TB; Terrific Broth culture medium; TEV; Tobacco etch virusLung surfactant; Protein folding; Inclusion body; Saposin-like protein; Lipid–protein interaction; Amphipathic protein

Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient by Andreas Fritze; Felicitas Hens; Andrea Kimpfler; Rolf Schubert; Regine Peschka-Süss (pp. 1633-1640).

This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependant drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 °C or 30% at 37 °C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.

Keywords: Doxorubicin; EPC/cholesterol liposome; Diammonium hydrogen phosphate; Cryo-electron microscopy; Remote loading; pH-triggered release

Role of kinases and G-proteins in the hyposmotic stimulation of cardiac I_Ks by Sergey Missan; Paul Linsdell; Terence F. McDonald (pp. 1641-1652).

Exposure of cardiac myocytes to hyposmotic solution stimulates slowly-activating delayed-rectifying K⁺ current (I_Ks) via unknown mechanisms. In the present study, I_Ks was measured in guinea-pig ventricular myocytes that were pretreated with modulators of cell signaling processes, and then exposed to hyposmotic solution. Pretreatment with compounds that (i) inhibit serine/threonine kinase activity (10–100 μM H89; 200 μM H8; 50 μM H7; 1 μM bisindolylmaleimide I; 10 μM LY294002; 50 μM PD98059), (ii) stimulate serine/threonine kinase activity (1–5 μM forskolin; 0.1 μM phorbol-12-myristate-13-acetate; 10 μM acetylcholine; 0.1 μM angiotensin II; 20 μM ATP), (iii) suppress G-protein activation (10 mM GDPβS), or (iv) disrupt the cytoskeleton (10 μM cytochalasin D), had little effect on the stimulation of I_Ks by hyposmotic solution. In marked contrast, pretreatment with tyrosine kinase inhibitor tyrphostin A25 (20 μM) strongly attenuated both the hyposmotic stimulation of I_Ks in myocytes and the hyposmotic stimulation of current in BHK cells co-expressing Ks channel subunits KCNQ1 and KCNE1. Since attenuation of hyposmotic stimulation was not observed in myocytes and cells pretreated with inactive tyrphostin A1, we conclude that TK has an important role in the response of cardiac Ks channels to hyposmotic solution.

Keywords: Guinea-pig ventricular myocytes; Ks channels hyposmotic solution; Kinase inhibitors; GDPβS; Cytochalasin D

Physicochemical characterization of a peptide deriving from the glycoprotein gp36 of the feline immunodeficiency virus and its lipoylated analogue in micellar systems by Cinzia Esposito; Gerardino D'Errico; Maria Rosaria Armenante; Simone Giannecchini; Mauro Bendinelli; Paolo Rovero; Anna M. D'Ursi (pp. 1653-1661).

P59 is the Trp-rich 20-mer peptide (⁷⁶⁷L–G⁷⁸⁶), partial sequence of the membrane-proximal external region (MPER) of the FIV gp36. It has potent antiviral activity, possibly due to a mechanism that inhibits the fusion of the virus with the cell membranes. In the hypothesis that a lipophilic tail could enhance the adhesion of P59 to the membrane so improving its antiviral activity, we synthesized its lipoylated analogue lipo-P59. Fluorescence, CD and NMR investigations in membrane mimicking environments (such as SDS and DPC micelles) were aimed to assess the potential of the lipo-P59 lipophilic tail to affect the biophysical and conformational behaviour of the peptide. In vitro inhibitory assays using lymphoid cell cultures to check the antiviral activity of peptides were also performed. The data show that the biophysical properties and the conformational preferences of the peptides are not dramatically affected by the hydrophobic tail, suggesting that the lipopeptide is capable of preserving all the biophysical peculiarities. Similarly, antiviral experimental data show that the membrane-anchored lipo-P59 peptide is also effective in inhibiting virus replication. Moreover, the lipophilic tail allows P59 to preserve its antiviral activity even in conditions in which the non lipoylated peptide is devoid of activity. In accordance with the unusual high Trp presence, the peptides confirm the preference to be positioned on the membrane interface. Furthermore, the data point out a peculiarity of interaction of the peptides with SDS as compared with DPC.

Keywords: FIV; Lipopeptide; Conformational analysis; Micelle interface

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers by Katja Rebolj; Nataša Poklar Ulrih; Peter Ma�?ek; Kristina Sep�?ić (pp. 1662-1670).

Ostreolysin, a cytolytic protein from the edible oyster mushroom ( Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins.

Keywords: Abbreviations; 7-DHC; 7-dehydrocholesterol; CDC; cholesterol-dependent cytolysin; Ch-Ac; cholesteryl acetate; Chol; cholesterol; Ch-one; 5-cholesten-3-one; DOPC; dioleoylphosphatidylcholine; DPH; 1,6-diphenyl-1,3,5-hexatriene; DPPC; dipalmitoylphosphatidylcholine; DRM; detergent-resistant membrane; DSC; differential scanning calorimetry; EDTA; ethylenediaminetetraacetic acid; Ergo; ergosterol; Lano; lanosterol; l; _d; liquid disordered domain; l; _o; lipid ordered domain; MLV; multilamellar vesicles; PC; phosphatidylcholine; POPC; palmitoyloleoylphosphatidylcholine; RU; resonance unit; Sito; β-sitosterol; SPR; surface plasmon resonance; Stig; stigmasterol; SV; sonicated vesicles; Oly; ostreolysin; SM; sphingomyelin; TRIS; (hydroxymethyl)aminomethane; VCC; Vibrio cholerae; cytolysinCholesterol; Cholesterol-dependent cytolysin; Liquid ordered phase; Pleurotus ostreatus; Pore forming protein; Sterol

A kinetic study of Rhodamine123 pumping by P-glycoprotein by Yulin Wang; DaCheng Hao; Wilfred D. Stein; Ling Yang (pp. 1671-1676).

The MDR1 P-glycoprotein (P-gp) actively extrudes a wide variety of structurally diverse cytotoxic compounds out of the cell, is widely expressed in the epithelial cells of kidney, liver and intestine, and in the endothelial cells of brain and placenta, and plays an important role in drug resistance. We measured the accumulation of Rhodamine 123 (Rho123), a substrate of P-gp, into a drug sensitive and a drug resistant strain of the human leukemia cell line K562, as function of Rho123 concentration. With the aid of a mathematical transformation, we used the accumulation of Rho123 into the sensitive cells as a surrogate measure for the internal concentration of the probe in the resistant cells, and were thus able to measure the kinetic parameters of drug efflux pumping by P-gp. Drug pumping was half-saturated at an external Rho123 concentration of 7.2E–06±1.1E–06 M, and displayed a co-operative behaviour with a Hill number of 1.94±0.32. Verapamil could be shown to inhibit Rho123 efflux uncompetitively.

Keywords: Abbreviations; P-gp; P-glycoprotein; ABC; ATP-binding cassette; TM; transmembrane; NBD; nucleotide-binding domain; Rho123; Rhodanmine123Pglycoprotein; Rhodamine 123; Kinetics; Resistance; Fluorescence; Quenching

Lipoplexes formed from sugar-based gemini surfactants undergo a lamellar-to-micellar phase transition at acidic pH. Evidence for a non-inverted membrane-destabilizing hexagonal phase of lipoplexes by Luc Wasungu; Marc C.A. Stuart; Marco Scarzello; Jan B.F.N. Engberts; Dick Hoekstra (pp. 1677-1684).

The present study aims at a better understanding of the mechanism of transfection mediated by two sugar-based gemini surfactants GS1 and GS2. Previously, these gemini surfactants have been shown to be efficient gene vectors for transfection both in vitro and in vivo. Here, using Nile Red, a solvatochromic fluorescent probe, we investigated the phase behavior of these gemini surfactants in complexes with plasmid DNA, so-called lipoplexes. We found that these lipoplexes undergo a lamellar-to-non-inverted micellar phase transition upon decreasing the pH from neutral to mildly acidic. This normal (non-inverted) phase at acidic pH is confirmed by the colloidal stability of the lipoplexes as shown by turbidity measurements. We therefore propose a normal hexagonal phase, H_I, for the gemini surfactant lipoplexes at acidic endosomal pH. Thus, we suggest that besides an inverted hexagonal (H_II) phase as reported for several transfection-potent cationic lipid systems, another type of non-inverted non-bilayer structure, different from H_II, may destabilize the endosomal membrane, necessary for cytosolic DNA delivery and ultimately, cellular transfection.

Keywords: Abbreviations; DNA; deoxyribonucleic acid; lipoplexes; complexes of DNA with cationic lipids; DOPE or PE; 1,2-dioleoyl-; sn; -glycero-3-phosphoethanolamine; PC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; PS; 1,2-dioleoyl-; sn; -glycero-3-[phospho-; l; -serine]; DOTAP; N-; [1-(2,3-dioleyl)propyl]-; N; ,; N; ,; N; -trimethylammonim chloride; CTAB; cetyltrimethylammonium bromide; EDLPC; ethyldilauroylphosphatidylcholine; EDOPC; ethyldioleoylphosphatidylcholine; SHN; sodium 3-hydroxy-2-naphthoate; SAINT-2; N; -methyl-4-(dioleyl)methylpyridinium chloride; λ; _max; emission; maximum emission wavelength; SAXS; small angle X-ray scattering; HEPES; N; -2-hydroxyethylpiperazine-; N'; -2-ethanesulfonic acid; MES; 2-[; N; -morpholino]ethanesulfonic acid; HBS solution; HEPES buffered saline solution; Lα; lamellar phase; L; _I; micellar phase; H; _II; inverted hexagonal phase; H; _I; normal hexagonal phaseCationic lipid; Gemini surfactant; pH sensitive; Transfection mechanism; Hexagonal phase; Nile Red fluorescence; Endosomal release

Grafting of polylysine with polyethylenoxide prevents demixing of O-pyromellitylgramicidin in lipid membranes by A.A. Pashkovskaya; E.P. Lukashev; P.E. Antonov; O.A. Finogenova; Yu.A. Ermakov; N.S. Melik-Nubarov; Yu.N. Antonenko (pp. 1685-1695).

Both natural and synthetic polycations can induce demixing of negatively charged components in artificial and possibly in natural membranes. This process can result in formation of clusters (binding of several components to a polycation chain) and/or domains (aggregation of clusters and formation of a separate phase enriched in some particular component). In order to distinguish between these two phenomena, a model lipid membrane system containing ion channels, formed by a negatively charged peptide, O-pyromellitylgramicidin, and polycations of different structures was used. Microelectrophoresis of liposomes, changes in boundary potential of planar bilayers, the shape of compression curves and potentials of lipid and lipid/peptide monolayers were used to monitor the electrostatic factors in polymer adsorption to the membrane and peptide–polymer interactions. The synthesized PEO-grafted polylysine, PLL–PEO20000, did not induce peptide demixing monitored by stabilization of the gramicidin channels, in contrast to parent polylysine (PLL). Both polymers were shown to bind effectively to negatively charged liposomes and lipid monolayers, suggesting that the ineffectiveness of PLL–PEO20000 was not due to reduction of its binding. It was hypothesized that PLL–PEO20000 could not induce domain formation due to steric hindrance of long PEO chains preventing lateral fusion of clusters. Another copolymer, PLL–PEO4000, having four PEO chains of 4000 Da, exhibited intermediate effect between PLL and PLL–PEO20000, which shows the importance of the copolymer architecture for the effect on the lateral distribution of OPg channels. The model system can be relevant to regulation of lateral organization of ion channels and other components in natural membrane systems.

Keywords: Abbreviations; BLM; bilayer lipid membrane; OPg; O-pyromellitylgramicidin; gA; gramicidin A; DPhPC; diphytanoylphosphatidylcholine; DPPC; dipalmitoylphosphatidylcoline; CL; cardiolipin; PLL; Poly-; l; -Lysine; PEO; polyethylene oxide; PLL–PEO4000; polyethylene oxide-grafted polylysine (PEO M.W. 4,000); PLL–PEO20000; polyethylene oxide-grafted polylysine (PEO M.W. 20,000); AlPcS; ₃; Aluminium trisulfophthalocyanine; EL; egg yolk lecithinIon channel; Domain; Cluster; Polymer; Phospholipid membrane

Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells by Beatrice Milon; Qin Wu; Jing Zou; Leslie C. Costello; Renty B. Franklin (pp. 1696-1701).

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H₁₅₈WHD₁₆₁. To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter as has been reported for the Zrt1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. The results support an intermolecular exchange mechanism of zinc transport.

Keywords: Zinc transporter; hZip1; Zinc transport mechanism; SLC39A1

Sections

Personal tools

BBA - Biomembranes (v.1758, #10)

Sections

Personal tools

Local resources

BBA - Biomembranes (v.1758, #10)