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BBA - Biomembranes (v.1720, #1-2)
Thermodynamics of membrane domains by Paulo F.F. Almeida; Antje Pokorny; Anne Hinderliter (pp. 1-13).
The concept of lipid rafts and the intense work toward their characterization in biological membranes has spurred a renewed interest in the understanding of domain formation, particularly in the case of cholesterol-containing membranes. The thermodynamic principles underlying formation of domains, rafts, or cholesterol/phospholipid complexes are reviewed here, along with recent work in model and biological membranes. A major motivation for this review was to present those concepts in a way appropriate for the broad readership that has been drawn to the field. Evidence from a number of different techniques points to the conclusion that lipid–lipid interactions are generally weak; therefore, in most cases, massive phase separations are not to be expected in membranes. On the contrary, small, dynamic lipid domains, possibly stabilized by proteins are the most likely outcome. The results on mixed lipid bilayers are used to discuss recent experiments in biological membranes. The clear indication is that proteins partition preferentially into fluid, disordered lipid domains, which is contrary to their localization in ordered, cholesterol/sphingomyelin rafts inferred from detergent extraction experiments on cell membranes. Globally, the evidence appears most consistent with a membrane model in which the majority of the lipid is in a liquid-ordered phase, with dispersed, small, liquid-disordered domains, where most proteins reside. Co-clustering of proteins and their concentration in some membrane areas may occur because of similar preferences for a particular domain but also because of simultaneous exclusion from other lipid phases. Specialized structures, such as caveolae, which contain high concentrations of cholesterol and caveolin are not necessarily similar to bulk liquid-ordered phase.
Keywords: Abbreviations; Chol; cholesterol; SM; sphingomyelin; PC; phosphatidylcholine; PS; phosphatidylserine; PG; phosphatidylglycerol; DAG; diacylglycerol; POPC; 1-palmitoyl-2-oleoylphosphatidylcholine; POPS; 1-palmitoyl-2-oleoylphosphatidylserine; DOPC; dioleoylphosphatidylcholine; 12:0; lauroyl; 13:0; tridecanoyl; 14:0; myristoyl; 16:0; palmitoyl; 18:0; stearoyl; 14:1; myristoleoyl; 16:1; palmitoleoyl; 18:1; oleoyl; 24:1; nervonoyl; GPI; glycosylphosphatidylinositol; ER; endoplasmic reticulum; DSC; differential scanning calorimetry; T; m; main transition temperature; ω; AB; lipid–lipid, nearest-neighbor interaction free energy; FRET; fluorescence resonance energy transfer; DRM; detergent-resistant membrane; GFP; green fluorescent protein; GUV; giant unilamellar vesicleRaft; Liquid-ordered; Phase separation; Lipid–lipid interaction; Protein–lipid interaction; Cholesterol complex
Lysophospholipid and fatty acid inhibition of pulmonary surfactant: Non-enzymatic models of phospholipase A2 surfactant hydrolysis by R. Duncan Hite; Michael C. Seeds; Randy B. Jacinto; Bonnie L. Grier; B. Moseley Waite; David A. Bass (pp. 14-21).
Secretory A2 phospholipases (sPLA2) hydrolyze surfactant phospholipids cause surfactant dysfunction and are elevated in lung inflammation. Phospholipase-mediated surfactant hydrolysis may disrupt surfactant function by generation of lysophospholipids and free fatty acids and/or depletion of native phospholipids. In this study, we quantitatively assessed multiple mechanisms of sPLA2-mediated surfactant dysfunction using non-enzymatic models including supplementation of surfactants with exogenous lysophospholipids and free fatty acids. Our data demonstrated lysophospholipids at levels ≥10 mol% of total phospholipid (i.e., ≥10% hydrolysis) led to a significant increase in minimum surface tension and increased the time to achieve a normal minimum surface tension. Lysophospholipid inhibition of surfactant function was independent of the lysophospholipid head group or total phospholipid concentration. Free fatty acids (palmitic acid, oleic acid) alone had little effect on minimum surface tension, but did increase the maximum surface tension and the time to achieve normal minimum surface tension. The combined effect of equimolar free fatty acids and lysophospholipids was not different from the effect of lysophospholipids alone for any measurement of surfactant function. Surfactant proteins did not change the percent lysophospholipids required to increase minimum surface tension. As a mechanism that causes surfactant dysfunction, depletion of native phospholipids required much greater change (equivalent to >80% hydrolysis) than generation of lysophospholipids. In summary, generation of lysophospholipids is the principal mechanism of phospholipase-mediated surfactant injury in our non-enzymatic models. These models and findings will assist in understanding more complex in vitro and in vivo studies of phospholipase-mediated surfactant injury.
Keywords: Abbreviations; BAL; bronchoalveolar lavage; sPLA; 2; secretory phospholipase A; 2; ARDS; acute respiratory distress syndrome; PL; phospholipid; PC; phosphatidylcholine; DPPC; dipalmitoylphosphatidylcholine; PG; phosphatidylglycerol; LPC; lysophosphatidylcholine; LPG; lysophosphatidylglycerolSurfactant; Lysophospholipid; Secretory phospholipase A2; Surface tension; Free fatty acid
Adsorption equilibria between liposome membrane formed of phosphatidylcholine and aqueous sodium chloride solution as a function of pH by J. Kotyńska; Z.A. Figaszewski (pp. 22-27).
The effect has been studied of the adsorption of ions (H+, Na+, OH−, Cl−) which are present in solution upon the electric charge of the liposome membrane formed of phosphatidylcholine (PC). The surface charge density of the membrane was determined as a function of pH and electrolyte concentration from electrophoretic mobility measurements. The measurements were carried out by the laser-Doppler microelectrophoresis method. A four-equilibria model has been proposed to describe the phenomena occurring on the membrane surface. The equilibria in which the adsorption of other ions on the liposome membrane surface was involved were assumed to exist beside the equilibria in which the H+ and OH− ions were engaged. The idea was confirmed by mathematical calculations. Association constants of the liposome membrane surface with ions of solution ( KAH, KANa, KBOH, KBCl) were determined. The proposed model has been proved to be correct by comparing the resulting theoretic charge variation curves of the lecithin membrane with the experimental data.
Keywords: Surface charge density; Liposome membrane; Phosphatidylcholine; Adsorption equilibria
Photoisomerisable cholesterol derivatives as photo-trigger of liposomes: Effect of lipid polarity, temperature, incorporation ratio, and cholesterol by Xin-Ming Liu; Bin Yang; Yu-Lu Wang; Jin-Ye Wang (pp. 28-34).
Three cholesterol derivatives containing an azobenzene moiety with different polarities were designed and synthesized (AB lipids1 to3). The effects of structure, temperature and incorporation ratio on liposomes were studied, with the results showing that the polarity in 4-substituent and in some cases, 4′-substituent may be important for their incorporation feasibility and photoisomerizability in liposomes. Liposomes incorporated with AB lipid3 could release multi-pulsatilely upon UV and visible light irradiation both in gel state and liquid crystal state of liposomes. An increase in the incorporation ratio of AB lipid3 enhanced the amount of drug released greatly. Unlike other azobenzene photo-triggers reported, AB lipid3 did not increase the spontaneous release of liposomes. Furthermore, cholesterol suppressed the spontaneous release of liposomes.
Keywords: Liposome; Cholesterol; Azobenzene; Drug delivery system
Length of C-terminus of rCx46 influences oligomerization and hemichannel properties by Carsten Zeilinger; Melanie Steffens; Hans-Albert Kolb (pp. 35-43).
Wild type connexin 46 of rat (wtrCx46), and human connexin 26 (wthCx26) and derivates from rCx46 elongated at the C-terminus by 25 amino acids (rCx46Ct) as well as C-terminal truncated constructs (rCx28.1, rCx45.3) were expressed in frog oocytes of Xenopus laevis. Single oocyte voltage-clamp analysis revealed that connexons or hemichannels of rCx46Ct exhibit similar conducting properties as those of wtrCx46. Insertion of a stop codon at C-terminal domains at position 243 and 409 resulted in a significant reduction in the corresponding hemichannel conductance. This result was also found for wthCx26, the shortest human connexin. Tagged connexin constructs rCx46Ct and hCx26Ct could be expressed in E. coli as monomers. The monomers of rCx46Ct and hCx26Ct were purified and electro-eluted from corresponding SDS gels. Studies of in vitro oligomerization showed that hexamers of these connexins were formed in presence of kinase and specific lipids. Purified rCx46Ct formed some oligomers in vitro if a lipid mixture of POPE/POPG and casein kinase I (CKI) was added, but in the presence of POPC, phosphorylated rCx46Ct monomers preferentially formed hexamers. Purified hCx26Ct formed hexamers in the presence of POPE/POPG. In addition, N-terminal truncated rCx46 (Cx35) oligomerized after phosphorylation. Reconstitution of purified recombinant connexin rCx46Ct in planar lipid bilayers mediated Ca2+-sensitive single channel activity. It is discussed whether the specific C-terminal end of the expressed connexins are responsible for hexamer formation as well as channel opening.
Keywords: rCx46; hCx26 voltage clamp; Heterologous expression; Recombinant membrane protein; Affinity purification; In vitro assembly; Lipid bilayer
Functional expression of the rat organic anion transporter 1 (rOAT1) in Saccharomyces cerevisiae by Suphansa Sawamiphak; Samaisukh Sophasan; Hitoshi Endou; Chuenchit Boonchird (pp. 44-51).
Organic anion transporter 1 (OAT1) is localized in the basolateral membrane of the proximal tubule in the kidney and plays an essential role in eliminating a wide range of organic anions, preventing their toxic effects on the body. Structural and functional studies of the transporter would be greatly assisted by inexpensive and rapid expression in the yeast Saccharomyces cerevisiae. The gene encoding rat OAT1 (rOAT1) contains many yeast non-preferred codons at the N-terminus and so was modified by fusion of the favored codon sequence of a hemagglutinin (HA) epitope preceding the start codon. The modified gene was cloned into several yeast expression plasmids, both integrative and multicopy, with either ADH1 promoter or GAL1 promoter in order to find a suitable expression system. Compared with the wild type gene, a substantial increase in rOAT1 expression was achieved by modification in the translational initiation region, suggesting that the codon chosen at the N-terminus influenced its expression. The highest inducible expression of rOAT1 was obtained under GAL1 promoter in 2 μ plasmid. A large fraction of rOAT1 was glycosylated in yeast, unaffected by growth temperature. The recombinant yeast expressing rOAT1 showed an increase in the uptake of p-aminohippurate (PAH) and this showed a positive correlation with rOAT1 expression level. Location of rOAT1 predominantly in the yeast plasma membrane confirmed correct processing. The importance of glycosylation for rOAT1 targeting was also shown. To our knowledge, this is the first successful functional expression of rOAT1 in the yeast S. cerevisiae.
Keywords: Organic anion transporter 1; Membrane protein; Saccharomyces cerevisiae; Heterologous gene expression
Phenothiazine maleates stimulate MRP1 transport activity in human erythrocytes by Olga Wesołowska; Daniela Mosiądz; Noboru Motohashi; Masami Kawase; Krystyna Michalak (pp. 52-58).
The expression of multidrug resistance-associated protein (MRP1) results in ATP-dependent reduction of drugs' concentration in cancer cells, i.e., multidrug resistance (MDR). Since the majority of projects are concentrated on the search of the new MDR modulators, there are very few reports on drug-induced stimulation of MDR transporters activity. In the present work, by means of functional fluorescence assay we have shown that MRP1-mediated efflux of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) out of human erythrocytes is stimulated by phenothiazine maleates that have been already identified as P-glycoprotein inhibitors. Phenothiazine maleates-induced stimulation of ATP-dependent uptake of 2′,7′-bis-(3-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) into inside-out membrane vesicles prepared from erythrocyte membranes has been also demonstrated. Moreover, it was shown that phenothiazine maleates exerted stimulating effect on ATPase activity measured in erythrocyte membranes. To our best knowledge, this report is the first one demonstrating that compounds able to inhibit transport activity of P-glycoprotein can stimulate MRP1 transporter. We conclude that phenothiazine maleates probably exert their stimulatory effect on MRP1 by direct interaction with the protein at the site different from the substrate binding site.
Keywords: Abbreviations; MRP1 (ABCC1); multidrug resistance-associated protein 1; MDR; multidrug resistance; BCECF; 2′,7′-bis-(3-carboxyethyl)-5-(and-6)-carboxyfluorescein; P-gp; P-glycoprotein; BCPCF; 2′,7′-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein; PhMs; phenothiazine maleates; PBS; phosphate-buffered saline; GSH; glutathioneMultidrug resistance (MDR); Multidrug resistance-associated protein (MRP); Transport stimulation; ATPase activity; Phenothiazine derivative; Erythrocyte
Structures of pulmonary surfactant films adsorbed to an air–liquid interface in vitro by H. Bachofen; U. Gerber; P. Gehr; M. Amrein; S. Schürch (pp. 59-72).
Phospholipid films can be preserved in vitro when adsorbed to a solidifiable hypophase. Suspensions of natural surfactant, lipid extract surfactants, and artificial surfactants were added to a sodium alginate solution and filled into a captive bubble surfactometer (CBS). Surfactant film was formed by adsorption to the bubble of the CBS for functional tests. There were no discernible differences in adsorption, film compressibility or minimal surface tension on quasi-static or dynamic compression for films formed in the presence or absence of alginate in the subphase of the bubble. The hypophase-film complex was solidified by adding calcium ions to the suspension with the alginate. The preparations were stained with osmium tetroxide and uranyl acetate for transmission electron microscopy. The most noteworthy findings are: (1) Surfactants do adsorb to the surface of the bubble and form osmiophilic lining layers. Pure DPPC films could not be visualized. (2) A distinct structure of a particular surfactant film depends on the composition and the concentration of surfactant in the bulk phase, and on whether or not the films are compressed after their formation. The films appear heterogeneous, and frequent vesicular and multi-lamellar film segments are seen associated with the interfacial films. These features are seen already upon film formation by adsorption, but multi-lamellar segments are more frequent after film compression. (3) The rate of film formation, its compressibility, and the minimum surface tension achieved on film compression appear to be related to the film structure formed on adsorption, which in turn is related to the concentration of the surfactant suspension from which the film is formed. The osmiophilic surface associated surfactant material seen is likely important for the surface properties and the mechanical stability of the surfactant film at the air–fluid interface.
Keywords: Lung surfactant; Surfactant film structure; Electron microscopy; Sodium alginate; Surface associated surfactant reservoir
Lipid composition determines interaction of liposome membranes with Pluronic L61 by Artem E. Zhirnov; Tatiana V. Demina; Oxana O. Krylova; Irina D. Grozdova; Nickolay S. Melik-Nubarov (pp. 73-83).
Triblock copolymers of ethylene oxide (EO) and propylene oxide (PO) of EOn/2POmEOn/2 type (Pluronics) demonstrate a variety of biological effects that are mainly due to their interaction with cell membranes. Previously, we have shown that Pluronics can bind to artificial lipid membranes and enhance accumulation of the anti-tumor drug doxorubicin (DOX) inside the pH-gradient liposomes and transmembrane migration (flip-flop) of NBD-labeled phosphatidylethanolamine in the liposomes composed from one component—lecithin. Here, we describe the effects caused by insertion of other natural lipids in lecithin liposomes and the significance of the lipid composition for interaction of Pluronic L61 with the membrane. We used binary liposomes consisting of lecithin and one of the following lipids: cholesterol, phosphatidylethanolamine, ganglioside GM1, sphingomyelin, cardiolipin or phosphatidic acid. The influence of the additives on (1) membrane microviscosity; (2) binding of Pluronic L61; (3) the copolymer effect on lipid flip-flop and membrane permeability towards DOX was studied. The results showed that insertion of sphingomyelin and cardiolipin did not influence membrane microviscosity and effects of Pluronic on the membrane permeability. Addition of phosphatidic acid led to a decrease in microviscosity of the bilayer and provoked its destabilization by the copolymer. On the contrary, cholesterol increased microviscosity of the membrane and decreased binding of Pluronic and its capacity to enhance flip-flop and DOX accumulation. Analogous tendencies were revealed upon incorporation of egg phosphatidylethanolamine or bovine brain ganglioside GM1. Thus, a reverse dependence between the microviscosity of membranes and their sensitivity to Pluronic effects was demonstrated. The described data may be relevant to mechanisms of Pluronic L61 interaction with normal and tumor cells.
Keywords: Liposome; Pluronic; Flip-flop; Doxorubicin; Permeability; Lipid composition
Bilayer thickness and thermal response of dimyristoylphosphatidylcholine unilamellar vesicles containing cholesterol, ergosterol and lanosterol: A small-angle neutron scattering study by Jeremy Pencer; Mu-Ping Nieh; Thad A. Harroun; Susan Krueger; Carl Adams; John Katsaras (pp. 84-91).
Small-angle neutron scattering (SANS) measurements are performed on pure dimyristoyl phosphatidylcholine (DMPC) unilamellar vesicles (ULV) and those containing either 20 or 47 mol% cholesterol, ergosterol or lanosterol. From the SANS data, we were able to determine the influence of these sterols on ULV bilayer thickness and vesicle area expansion coefficients. While these parameters have been determined previously for membranes containing cholesterol, to the best of our knowledge, this is the first time such results have been presented for membranes containing the structurally related sterols, ergosterol and lanosterol. At both molar concentrations and at temperatures ranging from 10 to 45 °C, the addition of the different sterols leads to increases in bilayer thickness, relative to pure DMPC. We observe large differences in the influence of these sterols on the membrane thermal area expansion coefficient. All three sterols, however, produce very similar changes to membrane thickness.
Keywords: Cholesterol; Ergosterol; Lanosterol; Unilamellar vesicle; Hydrophobic thickness; Small-angle neutron scattering; Dimyrisotylphosphatidylcholine (DMPC)
A small HSP, Lo18, interacts with the cell membrane and modulates lipid physical state under heat shock conditions in a lactic acid bacterium by Françoise Coucheney; Laurent Gal; Laurent Beney; Jeannine Lherminier; Patrick Gervais; Jean Guzzo (pp. 92-98).
The small heat shock proteins (sHSP) are characterized by a chaperone activity to prevent irreversible protein denaturation. This study deals with the sHSP Lo18 induced by multiple stresses in Oenococcus oeni, a lactic acid bacterium. Using in situ immunocytochemistry and cellular fractionation experiments, we demonstrated the association of Lo18 with the membrane in O. oeni cells submitted to heat shock. The same result was obtained after exposure of cells to ethanol or benzyl alcohol, agents known to have an influence on membranes. For the different stresses, the protein was located on the periphery of the cell at membrane level and was also found within the cytoplasm. In order to determine if Lo18 could interact with the phospholipids, we used model membranes made of lipids extracted from O. oeni cells. Using fluorescence anisotropy of diphenylhexatriene (DPH) and generalized polarization of Laurdan, we showed that purified Lo18 interacts with these liposomes, and increases the molecular order of the lipid bilayer in these membranes when the temperature reaches 33.8 °C. All these data suggest that Lo18 could be involved in an adaptive response allowing the maintenance of membrane integrity during stress conditions in O. oeni cells.
Keywords: Oenococcus oeni; Small HSP; Immunolocalization; Membrane fluidity; Lipochaperone; Lipid–protein interaction
Effects of pH-induced variations of the charge of the transmembrane α-helical peptide Ac-K2(LA)12K2-amide on the organization and dynamics of the host dimyristoylphosphatidylcholine bilayer membrane by Witold K. Subczynski; Anna Wisniewska; Akihiro Kusumi; Ronald N. McElhaney (pp. 99-109).
The effects of the transmembrane α-helical peptide Ac-K2(LA)12K2-amide ((LA)12) on the phase transition and dynamics of saturated dimyristoylphosphatidylcholine (DMPC) membranes were investigated at different pH using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels. At a peptide-to-DMPC ratio of 1/10, the main phase-transition temperature of the DMPC bilayer is decreased by 4.0 °C when measured at pH 7.0, by 1.6 °C when measured at pH 9.5, and not affected when measured at pH 11.5. This reversible pH effect is due to the subsequent neutralization of the positive charges of lysine side chains at both ends of (LA)12. Apparent p Kas of the lysine side chain amino groups of (LA)12 in DMPC bilayer are 8.6 and ∼10.9, as compared with the p Ka value of 10.5 for these groups when lysine is dissolved in water. Saturation-recovery curves as a function of oxygen concentration using phosphatidylcholine spin labels in DMPC bilayer containing (LA)12 are always mono-exponential when measured at pH 7.0 and 9.5. This observation is consistent with the hypothesis that the lipid exchange rates among the bulk, boundary, and (LA)12-rich regions are faster than 0.5 μs, the electron spin-lattice relaxation time in the presence of molecular oxygen, suggesting that stable oligomers of (LA)12 do not form. Neutralization of one lysine side chain positive charge on each end of the peptide significantly decreases the ordering effect of (LA)12 on the lipid hydrocarbon chains, while its effect on the reorientational motion of terminal groups of lipid hydrocarbon chains is rather moderate. It does not affect the local diffusion-solubility product of oxygen measured in the DMPC-(LA)12 membrane interior.
Keywords: Pulse EPR; Spin labeling; α-Helical transmembrane peptide; Model membrane; Oxygen transport
Stabilization of trypsin by association to plasma membranes: Implications for tryptic cleavage of membrane-bound Na,K-ATPase by Yasser A. Mahmmoud (pp. 110-116).
Tryptic cleavage has been a potential method for studying the structure and mechanism of many membrane transport proteins. Here, we report tight association of trypsin to pig kidney plasma membranes enriched in Na,K-ATPase. Trypsin also associated with protein-free vesicles prepared from plasma membrane lipids. Membrane-associated trypsin was found to be highly resistant to autolysis and insensitive to inhibition by PMSF. Na,K-ATPase substrate ions differentially influenced the level of trypsin membrane association. Thus, NaCl significantly increased trypsin membrane association compared to KCl. The ions seem to exert direct effects on the membrane independent of their effects on protein conformation. Bicarbonate anions, which detach peripheral membrane proteins, efficiently released trypsin from the membrane. Trypsin membrane association was found to enhance the cleavage of the Na,K-ATPase γ-subunit. Comparison between membranes from shark rectal gland and pig kidney showed that trypsin association was significantly higher in the former. This was found to be partly due to the presence of higher cholesterol levels in the membrane. In conclusion, the differential membrane association of trypsin may affect the outcome of proteolytic cleavage of membrane-bound proteins.
Keywords: Abbreviations; E1; enzyme conformation that has high affinity for Na; +; E2; enzyme conformation that has high affinity for K; +; ECL; enhanced chemiluminescence; PBS; phosphate-buffered saline; PMSF; Phenyl methyl sulfonyl fluoride; PVDF; polyvinylidene fluoride; SDS-PAGE; sodium dodecyl sulphate-poly acrylamide gel electrophoresis; Tricine; N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine TCA, trichloroacetic acid; Tween-20; polyoxyethylene sorbitan monolaurateMembrane-bound protein; Trypsin; Membrane; P-type ATPase; Na,K-ATPase
Intrinsic ionic conductances mediate the spontaneous electrical activity of cultured mouse myotubes by Marina Sciancalepore; Ramil Afzalov; Vanessa Buzzin; Mihaela Jurdana; Paola Lorenzon; Fabio Ruzzier (pp. 117-124).
Mouse skeletal myotubes differentiated in vitro exhibited spontaneous contractions associated with electrical activity. The ionic conductances responsible for the origin and modulation of the spontaneous activity were examined using the whole-cell patch-clamp technique and measuring [Ca2+]i transients with the Ca2+ indicator, fura 2-AM. Regular spontaneous activity was characterized by single TTX-sensitive action potentials, followed by transient increases in [Ca2+]i. Since the bath-application of Cd2+ (300 μM) or Ni2+ (50 μM) abolished the cell firing, T-type ( ICa,T) and L-type ( ICa,L) Ca2+ currents were investigated in spontaneously contracting myotubes. The low activation threshold (around −60 mV) and the high density of ICa,T observed in contracting myotubes suggested that ICa,T initiated action potential firing, by bringing cells to the firing threshold. The results also suggested that the activity of ICa,L could sustain the [Ca2+]i transients associated with the action potential, leading to the activation of apamin-sensitive SK-type Ca2+-activated K+ channels and the afterhyperpolarization (AHP) following single spikes. In conclusion, an interplay between voltage-dependent inward (Na+ and Ca2+) and outward (SK) conductances is proposed to mediate the spontaneous pacemaker activity in cultured muscle myotubes during the process of myogenesis.
Keywords: Skeletal muscle; Myotube; Spontaneous firing activity; T-type calcium current; L-type calcium current; SK K; +; current
Oligomerization of the Saccharomyces cerevisiae Na+/H+ antiporter Nha1p: Implications for its antiporter activity by Keiji Mitsui; Hidetomo Yasui; Norihiro Nakamura; Hiroshi Kanazawa (pp. 125-136).
The Na+/H+ antiporter (Nha1p) from the budding yeast Saccharomyces cerevisiae plays an important role in intracellular pH and Na+ homeostasis. Here, we show by co-precipitation of differently tagged Nha1p proteins expressed in the same cell that the yeast Nha1p l forms an oligomer. In vitro cross-linking experiments then revealed that Nha1p-FLAG is present in the membranes as a dimer. Differently tagged Nha1p proteins were also co-precipitated from sec18-1 mutant cells in which ER-to-Golgi traffic is blocked under non-permissive temperatures, suggesting that Nha1p may already dimerize in the ER membrane. When we over-expressed a mutant Nha1p with defective antiporter activity in cells that also express the wild-type Nha1p-EGFP fusion protein, we found impaired cell growth in highly saline conditions, even though the wild-type protein was appropriately expressed and localized correctly. Co-immunoprecipitation assays then showed the inactive Nha1p-FLAG mutant interacted with the wild-type Nha1p-EGFP protein. These results support the notion that Nha1p exists in membranes as a dimer and that the interaction of its monomers is important for its antiporter activity.
Keywords: Abbreviations; Nha1p; Na; +; /H; +; antiporter 1 protein; SDS-PAGE; SDS-polyacrylamide gel electrophoresis; DDM; n; -dodexyl β-; d; -maltoside; DSS; disuccinimidyl suberate; o; -PDM; N,N; ′-(; o; -phenylene) dimaleimmide; WT; wild-typeYeast Na; +; /H; +; antiporter; Salinity resistance; Oligomerization; Co-precipitation; In vitro cross-linking; Dominant negative effect
Effect of triorganotin compounds on membrane permeability by Antonio Ortiz; José A. Teruel; Francisco J. Aranda (pp. 137-142).
Organotin compounds are widely distributed toxicants. They are membrane-active molecules with broad biological toxicity. In this contribution, we study the effect of triorganotin compounds on membrane permeability using phospholipid model membranes and human erythrocytes. Tribultyltin and triphenyltin are able to induce the release of entrapped carboxyfluorescein from large unilamellar vesicles. The rate of release is similar for phosphatidylcholine and phosphatidylserine systems and the presence of equimolar cholesterol decreases the rate of the process. Release of carboxyfluorescein is almost abolished when a non-diffusible anion like gluconate is present in the external medium, and it is restored by addition of chloride. Tributyltin is able to cause hemolysis of human erythrocytes in a dose-dependent manner. Relative kinetics determination shows that potassium leakage occurs simultaneously with hemoglobin release. Hemolysis is reduced when erythrocytes are suspended in a gluconate medium. These results indicate that triorganotin compounds are able to transport organic anions like carboxyfluorescein across phospholipids bilayers by exchange diffusion with chloride and suggest that anion exchange through erythrocyte membrane could be related to the process of hemolysis.
Keywords: Abbreviations; CF; carboxyfluorescein; CHOL; cholesterol; FITC; fluorescein isothiocyanate; PC; phosphatidylcholine; PS; phosphatidylserine; TBT; tri-; n; -butyltin chloride; TPT; tri-; n; -phenyltin chlorideOrganotin compound; Carboxyfluorescein release; Model membrane; Hemolysis