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BBA - Biomembranes (v.1798, #5)
A comparative study on the interactions of SMAP-29 with lipid monolayers by Frances Neville; Andrey Ivankin; Oleg Konovalov; David Gidalevitz (pp. 851-860).
This work investigates the discrimination of lipid monolayers by the ovine antimicrobial peptide SMAP-29 and compares it to that of the human LL-37 peptide. Fluid phospholipid monolayers were formed in a Langmuir trough and subsequently studied with the X-ray scattering techniques of X-ray reflectivity and grazing incidence X-ray diffraction. Any changes in the phospholipid structure after injection of peptide under the monolayer were considered to be due to interactions between the peptides and lipids. The data show that SMAP-29 discriminates against negatively charged phospholipids in a similar way to LL-37. However, it is even more interesting to note that despite a higher concentration of SMAP-29 near the monolayer, ensured by its greater charge as compared to LL-37, the amount of SMAP-29 needed to observe monolayer disruption was around three and a half times the number of molecules of LL-37 used to see similar changes with the same system. This result suggests that the structure, amino acid sequence or size of the peptide may well be as important as electrical charge and therefore gives many implications for the further study of antimicrobial peptides with regards to novel drug design and development.
Keywords: Abbreviations; SMAP-29; sheep myeloid antimicrobial peptide-29; GIXD; grazing incidence X-ray diffraction; XR; X-ray reflectivity; ESRF; European Synchrotron Research Facility; DPPG; dipalmitoylphosphatidylglycerol; DPPC; dipalmitoylphosphatidylcholinePeptide–lipid interactions; Antimicrobial peptide; Langmuir monolayer; X-ray scattering
A comparative study on the interactions of SMAP-29 with lipid monolayers by Frances Neville; Andrey Ivankin; Oleg Konovalov; David Gidalevitz (pp. 851-860).
This work investigates the discrimination of lipid monolayers by the ovine antimicrobial peptide SMAP-29 and compares it to that of the human LL-37 peptide. Fluid phospholipid monolayers were formed in a Langmuir trough and subsequently studied with the X-ray scattering techniques of X-ray reflectivity and grazing incidence X-ray diffraction. Any changes in the phospholipid structure after injection of peptide under the monolayer were considered to be due to interactions between the peptides and lipids. The data show that SMAP-29 discriminates against negatively charged phospholipids in a similar way to LL-37. However, it is even more interesting to note that despite a higher concentration of SMAP-29 near the monolayer, ensured by its greater charge as compared to LL-37, the amount of SMAP-29 needed to observe monolayer disruption was around three and a half times the number of molecules of LL-37 used to see similar changes with the same system. This result suggests that the structure, amino acid sequence or size of the peptide may well be as important as electrical charge and therefore gives many implications for the further study of antimicrobial peptides with regards to novel drug design and development.
Keywords: Abbreviations; SMAP-29; sheep myeloid antimicrobial peptide-29; GIXD; grazing incidence X-ray diffraction; XR; X-ray reflectivity; ESRF; European Synchrotron Research Facility; DPPG; dipalmitoylphosphatidylglycerol; DPPC; dipalmitoylphosphatidylcholinePeptide–lipid interactions; Antimicrobial peptide; Langmuir monolayer; X-ray scattering
Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton by Karena L. Waller; Lisa M. Stubberfield; Valentina Dubljevic; Donna W. Buckingham; Narla Mohandas; Ross L. Coppel; Brian M. Cooke (pp. 861-871).
Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin ( Kd=0.60µM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.
Keywords: Abbreviations; RBC; red blood cell; IRBC; infected red blood cell; Pf332 or Ag332; Plasmodium falciparum; Antigen 332; K; d; half saturation concentration; 4.1R; red blood cell protein 4.1; GPC and GPD; glycophorins C and D, respectively; PfEMP1; Plasmodium falciparum; erythrocyte membrane protein 1; KAHRP; knob-associated histidine rich protein; MESA; mature parasite-infected erythrocyte surface antigen; RESA; ring-infected erythrocyte surface antigen; PfEMP3; Plasmodium falciparum; erythrocyte membrane protein 3; PV; parasitophorous vacuole; PEXEL; Plasmodium; export element; VTS; vacuolar transport signal; PVM; parasitophorous vacuolar membrane; MCs; Maurer's clefts; IOV and pIOV; inside-out vesicle prepared from normal RBCs or parasite-infected RBCs; PCR; polymerase chain reaction; MBP; maltose-binding protein; PBS; phosphate-buffered saline; IB; IOV incubation buffer; BSA; bovine serum albumin; PVDF; Polyscreen; ®; polyvinylidene difluoride; TX100; Triton X-100; DBL; Duffy binding-like domain Plasmodium falciparum; Malaria; Pf332; Actin; Red blood cell; Membrane skeleton
Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton by Karena L. Waller; Lisa M. Stubberfield; Valentina Dubljevic; Donna W. Buckingham; Narla Mohandas; Ross L. Coppel; Brian M. Cooke (pp. 861-871).
Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin ( Kd=0.60µM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.
Keywords: Abbreviations; RBC; red blood cell; IRBC; infected red blood cell; Pf332 or Ag332; Plasmodium falciparum; Antigen 332; K; d; half saturation concentration; 4.1R; red blood cell protein 4.1; GPC and GPD; glycophorins C and D, respectively; PfEMP1; Plasmodium falciparum; erythrocyte membrane protein 1; KAHRP; knob-associated histidine rich protein; MESA; mature parasite-infected erythrocyte surface antigen; RESA; ring-infected erythrocyte surface antigen; PfEMP3; Plasmodium falciparum; erythrocyte membrane protein 3; PV; parasitophorous vacuole; PEXEL; Plasmodium; export element; VTS; vacuolar transport signal; PVM; parasitophorous vacuolar membrane; MCs; Maurer's clefts; IOV and pIOV; inside-out vesicle prepared from normal RBCs or parasite-infected RBCs; PCR; polymerase chain reaction; MBP; maltose-binding protein; PBS; phosphate-buffered saline; IB; IOV incubation buffer; BSA; bovine serum albumin; PVDF; Polyscreen; ®; polyvinylidene difluoride; TX100; Triton X-100; DBL; Duffy binding-like domain Plasmodium falciparum; Malaria; Pf332; Actin; Red blood cell; Membrane skeleton
Structure and phase behavior of O-stearoylethanolamine: A combined calorimetric, spectroscopic and X-ray diffraction study by Pradip K. Tarafdar; Musti J. Swamy (pp. 872-881).
Recent studies show that O-acylethanolamines (OAEs), structural isomers of the putative stress-fighting lipids, namely N-acylethanolamines (NAEs), can be derived from NAEs and are present in biological membranes under physiological conditions. In view of this, we have synthesized O-stearoylethanolamine (OSEA) as a representative OAE and investigated its phase behavior and crystal structure. The thermotropic phase transitions of OSEA dispersed in water and in 150mM NaCl were characterized using calorimetric, spectroscopic, turbidimetric and X-ray diffraction studies. These studies have revealed that when dispersed in water OSEA undergoes a cooperative phase transition centered at 53.8°C from an ordered gel phase to a micellar structure whereas in presence of 150mM NaCl the transition temperature increases to 55.8°C and most likely the bilayer structure is retained above the phase transition. O-Stearoylethanolamine crystallized in the orthorhombic space group P212121 with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that OSEA molecules adopt a linear structure with all-trans conformation in the acyl chain region. The molecules are organized in a tail-to-tail fashion, similar to the arrangement in a bilayer membrane. These studies are relevant to understanding the role of salt on the phase properties of this new class of lipids.
Keywords: Abbreviations; OAEs; O; -acylethanolamines; NAEs; N; -acylethanolamines; OSEA; O; -stearoylethanolamine; NAPE; N; -acylphosphatidylethanolamine; NPEA; N; -palmitoylethanolamine; NMEA; N; -myristoylethanolamine; NSEA; N; -stearoylethanolamine; DMAP; 4-dimethylaminopyridine; DCC; N, N′; -dicyclohexylcarbodiimide; DSC; differential scanning calorimetry; T; t; transition temperature; Δ; H; t; enthalpy of transition; Δ; S; t; entropy of transition; T; 1/2; the width at half maximum of the transition; ESR; electron spin resonance; 2A; max; outer hyperfine splitting; LAXS; low-angle X-ray scattering; P; packing parameterDifferential scanning calorimetry; O; -acylethanolamine; Micelle; Hydrocarbon chain tilt; Outer hyperfine splitting
Structure and phase behavior of O-stearoylethanolamine: A combined calorimetric, spectroscopic and X-ray diffraction study by Pradip K. Tarafdar; Musti J. Swamy (pp. 872-881).
Recent studies show that O-acylethanolamines (OAEs), structural isomers of the putative stress-fighting lipids, namely N-acylethanolamines (NAEs), can be derived from NAEs and are present in biological membranes under physiological conditions. In view of this, we have synthesized O-stearoylethanolamine (OSEA) as a representative OAE and investigated its phase behavior and crystal structure. The thermotropic phase transitions of OSEA dispersed in water and in 150mM NaCl were characterized using calorimetric, spectroscopic, turbidimetric and X-ray diffraction studies. These studies have revealed that when dispersed in water OSEA undergoes a cooperative phase transition centered at 53.8°C from an ordered gel phase to a micellar structure whereas in presence of 150mM NaCl the transition temperature increases to 55.8°C and most likely the bilayer structure is retained above the phase transition. O-Stearoylethanolamine crystallized in the orthorhombic space group P212121 with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that OSEA molecules adopt a linear structure with all-trans conformation in the acyl chain region. The molecules are organized in a tail-to-tail fashion, similar to the arrangement in a bilayer membrane. These studies are relevant to understanding the role of salt on the phase properties of this new class of lipids.
Keywords: Abbreviations; OAEs; O; -acylethanolamines; NAEs; N; -acylethanolamines; OSEA; O; -stearoylethanolamine; NAPE; N; -acylphosphatidylethanolamine; NPEA; N; -palmitoylethanolamine; NMEA; N; -myristoylethanolamine; NSEA; N; -stearoylethanolamine; DMAP; 4-dimethylaminopyridine; DCC; N, N′; -dicyclohexylcarbodiimide; DSC; differential scanning calorimetry; T; t; transition temperature; Δ; H; t; enthalpy of transition; Δ; S; t; entropy of transition; T; 1/2; the width at half maximum of the transition; ESR; electron spin resonance; 2A; max; outer hyperfine splitting; LAXS; low-angle X-ray scattering; P; packing parameterDifferential scanning calorimetry; O; -acylethanolamine; Micelle; Hydrocarbon chain tilt; Outer hyperfine splitting
Restoring the charge and surface activity of bovine lung extract surfactants with cationic and anionic polysaccharides by Edgar J. Acosta; Zdenka Policova; Simon Lee; Andrew Dang; Michael L. Hair; A. Wilhelm Neumann (pp. 882-890).
Chitosan, a cationic polysaccharide, has been found to improve the surface activity of lung surfactant extracts in the presence of various inhibitors. It has been proposed that chitosan binds to anionic lipids (e.g. phosphatidyl glycerols) in lung surfactants, producing stable lipid films at the air–water interface. This binding also reverses the net charge of the surfactant aggregates, from negative to positive. Unfortunately, positively charged aggregates may adsorb or interact with the negatively charged epithelial tissue, leading to poor surfactant performance. To address this issue an anionic polysaccharide, dextran sulfate (dexS), was used as a secondary coating to reverse the charge of chitosan–lung surfactant extracts without affecting the surface activity of the preparation. The dynamic surface tension and zeta potential of bovine lipid extract surfactant (BLES) containing chitosan chloride (chiCl) and dexS were evaluated as a function of dexS concentration. These studies were conducted in the absence and presence of sodium bicarbonate buffer, and in the absence and presence of bovine serum used as model inhibitor. It was determined that using an appropriate concentration of dexS, especially at physiological pH, it is possible to restore the negative charge of the surfactant aggregates, and retain their surface activity, even in the presence of bovine serum. High concentrations of dexS affect the binding of chiCl to BLES, and the surface activity of the preparation.
Keywords: Chitosan chloride; Dextran sulfate; Self-assembly; Respiratory distress syndrome
Restoring the charge and surface activity of bovine lung extract surfactants with cationic and anionic polysaccharides by Edgar J. Acosta; Zdenka Policova; Simon Lee; Andrew Dang; Michael L. Hair; A. Wilhelm Neumann (pp. 882-890).
Chitosan, a cationic polysaccharide, has been found to improve the surface activity of lung surfactant extracts in the presence of various inhibitors. It has been proposed that chitosan binds to anionic lipids (e.g. phosphatidyl glycerols) in lung surfactants, producing stable lipid films at the air–water interface. This binding also reverses the net charge of the surfactant aggregates, from negative to positive. Unfortunately, positively charged aggregates may adsorb or interact with the negatively charged epithelial tissue, leading to poor surfactant performance. To address this issue an anionic polysaccharide, dextran sulfate (dexS), was used as a secondary coating to reverse the charge of chitosan–lung surfactant extracts without affecting the surface activity of the preparation. The dynamic surface tension and zeta potential of bovine lipid extract surfactant (BLES) containing chitosan chloride (chiCl) and dexS were evaluated as a function of dexS concentration. These studies were conducted in the absence and presence of sodium bicarbonate buffer, and in the absence and presence of bovine serum used as model inhibitor. It was determined that using an appropriate concentration of dexS, especially at physiological pH, it is possible to restore the negative charge of the surfactant aggregates, and retain their surface activity, even in the presence of bovine serum. High concentrations of dexS affect the binding of chiCl to BLES, and the surface activity of the preparation.
Keywords: Chitosan chloride; Dextran sulfate; Self-assembly; Respiratory distress syndrome
EPR and FTIR studies reveal the importance of highly ordered sterol-enriched membrane domains for ostreolysin activity by Katja Rebolj; Biserka Bakrač; Maja Garvas; Katja Ota; Marjeta Šentjurc; Cristina Potrich; Manuela Coraiola; Rossella Tomazzolli; Mauro Dalla Serra; Peter Maček; Kristina Sepčić (pp. 891-902).
Ostreolysin is a cytolytic protein from the edible oyster mushroom ( Pleurotus ostreatus), which recognizes specifically and binds to raft-like sterol-enriched membrane domains that exist in the liquid-ordered phase. Its binding can be abolished by micromolar concentrations of lysophospholipids and fatty acids. The membrane activity of ostreolysin, however, does not completely correlate with the ability of a certain sterol to induce the formation of a liquid-ordered phase, suggesting that the protein requires an additional structural organization of the membrane to exert its activity. The aim of this study was to further characterize the lipid membranes that facilitate ostreolysin binding by analyzing their lipid phase domain structure. Fourier-transformed infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) were used to analyze the ordering and dynamics of membrane lipids and the membrane domain structure of a series of unilamellar liposomes prepared by systematically changing the lipid components and their ratios. Our results corroborate the earlier conclusion that the average membrane fluidity of ostreolysin-susceptible liposomes alone cannot account for the membrane activity of the protein. Combined with previous data computer-aided interpretation of EPR spectra strongly suggests that chemical properties of membrane constituents, their specific distribution, and physical characteristics of membrane nanodomains, resulting from the presence of sterol and sphingomyelin (or a highly ordered phospholipid, dipalmitoylphosphatidylcholine), are essential prerequisites for ostreolysin membrane binding and pore-formation.
Keywords: Electron paramagnetic resonance; Cholesterol; Fourier-transformed infrared spectroscopy; Liquid ordered phase; Lipid raft; Ostreolysin; Steroid
EPR and FTIR studies reveal the importance of highly ordered sterol-enriched membrane domains for ostreolysin activity by Katja Rebolj; Biserka Bakrač; Maja Garvas; Katja Ota; Marjeta Šentjurc; Cristina Potrich; Manuela Coraiola; Rossella Tomazzolli; Mauro Dalla Serra; Peter Maček; Kristina Sepčić (pp. 891-902).
Ostreolysin is a cytolytic protein from the edible oyster mushroom ( Pleurotus ostreatus), which recognizes specifically and binds to raft-like sterol-enriched membrane domains that exist in the liquid-ordered phase. Its binding can be abolished by micromolar concentrations of lysophospholipids and fatty acids. The membrane activity of ostreolysin, however, does not completely correlate with the ability of a certain sterol to induce the formation of a liquid-ordered phase, suggesting that the protein requires an additional structural organization of the membrane to exert its activity. The aim of this study was to further characterize the lipid membranes that facilitate ostreolysin binding by analyzing their lipid phase domain structure. Fourier-transformed infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) were used to analyze the ordering and dynamics of membrane lipids and the membrane domain structure of a series of unilamellar liposomes prepared by systematically changing the lipid components and their ratios. Our results corroborate the earlier conclusion that the average membrane fluidity of ostreolysin-susceptible liposomes alone cannot account for the membrane activity of the protein. Combined with previous data computer-aided interpretation of EPR spectra strongly suggests that chemical properties of membrane constituents, their specific distribution, and physical characteristics of membrane nanodomains, resulting from the presence of sterol and sphingomyelin (or a highly ordered phospholipid, dipalmitoylphosphatidylcholine), are essential prerequisites for ostreolysin membrane binding and pore-formation.
Keywords: Electron paramagnetic resonance; Cholesterol; Fourier-transformed infrared spectroscopy; Liquid ordered phase; Lipid raft; Ostreolysin; Steroid
Mutation of His 834 in human anion exchanger 1 affects substrate binding by Shinya Takazaki; Yoshito Abe; Tomohiro Yamaguchi; Mikako Yagi; Tadashi Ueda; Dongchon Kang; Naotaka Hamasaki (pp. 903-908).
Anion exchanger 1 (AE1 or band 3) is responsible for Cl−–HCO3− exchange on erythrocyte membrane. Previously, we showed that band 3 is fixed in an inward-facing conformation by specific modification of His 834 with DEPC, resulting in a strong inhibition of its anion transport activity. To clarify the physiological role of His 834, we evaluated the sulfate transport activities of various band 3 mutants: different mutants at His 834 and alanine mutants of peripheral residues around 834 (Lys 829–Phe 836) in yeast cell membranes. The Km values of the His 834 mutants were 4–10 times higher than that of the wild type, while their Vmax values were barely lower than that of wild type. Meanwhile, the Km values of the peripheral alanine mutants were only slightly increased. These data suggest that His 834 is critically important for the efficient binding of sulfate anion, but not for the conformational change induced by substrate binding.
Keywords: Abbreviations; DEPC; diethyl pyrocarbonate; DIDS; 4,4-diisothiocyanostilbene-2,2-disulfonic acid; DNDS; 4,4-dinitrostilbene-2,2-disulfonic acid; ER; endoplasmic reticulum; GPA; glycophorin A; H; 2; DIDS; 4,4-diisothiocyanodihydrostilbene-2,2-disulfonic acid; PMSF; phenylmethanesulfonyl fluoride; TCA; trichloroacetic acid; TM; transmembrane spanning portionBand 3; Anion exchange; Membrane protein structure
Mutation of His 834 in human anion exchanger 1 affects substrate binding by Shinya Takazaki; Yoshito Abe; Tomohiro Yamaguchi; Mikako Yagi; Tadashi Ueda; Dongchon Kang; Naotaka Hamasaki (pp. 903-908).
Anion exchanger 1 (AE1 or band 3) is responsible for Cl−–HCO3− exchange on erythrocyte membrane. Previously, we showed that band 3 is fixed in an inward-facing conformation by specific modification of His 834 with DEPC, resulting in a strong inhibition of its anion transport activity. To clarify the physiological role of His 834, we evaluated the sulfate transport activities of various band 3 mutants: different mutants at His 834 and alanine mutants of peripheral residues around 834 (Lys 829–Phe 836) in yeast cell membranes. The Km values of the His 834 mutants were 4–10 times higher than that of the wild type, while their Vmax values were barely lower than that of wild type. Meanwhile, the Km values of the peripheral alanine mutants were only slightly increased. These data suggest that His 834 is critically important for the efficient binding of sulfate anion, but not for the conformational change induced by substrate binding.
Keywords: Abbreviations; DEPC; diethyl pyrocarbonate; DIDS; 4,4-diisothiocyanostilbene-2,2-disulfonic acid; DNDS; 4,4-dinitrostilbene-2,2-disulfonic acid; ER; endoplasmic reticulum; GPA; glycophorin A; H; 2; DIDS; 4,4-diisothiocyanodihydrostilbene-2,2-disulfonic acid; PMSF; phenylmethanesulfonyl fluoride; TCA; trichloroacetic acid; TM; transmembrane spanning portionBand 3; Anion exchange; Membrane protein structure
Three unrelated sphingomyelin analogs spontaneously cluster into plasma membrane micrometric domains by D. Tyteca; L. D'Auria; P. Van Der Smissen; T. Medts; S. Carpentier; J.C. Monbaliu; P. de Diesbach; P.J. Courtoy (pp. 909-927).
Micrometric lipid compartmentation at the plasma membrane is disputed. Using live confocal imaging, we found that three unrelated fluorescent sphingomyelin (SM) analogs spontaneously clustered at the outer leaflet into micrometric domains, contrasting with homogeneous labelling by DiIC18 and TMA-DPH. In erythrocytes, these domains were round, randomly distributed, and reversibly coalesced under hypotonicity. BODIPY-SM and -glucosylceramide showed distinct temperature-dependence, in the same ranking as Tm for corresponding natural lipids, indicating phase behaviour. Scanning electron microscopy excluded micrometric surface structural features. In CHO cells, similar surface micrometric patches were produced by either direct BODIPY-SM insertion or intracellular processing from BODIPY-ceramide, ruling out aggregation artefacts. BODIPY-SM surface micrometric patches were refractory to endocytosis block or actin depolymerization and clustered upon cholesterol deprivation, indicating self-clustering at the plasma membrane. BODIPY-SM excimers further suggested clustering in ordered domains. Segregation of BODIPY-SM and -lactosylceramide micrometric domains showed coexistence of distinct phases. Consistent with micrometric domain boundaries, fluorescence recovery after photobleaching (FRAP) revealed restriction of BODIPY-SM lateral diffusion over long-range, but not short-range, contrasting with comparable high mobile fraction of BODIPY-lactosylceramide in both ranges. Controlled perturbations of endogenous SM pool similarly affected BODIPY-SM domain size by confocal imaging and its mobile fraction by FRAP. The latter evidence supports the hypothesis that, as shown for BODIPY-SM, endogenous SM spontaneously clusters at the plasmalemma outer leaflet of living cells into ordered micrometric domains, defined in shape by liquid-phase coexistence and in size by membrane tension and cholesterol. This proposal remains speculative and calls for further investigations.
Keywords: Abbreviations; BODIPY; boron dipy*_r_*romethenedifluoride; BODIPY; 505; BODIPY “green”; BODIPY; 589; BODIPY “red”; Cer; ceramide; DF-BSA; defatted bovine serum albumin; DiI; dialkylindocarbocyanine; DRMs; detergent-resistant membranes; FB1; fumonisin B1; GlcCer; glucosylceramide; GPMVs; giant PM vesicles; GSL; glycosphingolipid; GUVs; giant unilamellar vesicles; LacCer; lactosylceramide; L; d; liquid-disordered; L; o; liquid-ordered; mβCD; methyl-β-cyclodextrin; mβCD:chol; methyl-β-cyclodextrin:cholesterol; NBD; 7-nitrobenz-2-oxa-1,3-diazol-4-yl; N; -Rh-PE; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-; N; -lissamine rhodamine B sulfonyl; PC; phosphatidylcholine; PM; plasma membrane; S; o; solid-ordered; SL; sphingolipid; SM; sphingomyelin; SMase; sphingomyelinase; Tm; phase transition temperature; TMA-DPH; 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatrieneSphingomyelin; Micrometric domain; CHO plasma membrane; Erythrocyte; Lateral diffusion; Phase coexistence
Three unrelated sphingomyelin analogs spontaneously cluster into plasma membrane micrometric domains by D. Tyteca; L. D'Auria; P. Van Der Smissen; T. Medts; S. Carpentier; J.C. Monbaliu; P. de Diesbach; P.J. Courtoy (pp. 909-927).
Micrometric lipid compartmentation at the plasma membrane is disputed. Using live confocal imaging, we found that three unrelated fluorescent sphingomyelin (SM) analogs spontaneously clustered at the outer leaflet into micrometric domains, contrasting with homogeneous labelling by DiIC18 and TMA-DPH. In erythrocytes, these domains were round, randomly distributed, and reversibly coalesced under hypotonicity. BODIPY-SM and -glucosylceramide showed distinct temperature-dependence, in the same ranking as Tm for corresponding natural lipids, indicating phase behaviour. Scanning electron microscopy excluded micrometric surface structural features. In CHO cells, similar surface micrometric patches were produced by either direct BODIPY-SM insertion or intracellular processing from BODIPY-ceramide, ruling out aggregation artefacts. BODIPY-SM surface micrometric patches were refractory to endocytosis block or actin depolymerization and clustered upon cholesterol deprivation, indicating self-clustering at the plasma membrane. BODIPY-SM excimers further suggested clustering in ordered domains. Segregation of BODIPY-SM and -lactosylceramide micrometric domains showed coexistence of distinct phases. Consistent with micrometric domain boundaries, fluorescence recovery after photobleaching (FRAP) revealed restriction of BODIPY-SM lateral diffusion over long-range, but not short-range, contrasting with comparable high mobile fraction of BODIPY-lactosylceramide in both ranges. Controlled perturbations of endogenous SM pool similarly affected BODIPY-SM domain size by confocal imaging and its mobile fraction by FRAP. The latter evidence supports the hypothesis that, as shown for BODIPY-SM, endogenous SM spontaneously clusters at the plasmalemma outer leaflet of living cells into ordered micrometric domains, defined in shape by liquid-phase coexistence and in size by membrane tension and cholesterol. This proposal remains speculative and calls for further investigations.
Keywords: Abbreviations; BODIPY; boron dipy*_r_*romethenedifluoride; BODIPY; 505; BODIPY “green”; BODIPY; 589; BODIPY “red”; Cer; ceramide; DF-BSA; defatted bovine serum albumin; DiI; dialkylindocarbocyanine; DRMs; detergent-resistant membranes; FB1; fumonisin B1; GlcCer; glucosylceramide; GPMVs; giant PM vesicles; GSL; glycosphingolipid; GUVs; giant unilamellar vesicles; LacCer; lactosylceramide; L; d; liquid-disordered; L; o; liquid-ordered; mβCD; methyl-β-cyclodextrin; mβCD:chol; methyl-β-cyclodextrin:cholesterol; NBD; 7-nitrobenz-2-oxa-1,3-diazol-4-yl; N; -Rh-PE; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-; N; -lissamine rhodamine B sulfonyl; PC; phosphatidylcholine; PM; plasma membrane; S; o; solid-ordered; SL; sphingolipid; SM; sphingomyelin; SMase; sphingomyelinase; Tm; phase transition temperature; TMA-DPH; 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatrieneSphingomyelin; Micrometric domain; CHO plasma membrane; Erythrocyte; Lateral diffusion; Phase coexistence
Effect of monolayer lipid charges on the structure and orientation of protein VAMP1 at the air–water interface by Wissam Yassine; Alexandra Milochau; Sebastien Buchoux; Jochen Lang; Bernard Desbat; Reiko Oda (pp. 928-937).
SNARE proteins are implicated in membrane fusion during neurotransmission and peptide hormone secretion. Relatively little is known about the molecular interactions of their trans- and juxtamembrane domains with lipid membranes. Here, we report the structure and the assembling behavior of one of the SNARE proteins, VAMP1/synaptobrevin1 incorporated in a lipid monolayer at an air–water interface which mimics the membrane environment. Our results show that the protein is extremely sensitive to surface pressure as well as the lipid composition. Monolayers of proteins alone or in the presence of the neutral phospholipid DMPC underwent structural transition from α-helix to β-sheet upon surface compression. In contrast, the anionic phospholipid DMPG inhibited this transition in a concentration-dependent manner. Moreover, the orientation of the proteins was highly sensitive to the charge density of the lipid layers. Thus, the structure of VAMP1 is clearly controlled by protein–lipid interactions.
Keywords: SNARE protein; Synaptobrevin; PMIRRAS; Brewster angle microscope; Protein–lipid interaction
Effect of monolayer lipid charges on the structure and orientation of protein VAMP1 at the air–water interface by Wissam Yassine; Alexandra Milochau; Sebastien Buchoux; Jochen Lang; Bernard Desbat; Reiko Oda (pp. 928-937).
SNARE proteins are implicated in membrane fusion during neurotransmission and peptide hormone secretion. Relatively little is known about the molecular interactions of their trans- and juxtamembrane domains with lipid membranes. Here, we report the structure and the assembling behavior of one of the SNARE proteins, VAMP1/synaptobrevin1 incorporated in a lipid monolayer at an air–water interface which mimics the membrane environment. Our results show that the protein is extremely sensitive to surface pressure as well as the lipid composition. Monolayers of proteins alone or in the presence of the neutral phospholipid DMPC underwent structural transition from α-helix to β-sheet upon surface compression. In contrast, the anionic phospholipid DMPG inhibited this transition in a concentration-dependent manner. Moreover, the orientation of the proteins was highly sensitive to the charge density of the lipid layers. Thus, the structure of VAMP1 is clearly controlled by protein–lipid interactions.
Keywords: SNARE protein; Synaptobrevin; PMIRRAS; Brewster angle microscope; Protein–lipid interaction
Membrane simulations mimicking acidic pH reveal increased thickness and negative curvature in a bilayer consisting of lysophosphatidylcholines and free fatty acids by Lahdesmaki Katariina Lähdesmäki; O.H. Samuli Ollila; Artturi Koivuniemi; Petri T. Kovanen; Hyvonen Marja T. Hyvönen (pp. 938-946).
Phospholipids are key components of biological membranes and their lipolysis with phospholipase A2 (PLA2) enzymes occurs in different cellular pH environments. Since no studies are available on the effect of pH on PLA2-modified phospholipid membranes, we performed 50-ns atomistic molecular dynamics simulations at three different pH conditions (pH 9.0, 7.5, and 5.5) using a fully PLA2-hydrolyzed phosphatidylcholine (PC) bilayer which consists solely of lysophosphatidylcholine and free fatty acid molecules. We found that a decrease in pH results in lateral squeezing of the membrane, i.e. in decreased surface area per headgroup. Thus, at the decreased pH, the lipid hydrocarbon chains had larger SCD order parameter values, and also enhanced membrane thickness, as seen in the electron density profiles across the membrane. From the lateral pressure profiles, we found that the values of spontaneous curvature of the two opposing monolayers became negative when the pH was decreased. At low pH, protonation of the free fatty acid headgroups reduces their mutual repulsion and accounts for the pH dependence of all the above-mentioned properties. The altered structural characteristics may significantly affect the overall surface properties of biomembranes in cellular vesicles, lipid droplets, and plasma lipoproteins, play an important role in membrane fission and fusion, and modify interactions between membrane lipids and the proteins embedded within them.
Keywords: MD-simulation; Phospholipid; pH; Phospholipase A; 2; Lateral pressure profile; Spontaneous curvature
Membrane simulations mimicking acidic pH reveal increased thickness and negative curvature in a bilayer consisting of lysophosphatidylcholines and free fatty acids by Lahdesmaki Katariina Lähdesmäki; O.H. Samuli Ollila; Artturi Koivuniemi; Petri T. Kovanen; Hyvonen Marja T. Hyvönen (pp. 938-946).
Phospholipids are key components of biological membranes and their lipolysis with phospholipase A2 (PLA2) enzymes occurs in different cellular pH environments. Since no studies are available on the effect of pH on PLA2-modified phospholipid membranes, we performed 50-ns atomistic molecular dynamics simulations at three different pH conditions (pH 9.0, 7.5, and 5.5) using a fully PLA2-hydrolyzed phosphatidylcholine (PC) bilayer which consists solely of lysophosphatidylcholine and free fatty acid molecules. We found that a decrease in pH results in lateral squeezing of the membrane, i.e. in decreased surface area per headgroup. Thus, at the decreased pH, the lipid hydrocarbon chains had larger SCD order parameter values, and also enhanced membrane thickness, as seen in the electron density profiles across the membrane. From the lateral pressure profiles, we found that the values of spontaneous curvature of the two opposing monolayers became negative when the pH was decreased. At low pH, protonation of the free fatty acid headgroups reduces their mutual repulsion and accounts for the pH dependence of all the above-mentioned properties. The altered structural characteristics may significantly affect the overall surface properties of biomembranes in cellular vesicles, lipid droplets, and plasma lipoproteins, play an important role in membrane fission and fusion, and modify interactions between membrane lipids and the proteins embedded within them.
Keywords: MD-simulation; Phospholipid; pH; Phospholipase A; 2; Lateral pressure profile; Spontaneous curvature
Effects of protons on macroscopic and single-channel currents mediated by the human P2X7 receptor by B. Flittiger; Klapperstuck M. Klapperstück; G. Schmalzing; F. Markwardt (pp. 947-957).
Human P2X7 receptors (hP2X7Rs) belong to the P2X family, which opens an intrinsic cation channel when challenged by extracellular ATP. hP2X7Rs are expressed in cells of the inflammatory and immune system. During inflammation, ATP and protons are secreted into the interstitial fluid. Therefore, we investigated the effect of protons on the activation of hP2X7Rs. hP2X7Rs were expressed in Xenopus laevis oocytes and activated by the agonists ATP or benzoyl-benzoyl-ATP (BzATP) at different pH values. The protons reduced the hP2X7R-dependent cation current amplitude and slowed the current deactivation depending on the type and concentration of the agonist used. These effects can be explained by (i) the protonation of ATP, which reduces the effective concentration of the agonist ATP4− at the high- and low-affinity ATP activation site of the hP2XR, and (ii) direct allosteric inhibition of the hP2X7R channel opening that follows ATP4− binding to the low-affinity activation site. Due to the hampered activation via the low-affinity activation site, a low pH (as observed in inflamed tissues) leads to a relative increase in the contribution of the high-affinity activation site for hP2X7R channel opening.
Keywords: Abbreviations; ATP; 4−; free form of ATP, not bound to cations; BzATP; 2′3′-O-(4-benzoyl)-benzoyl-ATP; hP2X7R; human purinergic P2X7 receptor; V; h; holding potentialP2 purinergic receptor; P2X7 receptor; pH dependence; Patch clamp
Effects of protons on macroscopic and single-channel currents mediated by the human P2X7 receptor by B. Flittiger; Klapperstuck M. Klapperstück; G. Schmalzing; F. Markwardt (pp. 947-957).
Human P2X7 receptors (hP2X7Rs) belong to the P2X family, which opens an intrinsic cation channel when challenged by extracellular ATP. hP2X7Rs are expressed in cells of the inflammatory and immune system. During inflammation, ATP and protons are secreted into the interstitial fluid. Therefore, we investigated the effect of protons on the activation of hP2X7Rs. hP2X7Rs were expressed in Xenopus laevis oocytes and activated by the agonists ATP or benzoyl-benzoyl-ATP (BzATP) at different pH values. The protons reduced the hP2X7R-dependent cation current amplitude and slowed the current deactivation depending on the type and concentration of the agonist used. These effects can be explained by (i) the protonation of ATP, which reduces the effective concentration of the agonist ATP4− at the high- and low-affinity ATP activation site of the hP2XR, and (ii) direct allosteric inhibition of the hP2X7R channel opening that follows ATP4− binding to the low-affinity activation site. Due to the hampered activation via the low-affinity activation site, a low pH (as observed in inflamed tissues) leads to a relative increase in the contribution of the high-affinity activation site for hP2X7R channel opening.
Keywords: Abbreviations; ATP; 4−; free form of ATP, not bound to cations; BzATP; 2′3′-O-(4-benzoyl)-benzoyl-ATP; hP2X7R; human purinergic P2X7 receptor; V; h; holding potentialP2 purinergic receptor; P2X7 receptor; pH dependence; Patch clamp
Temperature-induced structural transition in-situ in porcine lens — Changes observed in void size distribution by Petri Sane; Filip Tuomisto; Susanne K. Wiedmer; Tuula Nyman; Ilpo Vattulainen; Juha M. Holopainen (pp. 958-965).
The function of mammalian ocular lens is to provide a sharp image to the retina. Accordingly, the lens needs to be transparent and minimize light scattering. To do so the lens fiber cells first loose intracellular organelles, organize the cytoplasm and arrange the fiber cell membranes. Because the fiber cells are metabolically inactive, the plasma membrane becomes the only cellular organelle and consequently, the phase behavior of these membranes determines the physiological state of the lens. Previous studies have shown that lipids extracted from the nuclear and cortical region of human lens show a temperature-induced phase transition close to the body temperature. Yet, the physiological function of this phase transition is not known, and even the presence of the phase transition in intact lenses is unknown. Positron annihilation lifetime spectroscopy (PALS) was used to characterize the sub-nanometer-sized local structure of intact porcine lens and these studies were complemented with differential scanning calorimeter and mass spectrometric analysis in extracted porcine lens lipids. Using PALS, we present evidence for the presence of a temperature-dependent structural transition centered at 35.5°C in-situ in clear extracted porcine lenses. Further studies employing extracted lens lipids and purified egg-yolk sphingomyelin and cholesterol mixtures suggest that the nano-scale transition emerges from the phase behavior of lens lipids. Based on our results, PALS seems to be a viable method for gaining additional information on biological tissues, especially since it enables non-destructive studies on intact tissues.
Keywords: Lens; Lipid membranes; Positron annihilation lifetime spectroscopy; Differential scanning calorimetry; Sphingomyelin
Temperature-induced structural transition in-situ in porcine lens — Changes observed in void size distribution by Petri Sane; Filip Tuomisto; Susanne K. Wiedmer; Tuula Nyman; Ilpo Vattulainen; Juha M. Holopainen (pp. 958-965).
The function of mammalian ocular lens is to provide a sharp image to the retina. Accordingly, the lens needs to be transparent and minimize light scattering. To do so the lens fiber cells first loose intracellular organelles, organize the cytoplasm and arrange the fiber cell membranes. Because the fiber cells are metabolically inactive, the plasma membrane becomes the only cellular organelle and consequently, the phase behavior of these membranes determines the physiological state of the lens. Previous studies have shown that lipids extracted from the nuclear and cortical region of human lens show a temperature-induced phase transition close to the body temperature. Yet, the physiological function of this phase transition is not known, and even the presence of the phase transition in intact lenses is unknown. Positron annihilation lifetime spectroscopy (PALS) was used to characterize the sub-nanometer-sized local structure of intact porcine lens and these studies were complemented with differential scanning calorimeter and mass spectrometric analysis in extracted porcine lens lipids. Using PALS, we present evidence for the presence of a temperature-dependent structural transition centered at 35.5°C in-situ in clear extracted porcine lenses. Further studies employing extracted lens lipids and purified egg-yolk sphingomyelin and cholesterol mixtures suggest that the nano-scale transition emerges from the phase behavior of lens lipids. Based on our results, PALS seems to be a viable method for gaining additional information on biological tissues, especially since it enables non-destructive studies on intact tissues.
Keywords: Lens; Lipid membranes; Positron annihilation lifetime spectroscopy; Differential scanning calorimetry; Sphingomyelin
Role of glycine residues highly conserved in the S2–S3 linkers of domains I and II of voltage-gated calcium channel α1 subunits by Jinfeng Teng; Kazuko Iida; Masanori Ito; Hiroko Izumi-Nakaseko; Itaru Kojima; Satomi Adachi-Akahane; Hidetoshi Iida (pp. 966-974).
The pore-forming component of voltage-gated calcium channels, α1 subunit, contains four structurally conserved domains (I–IV), each of which contains six transmembrane segments (S1–S6). We have shown previously that a Gly residue in the S2–S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2–S3 linkers of domains I and II, which correspond positionally to the Gly in the S2–S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2–S3 linkers of domains I and II of Cav1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Cav1.2s; G182E, G182Q, G579E, G579Q, and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current–voltage relationships of the four mutants were the same as those of wild-type Cav1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly182 in domain I is involved in the membrane trafficking or surface expression of α1 subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Cav1.2, although G579Q showed a normal VDI, implying that Gly579 in domain II is involved in the regulation of VDI and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for α1 subunit to become functional.
Keywords: Abbreviations; VGCC; voltage-gated Ca; 2+; channel; AID; α; 1; -interaction domain; VDI; voltage-dependent inactivation; CDI; Ca; 2+; -dependent inactivationCa; 2+; channel; VGCC; VDCC; Ca; v; 1.2; S2–S3 linker; Glycine residue
Role of glycine residues highly conserved in the S2–S3 linkers of domains I and II of voltage-gated calcium channel α1 subunits by Jinfeng Teng; Kazuko Iida; Masanori Ito; Hiroko Izumi-Nakaseko; Itaru Kojima; Satomi Adachi-Akahane; Hidetoshi Iida (pp. 966-974).
The pore-forming component of voltage-gated calcium channels, α1 subunit, contains four structurally conserved domains (I–IV), each of which contains six transmembrane segments (S1–S6). We have shown previously that a Gly residue in the S2–S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2–S3 linkers of domains I and II, which correspond positionally to the Gly in the S2–S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2–S3 linkers of domains I and II of Cav1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Cav1.2s; G182E, G182Q, G579E, G579Q, and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current–voltage relationships of the four mutants were the same as those of wild-type Cav1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly182 in domain I is involved in the membrane trafficking or surface expression of α1 subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Cav1.2, although G579Q showed a normal VDI, implying that Gly579 in domain II is involved in the regulation of VDI and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for α1 subunit to become functional.
Keywords: Abbreviations; VGCC; voltage-gated Ca; 2+; channel; AID; α; 1; -interaction domain; VDI; voltage-dependent inactivation; CDI; Ca; 2+; -dependent inactivationCa; 2+; channel; VGCC; VDCC; Ca; v; 1.2; S2–S3 linker; Glycine residue
Lateral reorganization of plasma membrane is involved in the yeast resistance to severe dehydration by Sebastien Dupont; Laurent Beney; Jean-Francois Ritt; Jeannine Lherminier; Patrick Gervais (pp. 975-985).
In this study, we investigated the kinetic and the magnitude of dehydrations on yeast plasma membrane (PM) modifications because this parameter is crucial to cell survival. Functional (permeability) and structural (morphology, ultrastructure, and distribution of the protein Sur7-GFP contained in sterol-rich membrane microdomains) PM modifications were investigated by confocal and electron microscopy after progressive (non-lethal) and rapid (lethal) hyperosmotic perturbations. Rapid cell dehydration induced the formation of many PM invaginations followed by membrane internalization of low sterol content PM regions with time. Permeabilization of the plasma membrane occurred during the rehydration stage because of inadequacies in the membrane surface and led to cell death. Progressive dehydration conducted to the formation of some big PM pleats without membrane internalization. It also led to the modification of the distribution of the Sur7-GFP microdomains, suggesting that a lateral rearrangement of membrane components occurred. This event is a function of time and is involved in the particular deformations of the PM during a progressive perturbation. The maintenance of the repartition of the microdomains during rapid perturbations consolidates this assumption. These findings highlight that the perturbation kinetic influences the evolution of the PM organization and indicate the crucial role of PM lateral reorganization in cell survival to hydric perturbations.
Keywords: Dehydration kinetic; Cell survival; Microdomain; Confocal microscopy; Electron microscopy
Lateral reorganization of plasma membrane is involved in the yeast resistance to severe dehydration by Sebastien Dupont; Laurent Beney; Jean-Francois Ritt; Jeannine Lherminier; Patrick Gervais (pp. 975-985).
In this study, we investigated the kinetic and the magnitude of dehydrations on yeast plasma membrane (PM) modifications because this parameter is crucial to cell survival. Functional (permeability) and structural (morphology, ultrastructure, and distribution of the protein Sur7-GFP contained in sterol-rich membrane microdomains) PM modifications were investigated by confocal and electron microscopy after progressive (non-lethal) and rapid (lethal) hyperosmotic perturbations. Rapid cell dehydration induced the formation of many PM invaginations followed by membrane internalization of low sterol content PM regions with time. Permeabilization of the plasma membrane occurred during the rehydration stage because of inadequacies in the membrane surface and led to cell death. Progressive dehydration conducted to the formation of some big PM pleats without membrane internalization. It also led to the modification of the distribution of the Sur7-GFP microdomains, suggesting that a lateral rearrangement of membrane components occurred. This event is a function of time and is involved in the particular deformations of the PM during a progressive perturbation. The maintenance of the repartition of the microdomains during rapid perturbations consolidates this assumption. These findings highlight that the perturbation kinetic influences the evolution of the PM organization and indicate the crucial role of PM lateral reorganization in cell survival to hydric perturbations.
Keywords: Dehydration kinetic; Cell survival; Microdomain; Confocal microscopy; Electron microscopy
Methyl-β-cyclodextrin restores the structure and function of pulmonary surfactant films impaired by cholesterol by Lasantha C. Gunasekara; Ryan M. Pratt; W. Michael Schoel; Sherrie Gosche; Elmar J. Prenner; Matthias Walter Amrein (pp. 986-994).
Pulmonary surfactant, a defined mixture of lipids and proteins, imparts very low surface tension to the lung–air interface by forming an incompressible film. In acute respiratory distress syndrome and other respiratory conditions, this function is impaired by a number of factors, among which is an increase of cholesterol in surfactant. The current study shows in vitro that cholesterol can be extracted from surfactant and function subsequently restored to dysfunctional surfactant films in a dose-dependent manner by methyl-β-cyclodextrin (MβCD). Bovine lipid extract surfactant was supplemented with cholesterol to serve as a model of dysfunctional surfactant. Likewise, when cholesterol in a complex with MβCD (“water-soluble cholesterol”) was added in aqueous solution, surfactant films were rendered dysfunctional. Atomic force microscopy showed recovery of function by MβCD is accompanied by the re-establishment of the native film structure of a lipid monolayer with scattered areas of lipid bilayer stacks, whereas dysfunctional films lacked bilayers. The current study expands upon a recent perspective of surfactant inactivation in disease and suggests a potential treatment.
Keywords: Abbreviations; Chol-BLES; bovine lipid extract surfactant with cholesterol added; MST; minimum surface tension; IA; initial adsorption; QS; quasi-static cycle; D; dynamic cyclePulmonary surfactant dysfunction; Captive bubble surfactometer; Cholesterol; Methyl-β; -; cyclodextrin; Bovine lipid extract surfactant; Atomic force microscopy
Methyl-β-cyclodextrin restores the structure and function of pulmonary surfactant films impaired by cholesterol by Lasantha C. Gunasekara; Ryan M. Pratt; W. Michael Schoel; Sherrie Gosche; Elmar J. Prenner; Matthias Walter Amrein (pp. 986-994).
Pulmonary surfactant, a defined mixture of lipids and proteins, imparts very low surface tension to the lung–air interface by forming an incompressible film. In acute respiratory distress syndrome and other respiratory conditions, this function is impaired by a number of factors, among which is an increase of cholesterol in surfactant. The current study shows in vitro that cholesterol can be extracted from surfactant and function subsequently restored to dysfunctional surfactant films in a dose-dependent manner by methyl-β-cyclodextrin (MβCD). Bovine lipid extract surfactant was supplemented with cholesterol to serve as a model of dysfunctional surfactant. Likewise, when cholesterol in a complex with MβCD (“water-soluble cholesterol”) was added in aqueous solution, surfactant films were rendered dysfunctional. Atomic force microscopy showed recovery of function by MβCD is accompanied by the re-establishment of the native film structure of a lipid monolayer with scattered areas of lipid bilayer stacks, whereas dysfunctional films lacked bilayers. The current study expands upon a recent perspective of surfactant inactivation in disease and suggests a potential treatment.
Keywords: Abbreviations; Chol-BLES; bovine lipid extract surfactant with cholesterol added; MST; minimum surface tension; IA; initial adsorption; QS; quasi-static cycle; D; dynamic cyclePulmonary surfactant dysfunction; Captive bubble surfactometer; Cholesterol; Methyl-β; -; cyclodextrin; Bovine lipid extract surfactant; Atomic force microscopy
Regulation of human cardiac KCNQ1/KCNE1 channel by epidermal growth factor receptor kinase by Ming-Qing Dong; Hai-Ying Sun; Qiang Tang; Hung-Fat Tse; Chu-Pak Lau; Gui-Rong Li (pp. 995-1001).
The aim of the present study was to investigate whether/how the recombinant human cardiac IKs could be regulated by epidermal growth factor receptor kinase in HEK 293 cells stably expressing hKCNQ1/hKCNE1 genes using the approaches of perforated patch clamp technique, immunoprecipitation and Western blot analysis. It was found that the broad spectrum isoflavone tyrosine kinase inhibitor genistein and the selective epidermal growth factor receptor kinase inhibitor tyrphostin AG556 suppressed the recombinant IKs, and their inhibition was countered by the protein tyrosine phosphatase inhibitor orthovanadate. The Src-family kinase inhibitor PP2 reduced the current, but the effect was not antagonized by orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hKCNQ1 protein was decreased by genistein or AG556, but not by PP2. These results provide the novel information that epidermal growth factor receptor kinase, but not Src-family kinases, regulates the recombinant cardiac IKs stably expressed in HEK 293 cells via phosphorylating KCNQ1 protein of the channel.
Keywords: Epidermal growth factor receptor kinase; Protein tyrosine phosphorylation; Recombinant cardiac hKCNQ1/hKCNE1 channel; Electrophysiology
Regulation of human cardiac KCNQ1/KCNE1 channel by epidermal growth factor receptor kinase by Ming-Qing Dong; Hai-Ying Sun; Qiang Tang; Hung-Fat Tse; Chu-Pak Lau; Gui-Rong Li (pp. 995-1001).
The aim of the present study was to investigate whether/how the recombinant human cardiac IKs could be regulated by epidermal growth factor receptor kinase in HEK 293 cells stably expressing hKCNQ1/hKCNE1 genes using the approaches of perforated patch clamp technique, immunoprecipitation and Western blot analysis. It was found that the broad spectrum isoflavone tyrosine kinase inhibitor genistein and the selective epidermal growth factor receptor kinase inhibitor tyrphostin AG556 suppressed the recombinant IKs, and their inhibition was countered by the protein tyrosine phosphatase inhibitor orthovanadate. The Src-family kinase inhibitor PP2 reduced the current, but the effect was not antagonized by orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hKCNQ1 protein was decreased by genistein or AG556, but not by PP2. These results provide the novel information that epidermal growth factor receptor kinase, but not Src-family kinases, regulates the recombinant cardiac IKs stably expressed in HEK 293 cells via phosphorylating KCNQ1 protein of the channel.
Keywords: Epidermal growth factor receptor kinase; Protein tyrosine phosphorylation; Recombinant cardiac hKCNQ1/hKCNE1 channel; Electrophysiology
Functional impact of serial deletions at the C-terminus of the human GABAρ1 receptor by Jorge Mauricio Reyes-Ruiz; Lenin David Ochoa-de la Paz; Martinez-Torres Ataúlfo Martínez-Torres; Ricardo Miledi (pp. 1002-1007).
GABAρ1 receptors are formed by homopentameric assemblies that gate a chloride ion-channel upon activation by the neurotransmitter. Very little is known about the structural and functional roles played by the different domains that form each subunit; but one of them, the fourth transmembrane segment (TM4), is known to form a hydrophobic bundle together with three other TM segments that are necessary to stabilize the structure of the receptor. In this study we progressively removed amino acid residues from the C-terminus of the human GABAρ1 and studied the functional properties of the receptor mutants expressed in X. laevis oocytes. We found that deletions of up to the last four residues gave rise to receptors that were still functional, generating currents of 3.92µA for the wt, 5.75μA for S479X, 1.82μA for F478X, 0.52μA for I477X and 0.27μA for S476X when exposed to 5µM GABA; surprisingly, the mutant with one residue removed resulted more sensitive to the agonists. Further deletions, up to residue W475, resulted in receptors that did not gate an ion-channel. In addition, deleting the signal sequence, from R2-A15, in the N-terminus produced non-functional receptors. This study reveals that GABAρ1 can tolerate removal of several residues that form the fourth transmembrane segment up to a critical point, signaled by W475, beyond which the mutant protein is translated but does not form functional receptors. A comparative study is presented of some electrophysiological and pharmacological properties of the deletion mutants that were able to generate GABA currents.
Keywords: GFP; LGIC; TM4; Site-directed mutagenesis; Xenopus; oocytes
Functional impact of serial deletions at the C-terminus of the human GABAρ1 receptor by Jorge Mauricio Reyes-Ruiz; Lenin David Ochoa-de la Paz; Martinez-Torres Ataúlfo Martínez-Torres; Ricardo Miledi (pp. 1002-1007).
GABAρ1 receptors are formed by homopentameric assemblies that gate a chloride ion-channel upon activation by the neurotransmitter. Very little is known about the structural and functional roles played by the different domains that form each subunit; but one of them, the fourth transmembrane segment (TM4), is known to form a hydrophobic bundle together with three other TM segments that are necessary to stabilize the structure of the receptor. In this study we progressively removed amino acid residues from the C-terminus of the human GABAρ1 and studied the functional properties of the receptor mutants expressed in X. laevis oocytes. We found that deletions of up to the last four residues gave rise to receptors that were still functional, generating currents of 3.92µA for the wt, 5.75μA for S479X, 1.82μA for F478X, 0.52μA for I477X and 0.27μA for S476X when exposed to 5µM GABA; surprisingly, the mutant with one residue removed resulted more sensitive to the agonists. Further deletions, up to residue W475, resulted in receptors that did not gate an ion-channel. In addition, deleting the signal sequence, from R2-A15, in the N-terminus produced non-functional receptors. This study reveals that GABAρ1 can tolerate removal of several residues that form the fourth transmembrane segment up to a critical point, signaled by W475, beyond which the mutant protein is translated but does not form functional receptors. A comparative study is presented of some electrophysiological and pharmacological properties of the deletion mutants that were able to generate GABA currents.
Keywords: GFP; LGIC; TM4; Site-directed mutagenesis; Xenopus; oocytes
Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length by Thomas K.M. Nyholm; Pia-Maria Grandell; Bodil Westerlund; J. Peter Slotte (pp. 1008-1013).
It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-β-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37°C addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. At 23°C the 18:1-ceramide was not as effective at inhibiting sterol partitioning into the vesicles as its saturated equivalent, but at 37°C the additional double bond had no effect. The longer 24:1-ceramide behaved as 24:0-ceramide at both temperatures. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with sphingomyelin.
Keywords: Abbreviations; CTL; cholesta-5,7,9(11)-trien-3-beta-ol; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; PCer; D-; erythro-N; -palmitoyl ceramide; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; D-; erythro-N; -palmitoyl sphingomyelinDioleoyl phosphatidylcholine; Palmitoyl sphingomyelin; Cholesterol; Cholestatrienol; Lateral domain; Membrane structure
Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length by Thomas K.M. Nyholm; Pia-Maria Grandell; Bodil Westerlund; J. Peter Slotte (pp. 1008-1013).
It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-β-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37°C addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. At 23°C the 18:1-ceramide was not as effective at inhibiting sterol partitioning into the vesicles as its saturated equivalent, but at 37°C the additional double bond had no effect. The longer 24:1-ceramide behaved as 24:0-ceramide at both temperatures. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with sphingomyelin.
Keywords: Abbreviations; CTL; cholesta-5,7,9(11)-trien-3-beta-ol; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; PCer; D-; erythro-N; -palmitoyl ceramide; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; D-; erythro-N; -palmitoyl sphingomyelinDioleoyl phosphatidylcholine; Palmitoyl sphingomyelin; Cholesterol; Cholestatrienol; Lateral domain; Membrane structure
Preferential insertion of lactose permease in phospholipid domains: AFM observations by Laura Picas; Adrián Carretero-Genevrier; M. Teresa Montero; Vazquez-Ibar J.L. Vázquez-Ibar; Bastien Seantier; Pierre-Emmanuel Milhiet; Hernandez-Borrell Jordi Hernández-Borrell (pp. 1014-1019).
We report the insertion of a transmembrane protein, lactose permease (LacY) from Escherichia coli ( E. coli), in supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol (POPG), in biomimetic molar proportions. We provide evidence of the preferential insertion of LacY in the fluid domains. Analysis of the self-assembled protein arrangements showed that LacY: (i) is inserted as a monomer within fluid domains of SLBs of POPE:POPG (3:1, mol/mol), (ii) has a diameter of approx. 7.8nm; and (iii) keeps an area of phospholipids surrounding the protein that is compatible with shells of phospholipids.
Keywords: Lactose permease; AFM; Phospholipid domains
Preferential insertion of lactose permease in phospholipid domains: AFM observations by Laura Picas; Adrián Carretero-Genevrier; M. Teresa Montero; Vazquez-Ibar J.L. Vázquez-Ibar; Bastien Seantier; Pierre-Emmanuel Milhiet; Hernandez-Borrell Jordi Hernández-Borrell (pp. 1014-1019).
We report the insertion of a transmembrane protein, lactose permease (LacY) from Escherichia coli ( E. coli), in supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol (POPG), in biomimetic molar proportions. We provide evidence of the preferential insertion of LacY in the fluid domains. Analysis of the self-assembled protein arrangements showed that LacY: (i) is inserted as a monomer within fluid domains of SLBs of POPE:POPG (3:1, mol/mol), (ii) has a diameter of approx. 7.8nm; and (iii) keeps an area of phospholipids surrounding the protein that is compatible with shells of phospholipids.
Keywords: Lactose permease; AFM; Phospholipid domains