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BBA - Biomembranes (v.1717, #1)

Editorial Board (pp. ii).

Type 1 ryanodine receptor in cardiac mitochondria: Transducer of excitation–metabolism coupling by Gisela Beutner; Virendra K. Sharma; Lin Lin; Shin-Young Ryu; Robert T. Dirksen; Shey-Shing Sheu (pp. 1-10).

Mitochondria in a variety of cell types respond to physiological Ca²⁺ oscillations in the cytosol dynamically with Ca²⁺ uptakes. In heart cells, mitochondrial Ca²⁺ uptakes occur by a ruthenium red-sensitive Ca²⁺ uniporter (CaUP), a rapid mode of Ca²⁺ uptake (RaM) and a ryanodine receptor (RyR) localized in the inner mitochondrial membrane (IMM). Three subtypes of RyRs have been described and cloned, however, the subtype identity of the mitochondrial ryanodine receptor (mRyR) is unknown. Using subtype specific antibodies, we characterized the mRyR in the IMM from rat heart as RyR1. These results are substantiated by the absence of RyR protein in heart mitochondria from RyR1 knockout mice. The bell-shape Ca²⁺-dependent [³H]ryanodine binding curve and its modulation by caffeine and adenylylmethylenediphosphonate (AMPPCP) give further evidence that mRyR functions pharmacologically like RyR1. Ryanodine prevents mitochondrial Ca²⁺ uptake induced by raising extramitochondrial Ca²⁺ to 10 μM. Similarly, ryanodine inhibits oxidative phosphorylation stimulated by 10 μM extramitochondrial Ca²⁺. In summary, our results show that the mRyR in cardiac muscle has similar biochemical and pharmacological properties to the RyR1 in the sarcoplasmic reticulum (SR) of skeletal muscle. These results could also suggest an efficient mechanism by which mitochondria sequesters Ca²⁺ via mRyR during excitation–contraction coupling to stimulate oxidative phosphorylation for ATP production to meet metabolic demands. Thus, the mRyR functions as a transducer for excitation–metabolism coupling.

Keywords: Abbreviations; Ca; ²⁺; calcium; CaUP; mitochondrial calcium uniporter; RR; ruthenium red; SR; sarcoplasmic reticulum; RyR; ryanodine receptor; SR-RyR; sarcoplasmic RyR; mRyR; mitochondrial RyR; SERCA; sarco- and endoplasmic reticulum Ca; ²⁺; ATPase; IMM; inner mitochondrial membrane; VDAC; voltage gated anion channel; AMPPCP; adenylylmethylenedi-phosphonate; RCI; respiratory control indexHeart; Mitochondria; Mitochondrial ryanodine receptor; Calcium; Oxygen consumption

α-Lactalbumin binding and membrane integrity—effect of charge and degree of unsaturation of glycerophospholipids by Ingunn R�dland; �yvind Halskau; Aurora Mart�nez; Holm Holmsen (pp. 11-20).

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein–membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, ( apo)-conformation of bovine α-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation—leading to looser packing—promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA–membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human α-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.

Keywords: Abbreviations; BLA; bovine α-lactalbumin; CMC; critic micelle concentration; HAMLET; human lactalbumin made lethal to tumour cells; DOPG; 1,2-dioleylphosphatidylglycerol; DSPS; 1,2-distearoyl-sn-glycero-3-[phospho-; l; -serine]; EYPC; egg yolk phosphatidylcholine; LUV; large unilamellar vesicles; SOPS; 1-stearoyl-2-oleyl-sn-glycero-3-[phospho-; l; -serine]; SUV; small unilamellar lipid vesiclesAlpha lactalbumin; Protein–membrane interaction; Liposome; Langmuir; Vesicular leakage; Fluorescence

Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria by Xiang-Hong Liu; Alptekin Aksan; Michael A. Menze; Steven C. Hand; Mehmet Toner (pp. 21-26).

Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (∼190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.

Keywords: Trehalose; Desiccation; Mitochondria; Permeabilization; Mitochondrial membrane potential; Mitochondrial permeability transition pore

Inhibition of crotoxin binding to synaptosomes by a receptor-like protein from Crotalus durissus terrificus (the South American rattlesnake) by Roberta M�rcia Marques dos Santos; Leida Caleg�rio Oliveira; Maria In�cia Estev�o-Costa; Maria Elena de Lima; Marcelo Matos Santoro; Consuelo Latorre Fortes-Dias (pp. 27-33).

Crotoxin (Ctx) is a potent neurotoxin of the venom of Crotalus durissus terrificus (the South American rattlesnake). Ctx is a heterodimer composed of CB, a toxic PLA₂ subunit, and CA, a non-toxic and non-enzymatic subunit, that potentiates the neurotoxicity of CB in vivo. The deleterious action of Ctx upon C. d. terrificus snakes themselves is known to be prevented by a PLA₂ inhibitor (CNF) present in their blood serum. CNF acts by replacing CA in Ctx, thus forming a new stable complex CNF–CB. This complex no longer interacts with the target receptor (TR) to deliver CB to cause its lethal effect. Furthermore, CNF–CB seems to be reminiscent of the interaction Ctx–TR at the pre-synaptic site. In the present work, the binding competition between rat brain synaptosomes (TR) and CNF for Ctx was investigated. Radiolabeled Ctx, made of CA and one isoform of CB (CA–¹²⁵ICB₂), was used as ligand. The competition by unlabeled Ctx was taken as a reference. The potency of CNF as a competitor was evaluated under different incubation conditions with varying time scale addition of reagents (CA–¹²⁵ICB₂, synaptosomes and CA–CB₂ or CNF). CNF was able to inhibit the binding of the toxin to synaptosomes as well as to partially displace the toxin already bound to its membrane target. The mechanisms of competition involved were discussed and a previous schematic model of interactions between Ctx, TR and CNF was updated.

Keywords: Crotoxin; Phospholipase A; ₂; Phospholipase A; ₂; inhibitor; Synaptosome; CNF; Crotalus

Identification of a major protein on the cytosolic face of caveolae by J. Vinten; A.H. Johnsen; P. Roepstorff; J. Harp�th; J. Tranum-Jensen (pp. 34-40).

Cav-p60, a specific and ubiquitous caveolar protein, was immunoprecipitated from solubilized rat adipocyte plasma membranes and identified as similar to a GeneBank entry annotated mouse polymerase transcript release factor (PTRF) by MALDI-TOF and MS-MS of major fragments. Cloning and virtual translation of the corresponding rat adipocyte cDNA sequence revealed 98.7% identity with mouse PTRF. In vitro translation of this sequence produced a protein, which was recognized by antibodies to both cav-p60 and PTRF. EM gold labeling studies showed that a rabbit antiserum against murine PTRF immunolabeled caveolae specifically in adipocytes from both mouse and rat. In view of the reported function of the protein, which is exerted in the cell nucleus, its subcellular localization was investigated. We found that the protein could be purified by differential solubilization of a plasma membrane fraction followed by SDS-PAGE, and that the protein was as abundant as caveolin in this fraction. We were unable to detect the protein in cell nuclei by subcellular fractionation or fluorescence microscopy. The results show that in a large number of cell types, PTRF is essentially located to caveolae, and that each caveola harbors many copies of the protein. Consequently, we suggest the name Cavin for this protein.

Keywords: Rat adipocyte; Caveolin; cav-p60; Electron microscopy; Polymerase and Transcript Release Factor; Mass spectrometry

Effects of gramicidin-A on the adsorption of phospholipids to the air–water interface by Samares C. Biswas; Shankar B. Rananavare; Stephen B. Hall (pp. 41-49).

Prior studies suggest that the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of the lipids in pulmonary surfactant to an air–water interface by stabilizing a negatively curved rate-limiting structure that is intermediate between bilayer vesicles and the surface film. This model predicts that other peptides capable of stabilizing negative curvature should also promote lipid adsorption. Previous reports have shown that under appropriate conditions, gramicidin-A (GrA) induces dioleoyl phosphatidylcholine (DOPC), but not dimyristoyl phosphatidylcholine (DMPC), to form the negatively curved hexagonal-II (H_II) phase. The studies reported here determined if GrA would produce the same effects on adsorption of DMPC and DOPC that the hydrophobic surfactant proteins have on the surfactant lipids. Small angle X-ray scattering and³¹P-nuclear magnetic resonance confirmed that at the particular conditions used to study adsorption, GrA induced DOPC to form the H_II phase, but DMPC remained lamellar. Measurements of surface tension showed that GrA in vesicles produced a general increase in the rate of adsorption for both phospholipids. When restricted to the interface, however, in preexisting films, GrA with DOPC, but not with DMPC, replicated the ability of the surfactant proteins to promote adsorption of vesicles containing only the lipids. The correlation between the structural and functional effects of GrA with the two phospholipids, and the similar effects on adsorption of GrA with DOPC and the hydrophobic surfactant proteins with the surfactant lipids fit with the model in which SP-B and SP-C facilitate adsorption by stabilizing a rate-limiting intermediate with negative curvature.

Keywords: Gramicidin; Lipid polymorphism; ³¹; P nuclear magnetic resonance; Pulmonary surfactant; Hydrophobic surfactant protein; Small angle x-ray scattering

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout ( Oncorhynchus mykiss) gill cells by Kristine Rom�ren; Xuan T.L. Fjeld; Antonio B.S. Pol�o; Gro Smistad; Beate J. Thu; �ystein Evensen (pp. 50-57).

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.

Keywords: DNA vaccine; Immersion; Rainbow trout gill cell

Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing³¹P and²H solid-state NMR spectroscopy by Shadi Abu-Baker; Xiaoyang Qi; Justin Newstadt; Gary A. Lorigan (pp. 58-66).

Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using²H and³¹P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 °C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (S_CD) were calculated from the dePaked²H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S_CD profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S_CD profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S_CD profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1–C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The³¹P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010–27017] [1].

Keywords: Abbreviations; Sap C; Saposin C; S; _CD; Molecular Order Parameters; CSA; Chemical Shift Anisotropy; GCase; Glucosylceramidase; CL; Cardiolipin; DOPG; Dioleoylphosphatidylglycerol; DOPS; Dioleoylphosphatidylserine; PC; Phosphoatidyecholines; PG; Phosphatidylglycerol; PS; Phosphatidylserine; DSPG-d70; Distearoyl-d70-phosphatidylglycerol; DMPC; Dimyristylphosphatidylcholine; HCL; Hydrochloric Acid; TFE; 2,2,2 Triflouroethanol; PLL; Poly(; l; -lysine); NMR; Nuclear Magnetic Resonance; CP-MAS; Cross-Polarization Magic-Angle Spinning; HPLC; High Performance Liquid Chromatography; MALDI-TOF; Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry; MLVs; Multilamellar vesiclesSaposin C; Dioleoylphosphatidylglycerol; Dioleoylphosphatidylserine; Solid-state NMR spectroscopy; Molecular order parameter; Mixed bilayer

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BBA - Biomembranes (v.1717, #1)