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BBA - Biomembranes (v.1798, #8)

Editorial Board (pp. i).

CLC channels and transporters: Proteins with borderline personalities by Alessio Accardi; Alessandra Picollo (pp. 1457-1464).

Controlled chloride movement across membranes is essential for a variety of physiological processes ranging from salt homeostasis in the kidneys to acidification of cellular compartments. The CLC family is formed by two, not so distinct, sub-classes of membrane transport proteins: Cl^- channels and H⁺/Cl^- exchangers. All CLC's are homodimers with each monomer forming an individual Cl- permeation pathway which appears to be largely unaltered in the two CLC sub-classes. Key residues for ion binding and selectivity are also highly conserved. Most CLC's have large cytosolic carboxy-terminal domains containing two cystathionine β-synthetase (CBS) domains. The C-termini are critical regulators of protein trafficking and directly modulate Cl- by binding intracellular ATP, H+ or oxidizing compounds. This review focuses on the recent mechanistic insights on the how the structural similarities between CLC channels and transporters translate in unexpected mechanistic analogies between these two sub-classes.

Keywords: Chloride; Channel; Transporter; Structure

Oxidative protein folding in the endoplasmic reticulum: Tight links to the mitochondria-associated membrane (MAM) by Thomas Simmen; Emily M. Lynes; Kevin Gesson; Gary Thomas (pp. 1465-1473).

The production of secretory proteins at the ER (endoplasmic reticulum) depends on a ready supply of energy and metabolites as well as the close monitoring of the chemical conditions that favor oxidative protein folding. ER oxidoreductases and chaperones fold nascent proteins into their export-competent three-dimensional structure. Interference with these protein folding enzymes leads to the accumulation of unfolded proteins within the ER lumen, causing an acute organellar stress that triggers the UPR (unfolded protein response). The UPR increases the transcription of ER chaperones commensurate with the load of newly synthesized proteins and can protect the cell from ER stress. Persistant stress, however, can force the UPR to commit cells to undergo apoptotic cell death, which requires the emptying of ER calcium stores. Conversely, a continuous ebb and flow of calcium occurs between the ER and mitochondria during resting conditions on a domain of the ER that forms close contacts with mitochondria, the MAM (mitochondria-associated membrane). On the MAM, ER folding chaperones such as calnexin and calreticulin and oxidoreductases such as ERp44, ERp57 and Ero1α regulate calcium flux from the ER through reversible, calcium and redox-dependent interactions with IP3Rs (inositol 1,4,5-trisphophate receptors) and with SERCAs (sarcoplasmic/endoplasmic reticulum calcium ATPases). During apoptosis progression and depending on the identity of the ER chaperone and oxidoreductase, these interactions increase or decrease, suggesting that the extent of MAM targeting of ER chaperones and oxidoreductases could shift the readout of ER–mitochondria calcium exchange from housekeeping to apoptotic. However, little is known about the cytosolic factors that mediate the on/off interactions between ER chaperones and oxidoreductases with ER calcium channels and pumps. One candidate regulator is the multi-functional molecule PACS-2 (phosphofurin acidic cluster sorting protein-2). Recent studies suggest that PACS-2 mediates localization of a mobile pool of calnexin to the MAM in addition to regulating homeostatic ER calcium signaling as well as MAM integrity. Together, these findings suggest that cytosolic, membrane and lumenal proteins combine to form a two-way switch that determines the rate of protein secretion by providing ions and metabolites and that appears to participate in the pro-apoptotic ER–mitochondria calcium transfer.

Keywords: Endoplasmic reticulum (ER); Mitochondria; Mitochondria-associated membrane (MAM); Oxidative protein folding; Chaperone; Apoptosis

Hydration valve controlled non-selective conduction of Na⁺ and K⁺ in the NaK channel by Rong Shen; Wanlin Guo; Wenyu Zhong (pp. 1474-1479).

The Na⁺ and K⁺ channels are essential to neural signaling, but our current knowledge at the atomic level is mainly limited to the conducting mechanism of K⁺. Unlike a K⁺ channel having four equivalent K⁺-binding sites in its selectivity filter, a NaK channel has a vestibule in the middle part of its selectivity filter, and can conduct both Na⁺ and K⁺ ions. However, the underlying mechanism for non-selective ion conduction in NaK remains elusive. Here we find four small grottos connecting with the vestibule of the NaK selectivity filter, which form a vestibule-grotto complex perpendicular to the filter pore with a few water molecules within it. It is shown that two or more of the water molecules coming to the vestibule to coordinate the cation are necessary for conducting both Na⁺ and K⁺ ions, while only one water molecule in the vestibule will obstruct ion permeation. Thus, the complex with the aid of interior water movement forms a dynamic hydration valve which is flexible in conveying different cations through the vestibule. Similar exquisite hydration valve mechanisms are expected to be utilized by other non-selective cation channels, and the results should shed new light on the importance of water in neural signaling.

Keywords: Hydration valve; Ion permeation; NaK channel; Water dynamics

Oligo-( R)-3-hydroxybutyrate modification of sorting signal enables pore formation by Escherichia coli OmpA by A. Negoda; E. Negoda; R.N. Reusch (pp. 1480-1484).

The outer membrane protein A (OmpA) of Escherichia coli is a well-known model for protein targeting and protein folding. Wild-type OmpA, isolated either from cytoplasmic inclusion bodies or from outer membranes, forms narrow pores of ∼80 pS in planar lipid bilayers at room temperature. The pores are well structured with narrow conductance range when OmpA is isolated using lithium dodecyl sulfate (LDS) or RapiGest surfactant but display irregular conductance when OmpA is isolated with urea or guanidine hydrochloride. Previous studies have shown that serine residues S163 and S167 of the sorting signal of OmpA (residues 163–169), i.e., the essential sequence for outer membrane incorporation, are covalently modified by oligomers of ( R)-3-hydroxybutyrate (cOHB). Here we find that single-mutants S163 and S167 of OmpA, which still contain cOHB on one serine of the sorting signal, form narrow pores in planar lipid bilayers at room temperature with lower and more irregular conductance than wild-type OmpA, whereas double mutants S163:S167 and S163:V166 of OmpA, with no cOHB on the sorting signal, are unable to form stable pores in planar lipid bilayers. Our results indicate that modification of serines in the sorting signal of OmpA by cOHB in the cytoplasm enables OmpA to incorporate into lipid bilayers at room temperature as a narrow pore. They further suggest that cOHB modification may be an important factor in protein targeting and protein folding.

Keywords: OmpA; Oligo-(; R; )-3-hydroxybutyrate (OHB); Complexed OHB (cOHB); Outer membrane sorting; Sorting signal; Bilayer incorporation; Pore formation; Protein modification; Amphipathic polymer

Antimicrobial peptides in toroidal and cylindrical pores by Maja Mihajlovic; Themis Lazaridis (pp. 1485-1493).

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH ∼7, the net charge of alamethicin is −1 whereas that of melittin is +5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions.

Keywords: Molecular dynamics simulation; Antimicrobial peptide; Toroidal pore; Barrel-stave pore; Melittin; Alamethicin

Antimicrobial peptides bind more strongly to membrane pores by Maja Mihajlovic; Themis Lazaridis (pp. 1494-1502).

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis.

Keywords: Molecular dynamics simulation; Antimicrobial peptide; Toroidal pore; Barrel-stave pore; Implicit solvent

Specific anchoring modes of two distinct dystrophin rod sub-domains interacting in phospholipid Langmuir films studied by atomic force microscopy and PM-IRRAS by Vie V. Vié; S. Legardinier; L. Chieze; O. Le Bihan; Y. Qin; J. Sarkis; J.-F. Hubert; A. Renault; B. Desbat; E. Le Rumeur (pp. 1503-1511).

Dystrophin rod repeats 1–3 sub-domain binds to acidic phosphatidylserine in a small vesicle binding assay, while the repeats 20–24 sub-domain does not. In the present work, we studied the adsorption behaviour of both sub-domains at the air/liquid interface and at the air/lipid interface in a Langmuir trough in order to highlight differences in interfacial properties. The adsorption behaviour of the two proteins at the air/liquid interface shows that they display surface activity while maintaining their alpha-helical secondary structure as shown by PM-IRRAS. Strikingly, R20–24 needs to be highly hydrated even at the interface, while this is not the case for R1–3, indicating that the surface activity is dramatically higher for R1–3 than R20–24. Surface-pressure measurements, atomic force microscopy and PM-IRRAS are used in a Langmuir experiment with DOPC–DOPS monolayers at two different surface pressures, 20mN/m and 30mN/m. At the lower surface pressure, the proteins are adsorbed at the lipid film interface while maintaining its alpha-helical structure. After an increase of the surface pressure, R1–3 subsequently produces a stable film, while R20–24 induces a reorganization of the lipid film with a subsequent decrease of the surface pressure close to the initial value. AFM and PM-IRRAS show that R1–3 is present in high amounts at the interface, being arranged in clusters representing 3.3% of the surface at low pressure. By contrast, R20–24 is present at the interface in small amounts bound only by a few electrostatic residues to the lipid film while the major part of the molecule remains floating in the sub-phase. Then for R1–3, the electrostatic interaction between the proteins and the film is enhanced by hydrophobic interactions. At higher surface pressure, the number of protein clusters increases and becomes closer in both cases implying the electrostatic character of the binding. These results indicate that even if the repeats exhibit large structural similarities, their interfacial properties are highly contrasted by their differential anchor mode in the membrane. Our work provides strong support for distinct physiological roles for the spectrin-like repeats and may partly explain the effects of therapeutic replacement of dystrophin deficiency by minidystrophins.

Keywords: Abbreviations; AFM; atomic force microscopy; LB film; Langmuir–Blodgett film; PM-IRRAS; polarization modulated-infrared reflection–absorption spectroscopyDystrophin rod domain; Spectrin-like repeats; Langmuir films; Atomic force microscopy; Polarization modulation infrared spectroscopy

The secretory pathway Ca²⁺-ATPase 1 is associated with cholesterol-rich microdomains of human colon adenocarcinoma cells by Szilvia Baron; Peter Vangheluwe; Maria Rosario Sepúlveda; Frank Wuytack; Luc Raeymaekers; Jo Vanoevelen (pp. 1512-1521).

Lipid rafts are often considered as microdomains enriched in sphingomyelin and cholesterol, predominantly residing in the plasma membrane but which originate in earlier compartments of the cellular secretory pathway. Within this pathway, the membranes of the Golgi complex represent a transition stage between the cholesterol-poor membranes of the endoplasmic reticulum (ER) and the cholesterol-rich plasma membrane. The rafts are related to detergent-resistant membranes, which because of their ordered structure are poorly penetrated by cold non-ionic detergents and float in density gradient centrifugation. In this study the microdomain niche of the Golgi-resident SPCA Ca²⁺/Mn²⁺ pumps was investigated in HT29 cells by Triton X-100 detergent extraction and density-gradient centrifugation. Similarly to cholesterol and the raft-resident flotillin-2, SPCA1 was found mainly in detergent-resistant fractions, while SERCA3 was detergent-soluble. Furthermore, cholesterol depletion of cells resulted in redistribution of flotillin-2 and SPCA1 to the detergent-soluble fractions of the density gradient. Additionally, the time course of solubilization by Triton X-100 was investigated in live COS-1 and HT29 cells expressing fluorescent SERCA2b, SPCA1d or SPCA2. In both cell types, the ER-resident SERCA2b protein was gradually solubilized, while SPCA1d resisted to detergent solubilization. SPCA2 was more sensitive to detergent extraction than SPCA1d. To investigate the functional impact of cholesterol on SPCA1, ATPase activity was monitored. Depletion of cholesterol inhibited the activity of SPCA1d, while SERCA2b function was not altered. From these results we conclude that SPCA1 is associated with cholesterol-rich domains of HT29 cells and that the cholesterol-rich environment is essential for the functioning of the pump.

Keywords: Golgi; SPCA1; Triton X-100; Detergent-resistant membrane; Flotillin-2; Methyl-β-cyclodextrin

Interaction of a new anticancer prodrug, gemcitabine–squalene, with a model membrane by Barbara Pili; Claudie Bourgaux; Heinz Amenitsch; Gérard Keller; Lepetre-Mouelhi Sinda Lepêtre-Mouelhi; Desmaele Didier Desmaële; Patrick Couvreur; Michel Ollivon (pp. 1522-1532).

Gemcitabine is an anticancer nucleoside analogue active against a wide variety of solid tumors. However it is rapidly deaminated to an inactive metabolite, leading to short biological half-life and induction of resistance. A new prodrug of gemcitabine, coupling squalene to gemcitabine (GemSq), has been designed to overcome the above drawbacks. It has been previously shown that this prodrug displays significantly higher anticancer activity than gemcitabine against leukemia. In the present study the structural modifications of dipalmitoylphosphatidylcholine (DPPC) model membranes induced by increasing concentrations of GemSQ have been investigated using small and wide angle X-ray scattering (SWAXS) and differential scanning calorimetry (DSC). At room temperature an unusual inverse bicontinuous cubic phase formed over a broad composition range. The basic bilayer structure displayed an intermediate order between those of the gel and fluid phases of DPPC. A reversible transition to a fluid lamellar phase occurred upon heating. The transitions between these two phases were governed by different mechanisms depending on the GemSq concentration in the membrane. Finally, the biological relevance of these observations for the cytotoxic activity of GemSq has been discussed.

Keywords: Gemcitabine–squalene; Anticancer prodrug; Prodrug–membrane interaction; Inverse bicontinue cubic phase; Small and wide angle X-ray scattering; Differential scanning calorimetry

Influence of lysophospholipid hydrolysis by the catalytic domain of neuropathy target esterase on the fluidity of bilayer lipid membranes by Aaron J. Greiner; Rudy J. Richardson; R. Mark Worden; Robert Y. Ofoli (pp. 1533-1539).

Neuropathy target esterase (NTE) is an integral membrane protein localized in the endoplasmic reticulum in neurons. Irreversible inhibition of NTE by certain organophosphorus compounds produces a paralysis known as organophosphorus compound-induced delayed neuropathy. In vitro, NTE has phospholipase/lysophospholipase activity that hydrolyses exogenously added single-chain lysophospholipids in preference to dual-chain phospholipids, and NTE mutations have been associated with motor neuron disease. NTE's physiological role is not well understood, although recent studies suggest that it may control the cytotoxic accumulation of lysophospholipids in membranes. We used the NTE catalytic domain (NEST) to hydrolyze palmitoyl-2-hydroxy- sn-glycero-3-phosphocholine (p-lysoPC) to palmitic acid in bilayer membranes comprising 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and the fluorophore 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]- sn-glycero-3-phosphocholine (NBD-PC). Translational diffusion coefficients ( D_L) in supported bilayer membranes were measured by fluorescence recovery after pattern photobleaching (FRAPP). The average D_L for DOPC/p-lysoPC membranes without NEST was 2.44µm²s^-1±0.09; the D_L for DOPC/p-lysoPC membranes containing NEST and diisopropylphosphorofluoridate, an inhibitor, was nearly identical at 2.45±0.08. By contrast, the D_L for membranes comprising NEST, DOPC, and p-lysoPC was 2.28±0.07, significantly different from the system with inhibited NEST, due to NEST hydrolysis. Likewise, a system without NEST containing the amount of palmitic acid that would have been produced by NEST hydrolysis of p-lysoPC was identical at 2.26±0.06. These results indicate that NTE's catalytic activity can alter membrane fluidity.

Keywords: Bilayer membrane fluidity; Lysophospholipid hydrolysis; Neuropathy target esterase (NTE); Neuropathy target esterase catalytic domain (NEST); Organophosphorus inhibition; Supported bilayer lipid membrane

Crystallization of the membrane protein hVDAC1 produced in cell-free system by A. Deniaud; L. Liguori; I. Blesneac; J.L. Lenormand; E. Pebay-Peyroula (pp. 1540-1546).

Structural studies of membrane proteins are in constant evolution with the development of new improvements for their expression, purification, stabilization and crystallization. However, none of these methods still provides a universal approach to solve the structure of membrane proteins. Here we describe the crystallization of the human voltage-dependent anion channel-1 produced by a bacterial cell-free expression system. While VDAC structures have been recently solved, we propose an alternative strategy for producing the recombinant protein, which can be applied to other membrane proteins reluctant to expression, purification and crystallization by classical approaches. Despite a lot of efforts to crystallize a cell-free expressed membrane protein, this study is to our knowledge one of the first reports of a successful crystallization. Focusing on expression in a soluble and functional state, in a detergent environment, is the key to get crystals. Although the diffraction of VDAC crystals is limited, the simplicity and the rapidity to set-up and optimize this technology are drastic advantages in comparison to other methods.

Keywords: Membrane protein; Voltage-dependent anion channel; Cell-free expression; Crystallization

The specificity of frutalin lectin using biomembrane models by Thatyane M. Nobre; Felippe J. Pavinatto; Márcia R. Cominetti; Selistre de-Araujo Heloísa S. Selistre de-Araújo; Maria E.D. Zaniquelli; Leila M. Beltramini (pp. 1547-1555).

Frutalin is a homotetrameric α-d-galactose (d-Gal)-binding lectin that activates natural killer cells in vitro and promotes leukocyte migration in vivo. Because lectins are potent lymphocyte stimulators, understanding the interactions that occur between them and cell surfaces can help to the action mechanisms involved in this process. In this paper, we present a detailed investigation of the interactions of frutalin with phospho- and glycolipids using Langmuir monolayers as biomembrane models. The results confirm the specificity of frutalin ford-Gal attached to a biomembrane. Adsorption of frutalin was more efficient for the galactose polar head lipids, in contrast to the one for sulfated galactose, in which a lag time is observed, indicating a rearrangement of the monolayer to incorporate the protein. Regarding ganglioside GM1 monolayers, lower quantities of the protein were adsorbed, probably due to the farther apart position ofd-galactose from the interface. Binary mixtures containing galactocerebroside revealed small domains formed at high lipid packing in the presence of frutalin, suggesting that lectin induces the clusterization and the forming of domains in vitro, which may be a form of receptor internalization. This is the first experimental evidence of such lectin effect, and it may be useful to understand the mechanism of action of lectins at the molecular level.

Keywords: Lectin; Frutalin; Langmuir monolayers; Glycolipids; Dynamic surface elasticity; Brewster angle microscopy

Identification of an “α-helix-extended segment-α-helix” conformation of the sixth transmembrane domain in DMT1 by Shuyan Xiao; Jiantao Li; Yuxia Wang; Chunyu Wang; Rong Xue; Shuo Wang; Fei Li (pp. 1556-1564).

DMT1 (divalent metal ion transporter 1) is one member of a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions. A pair of mutation-sensitive and highly conserved histidines in the sixth transmembrane domain (TM6) of DMT1 was found to be important for proton-metal ion cotransport. In the present work, we investigate the structures and locations of the peptides from TM6 of DMT1 and its H267A and H272A mutants in SDS micelles by CD and NMR methods. The circular dichroism studies show that the α-helix is a predominant conformation for the wildtype peptide and H267A mutant in SDS micelles, whereas the helicity is evidently decreased for H272A mutant. The pH value has little effect on the α-helical contents of the three peptides. The NMR studies indicate that the wildtype peptide in SDS micelles forms an “α-helix-extended segment-α-helix” structure in which the His267 locates near the central part of the extended segment, while the His272 is involved in the α-helical folding. Both histidines are buried in SDS micelles as evidenced by their p K_a values. The structure of the wildtype peptide is evidently changed by the mutations of H267A and H272A. The H267A mutant forms an ordered structure consisting of an α-helix from the C-terminus to the central part and continuous turns in the residual part. The extended structure in the central part of the wildtype peptide is abolished by H267A mutation. The H272A mutation mainly induces unfolding of the short helix in the N-terminal side, while the short helix in the C-terminal side and unordered conformation in the central part remain. All the three peptides are embedded in SDS micelles, and the H267A mutant is inserted more deeply due to increasing hydrophobicity in the central part of the peptide. The specific “α-helix-extended segment-α-helix” structure of TM6 may have an important implication for the binding of the transporter to H⁺ and metal ions and the conformation change induced by the mutations of two highly conserved histidines may be correlated to the deficiency of the transport activity of DMT1.

Keywords: DMT1-TM6; discontinuous structure; histidine; NMR

Sustained intracellular acidosis activates the myocardial Na⁺/H⁺ exchanger independent of amino acid Ser⁷⁰³ and p90^rsk by Pratap Karki; Ersilia Coccaro; Larry Fliegel (pp. 1565-1576).

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the myocardium and other tissues. It is an important mediator of the myocardial damage that occurs after ischemia–reperfusion injury and is implicated in heart hypertrophy. Regulation of NHE1 has been proposed as a therapeutic target for cardioprotection. We therefore examined mechanisms of control of NHE1 in the myocardium. Several different amino acids have been implicated as a being critical to NHE1 regulation in a number of tissues including Ser⁷⁰³, Ser⁷⁷⁰, and Ser⁷⁷¹. In the myocardium, NHE1 is activated in response to a variety of stimuli including activation by an ERK-dependent sustained intracellular acidosis. In this study, we determined whether Ser⁷⁰³ and p90^rsk activity are critical in activation of NHE1 by sustained intracellular acidosis. In vitro phosphorylation of NHE1 C-terminal fusion proteins determined that ERK-dependent phosphorylation of the cytoplasmic region was not dependent on Ser⁷⁰³; however, phosphorylation by p90^rsk required Ser⁷⁰³. A Ser703Ala mutation decreased basal NHE1 activity in CHO cells but not in cardiomyocytes. NHE1 with a Ser703Ala mutation was activated in response to sustained intracellular acidosis in CHO cells. In addition, sustained intracellular acidosis also activated the Ser703Ala mutant protein in isolated cardiomyocytes and phosphorylation levels were also increased by acidosis. The presence of a dominant-negative p90^rsk kinase also did not prevent activation and phosphorylation of NHE1 by sustained intracellular acidosis in isolated cardiomyocytes. We conclude that Ser⁷⁰³ and p90^rsk are not required for activation by sustained intracellular acidosis and that p90^rsk phosphorylation of Ser⁷⁰³ is independent of this type of activation.

Keywords: Abbreviations; CHO; Chinese hamster ovary; ERK; extracellular signal-regulated kinase; NHE1; Na; ⁺; /H; ⁺; exchanger type 1 isoform; GSK3β; glycogen synthase kinase-3beta; HA; hemagglutinin; RSK; ribosomal S6 kinase; SIA; sustained intracellular acidosisCardiomyocyte; ERK1/2; Na; ⁺; /H; ⁺; exchanger; Phosphorylation; p90; ^rsk; Sustained intracellular acidosis

The dynamics of cardiolipin synthesis post-mitochondrial fusion by Fred Y. Xu; Heidi McBride; Devrim Acehan; Frédéric M. Vaz; Riekelt H. Houtkooper; Raymond M. Lee; Michael A. Mowat; Grant M. Hatch (pp. 1577-1585).

Alteration in mitochondrial fusion may regulate mitochondrial metabolism. Since the phospholipid cardiolipin (CL) is required for function of the mitochondrial respiratory chain, we examined the dynamics of CL synthesis in growing Hela cells immediately after and 12h post-fusion. Cells were transiently transfected with Mfn-2, to promote fusion, or Mfn-2 expressing an inactive GTPase for 24h and de novo CL biosynthesis was examined immediately after or 12h post-fusion. Western blot analysis confirmed elevated Mfn-2 expression and electron microscopic analysis revealed that Hela cell mitochondrial structure was normal immediately after and 12h post-fusion. Cells expressing Mfn-2 exhibited reduced CL de novo biosynthesis from [1,3-³H]glycerol immediately after fusion and this was due to a decrease in phosphatidylglycerol phosphate synthase (PGPS) activity and its mRNA expression. In contrast, 12h post-mitochondrial fusion cells expressing Mfn-2 exhibited increased CL de novo biosynthesis from [1,3-³H]glycerol and this was due to an increase in PGPS activity and its mRNA expression. Cells expressing Mfn-2 with an inactive GTPase activity did not exhibit alterations in CL de novo biosynthesis immediately after or 12h post-fusion. The Mfn-2 mediated alterations in CL de novo biosynthesis were not accompanied by alterations in CL or monolysoCL mass. [1-¹⁴C]Oleate incorporation into CL was elevated at 12h post-fusion indicating increased CL resynthesis. The reason for the increased CL resynthesis was an increased mRNA expression of tafazzin, a mitochondrial CL resynthesis enzyme. Ceramide-induced expression of PGPS in Hela cells or in CHO cells did not alter expression of Mfn-2 indicating that Mfn-2 expression is independent of altered CL synthesis mediated by elevated PGPS. In addition, Mfn-2 expression was not altered in Hela cells expressing phospholipid scramblase-3 or a disrupted scramblase indicating that proper CL localization within mitochondria is not essential for Mfn-2 expression. The results suggest that immediately post-mitochondrial fusion CL de novo biosynthesis is “slowed down” and then 12h post-fusion it is “upregulated”. The implications of this are discussed.

Keywords: Cardiolipin; Phospholipid synthesis; Mitofusion-2; Mitochondria; Metabolism

Bilayer structural destabilization by low amounts of chlorophyll a by Radka Vladkova; Rumiana Koynova; Klaus Teuchner; Boris Tenchov (pp. 1586-1592).

The present study shows that small admixtures of one chlorophyll a (Chl a) molecule per several hundred lipid molecules have strong destabilizing effect on lipid bilayers. This effect is clearly displayed in the properties of the L_α–H_II transformations and results from a Chl a preference for the H_II relative to the L_α phase. Chl a disfavors the lamellar liquid crystalline phase L_α and induces its replacement with inverted hexagonal phase H_II, as is consistently demonstrated by DSC and X-ray diffraction measurements on phosphatidylethanolamine (PE) dispersions. Chl a lowers the L_α–H_II transition temperature (42°C) of the fully hydrated dipalmitoleoyl PE (DPoPE) by ∼8°C and ∼17°C at Chl a/DPoPE molar ratios of 1:500 and 1:100, respectively. Similar Chl a effect was recorded also for dielaidoyl PE dispersions. The lowering of the transition temperature and the accompanying significant loss of transition cooperativity reflect the Chl a repartitioning and preference for the H_II phase. The reduction of the H_II phase lattice constant in the presence of Chl a is an indication that Chl a favors H_II phase formation by decreasing the radius of spontaneous monolayer curvature, and not by filling up the interstitial spaces between the H_II phase cylinders. The observed Chl a preference for H_II phase and the substantial bilayer destabilization in the vicinity of a bilayer-to-nonbilayer phase transformation caused by low Chl a concentrations can be of interest as a potential regulatory or membrane-damaging factor.

Keywords: Abbreviations; Chl; a; chlorophyll; a; PE; phosphatidylethanolamine; DPoPE; 1,2-Dipalmitoleoyl-; sn; -glycero-3-phosphoethanolamine; DEPE; 1,2-Dielaidoyl-; sn; -glycero-3-phosphoethanolamine; DSC; differential scanning calorimetry; MeOH; methanol; MGDG; monogalactosyldiacylglycerolChlorophyll; a; Nonbilayer lipid; X-ray diffraction; Differential scanning calorimetry

Activation of phospholipase A2 by 1-palmitoyl-2-(9'-oxo-nonanoyl)- sn-glycero-3-phosphocholine in vitro by Christian Code; Ajay K. Mahalka; Kristian Bry; Paavo K.J. Kinnunen (pp. 1593-1600).

Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of oxidatively modified lipids. To this end, oxidized phospholipid derivatives bearing an aldehyde function, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)- sn-glycero-3-phosphocholine (PoxnoPC) can covalently react with proteins that come into direct contact. Intriguingly, we observed PoxnoPC in a 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) matrix to shorten and abolish the lag time in the action of phospholipase A2 (PLA2) on this composite substrate, with concomitant augmented decrement in pH, indicating more extensive hydrolysis, which was in keeping with enhanced 90° light scattering. The latter was abolished by the aldehyde scavenger methoxyamine, thus suggesting the involvement of Schiff base. Enhanced hydrolysis of a fluorescent phospholipid analogue was seen for PLA2 preincubated with PoxnoPC. Mixing PLA2 with submicellar (22µM) PoxnoPC caused a pronounced increase in Thioflavin T fluorescence, in keeping with the formation of amyloid-type fibers, which were seen also by electron microscopy.

Keywords: Abbreviations; Aβ; amyloid beta; C28-OPHPM; 1-octosanyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatidylmonomethyl ester; CMC; critical micelle concentration; CD; circular dichroism; DPPC; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; EDTA; ethylenediaminetetraacetic acid; EM; electron microscopy; FFA; free fatty acid; FRET; Förster-type resonance energy transfer; FTIR; Fourier transform infared spectroscopy; LUV; large unilamellar vesicles; lysoPC; 1-palmitoyl-sn-glycero-3-phosphocholine; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, PoxnoPC, 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine, PLA2, phospholipase A2; PLA2A; labeled with Alexa 568 (acceptor); PLA2D; labeled with Alexa 488 (donor); PUFA; polyunsaturated fatty acids; T; _m; main phase transition temperature; ThT; thioflavin T; UV; ultraviolet; X; mol %Phospholipase A2; Oligomerization; Oxidized phospholipids; PoxnoPC

Behavior of plant plasma membranes under hydrostatic pressure as monitored by fluorescent environment-sensitive probes by Yann Roche; Andrey S. Klymchenko; Patricia Gerbeau-Pissot; Patrick Gervais; Mely Yves Mély; Françoise Simon-Plas; Jean-Marie Perrier-Cornet (pp. 1601-1607).

We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5kbar at 30°C, by steady-state fluorescence spectroscopy using the newly introduced environment-sensitive probe F2N12S and also Laurdan and di-4-ANEPPDHQ. The consequences of sterol depletion by methyl-β-cyclodextrin were also studied. We found that application of hydrostatic pressure led to a marked decrease of hydration as probed by F2N12S and to an increase of the generalized polarization excitation (GPex) of Laurdan. We observed that the hydration effect of sterol depletion was maximal between 1 and 1.5kbar but was much less important at higher pressures (above 2kbar) where both parameters reached a plateau value. The presence of a highly dehydrated gel state, insensitive to the sterol content, was thus proposed above 2.5kbar. However, the F2N12S polarity parameter and the di-4-ANEPPDHQ intensity ratio showed strong effect on sterol depletion, even at very high pressures (2.5–3.5kbar), and supported the ability of sterols to modify the electrostatic properties of membrane, notably its dipole potential, in a highly dehydrated gel phase. We thus suggested that BY-2 PM undergoes a complex phase behavior in response to the hydrostatic pressure and we also emphasized the role of phytosterols to regulate the effects of high hydrostatic pressure on plant PM.

Keywords: High hydrostatic pressure; Fluorescence spectroscopy; Phytosterol; Plasma membrane; Methyl-β-cyclodextrin; 3-Hydroxyflavone

NMR structure of the transmembrane domain of the n-acetylcholine receptor β2 subunit by Vasyl Bondarenko; Tommy Tillman; Yan Xu; Pei Tang (pp. 1608-1614).

Nicotinic acetylcholine receptors (nAChRs) are involved in fast synaptic transmission in the central and peripheral nervous system. Among the many different types of subunits in nAChRs, the β2 subunit often combines with the α4 subunit to form α4β2 pentameric channels, the most abundant subtype of nAChRs in the brain. Besides computational predictions, there is limited experimental data available on the structure of the β2 subunit. Using high-resolution NMR spectroscopy, we solved the structure of the entire transmembrane domain (TM1234) of the β2 subunit. We found that TM1234 formed a four-helix bundle in the absence of the extracellular and intracellular domains. The structure exhibited many similarities to those previously determined for the Torpedo nAChR and the bacterial ion channel GLIC. We also assessed the influence of the fourth transmembrane helix (TM4) on the rest of the domain. Although secondary structures and tertiary arrangements were similar, the addition of TM4 caused dramatic changes in TM3 dynamics and subtle changes in TM1 and TM2. Taken together, this study suggests that the structures of the transmembrane domains of these proteins are largely shaped by determinants inherent in their sequence, but their dynamics may be sensitive to modulation by tertiary and quaternary contacts.

Keywords: Nicotinic acetylcholine receptor; nAChR; NMR; HFIP; Ion channel structure; Cys-loop structure

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols by Shishir Jaikishan; Bjorkbom Anders Björkbom; J. Peter Slotte (pp. 1615-1622).

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼49°C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼40–41°C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10–50°C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.

Keywords: Abbreviations; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; CTL; cholesta-5,7(11)-trien-3-beta-ol; DPH; 1,6-diphenyl-1,3,5-hexatriene; DSC; differential scanning calorimetry; DPPA; 1,2-dipalmitoyl-; sn; -glycero-3-phosphate; DPPBu; 1,2-dipalmitoyl-; sn; -glycero-3-phosphobutanol; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DPPEt; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanol; DPPMe; 1,2-dipalmitoyl-; sn; -glycero-3-phosphomethanol; DPPPr; 2-dipalmitoyl-; sn; -glycero-3-phosphopropanol; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; T; _m; gel–liquid transition temperature; ΔT; _1/2; full peak width at half maximum of T; _mPhospholipid interactions; Cholesterol interactions; Membrane domain; Calorimetry; Fluorescence quenching; DPH anisotropy

Corrigendum to “Structure of the antimicrobial β-hairpin peptide protegrin-1 in a DLPC lipid bilayer investigated by molecular dynamics simulation” [Biochim. Biophys. Acta 1768 (2007) 509–520] by Himanshu Khandelia; Yiannis N. Kaznessis (pp. 1623-1623).

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BBA - Biomembranes (v.1798, #8)

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BBA - Biomembranes (v.1798, #8)