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BBA - Biomembranes (v.1668, #2)

Editorial Board (pp. ii).

Publisher's note (pp. 157-157).

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy by Jonas Korlach; Tobias Baumgart; Watt W. Webb; Gerald W. Feigenson (pp. 158-163).

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.

Keywords: Fluorescence correlation spectroscopy; Membrane phase behavior; Lipid bilayer phase diagram; Ternary lipid mixture

Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions by Jamie L. Weiss; Nicholas A. Evans; Tanweer Ahmed; Jonathan D.J. Wrigley; Shukria Khan; Charles Wright; Jeffrey N. Keen; Andreas Holzenburg; John B.C. Findlay (pp. 164-174).

We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

Keywords: Abbreviations; DDAPS; N; -Dodecyl-; N; ,; N; -dimethyl-3-ammonio-propane sulfonate; HEK; human embryonic kidney; IP; ₃; inositol 1,4,5-triphosphate; LDAO; N; ,; N; -Dimethyl dodecylamine-; N; -oxide; PMSF; phenylmethylsulfonyl fluoride; RACE; rapid amplification of cDNA endsSquid; Loligo forbesi; Loligo pealei; Eye; Membrane protein

The interactions of antimicrobial peptides derived from lysozyme with model membrane systems by Howard N. Hunter; Weiguo Jing; David J. Schibli; Tony Trinh; In Yup Park; Sun Chang Kim; Hans J. Vogel (pp. 175-189).

Two peptides, RAWVAWR-NH₂ and IVSDGNGMNAWVAWR-NH₂, derived from human and chicken lysozyme, respectively, exhibit antimicrobial activity. A comparison between the L-RAWVAWR, D-RAWVAWR, and the longer peptide has been carried out in membrane mimetic conditions to better understand how their interaction with lipid and detergent systems relates to the reported higher activity for the all L-peptide. Using CD and 2D¹H NMR spectroscopy, the structures were studied with DPC and SDS micelles. Fluorescence spectroscopy was used to study peptide interactions with POPC and POPG vesicles and DOPC, DOPE, and DOPG mixed vesicle systems. Membrane–peptide interactions were also probed by ITC and DSC. The ability of fluorescein-labeled RAWVAWR to rapidly enter both E. coli and Staphylococcus aureus was visualized using confocal microscopy. Reflecting the bactericidal activity, the long peptide interacted very weakly with the lipids. The RAWVAWR-NH₂ peptides preferred lipids with negatively charged headgroups and interacted predominantly in the solvent–lipid interface, causing significant perturbation of membrane mimetics containing PG headgroups. Peptide structures determined by¹H NMR indicated a well-ordered coiled structure for the short peptides and the C-terminus of the longer peptide. Using each technique, the two enantiomers of RAWVAWR-NH₂ interacted in an identical fashion with the lipids, indicating that any difference in activity in vivo is limited to interactions not involving the membrane lipids.

Keywords: Abbreviations; 1D; one dimensional; 2D; two dimensional; CD; circular dichroism; CFU; colony forming unit; CSI; chemical shift index; DSC; differential scanning calorimetry; nuclear magnetic resonance; DSS; sodium 3-(trimethylsilyl)-1-propanesulfonate; DPC; dodecylphosphocholine; DPPG; 1,2-dipalmitoyl-; sn; -3-phosphatidylglycerol; DPPC; dipalmitoylphosphatidylcholine; DPPE; dipalmitoylphosphatidylethanolamine; DO; dioleoyl; DOPC; dioleoylphosphatidylcholine; DOPG; dioleoylphosphatidylglycerol; DOPE; dioleoylphosphatidylethanolamine; HPLC; high performance liquid chromatography; ITC; isothermal titration calorimetry; LUVs; large unilamellar vesicles; MLVs; multilamellar vesicles; NAPB; sodium phosphate buffer; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser enhancement spectroscopy; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PO; 1-palmitoyl-2-oleoyl; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-[(phospho-rac-(1-glycerol)] (sodium salt); rmsd; root-mean-square deviation; SDS; sodium dodecyl sulfate; TOCSY; total correlation spectroscopyLysozyme; Antimicrobial peptide; Phospholipid; Membrane mimetic; Structure

Intestinal uptake of nateglinide by an intestinal fluorescein transporter by Shirou Itagaki; Yukio Otsuka; Sayaka Kubo; Hideo Okumura; Yoshitaka Saito; Masaki Kobayashi; Takeshi Hirano; Ken Iseki (pp. 190-194).

Nateglinide, a novel oral hypoglycemic agent, rapidly reaches its maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the intestine. However, nateglinide itself is not transported by MCT1 or PEPT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. It has been reported that the uptake of fluorescein by Caco-2 cells occurs via an H⁺-driven transporter and that the intestinal fluorescein transporter is probably not MCT1. We examined the contribution of the fluorescein transporter to the uptake of nateglinide by Caco-2 cells. Fluorescein competitively inhibited H⁺-dependent nateglinide uptake. All of fluorescein transporter inhibitors examined reduced the uptake of nateglinide. Furthermore, nateglinide inhibited fluorescein uptake. We conclude that the intestinal nateglinide/H⁺ cotransport system is identical to the intestinal fluorescein/H⁺ cotransport system.

Keywords: Nateglinide; Intestine; Monocarboxylate transporter; Fluorescein; Absorption; Caco-2

Pertussis toxin-sensitive modulation of glutamate transport by endothelin-1 type A receptors in glioma cells by Mustapha Najimi; Jean-Marie Maloteaux; Emmanuel Hermans (pp. 195-202).

Endothelin-1 (ET-1) is a 21 amino acids peptide that exerts several biological activities through interaction with specific G-protein coupled receptors. Increased ET-1 expression is frequently associated with pathological situations involving alterations in glutamate levels. In the present study, a brief exposure to ET-1 was found to increase aspartate uptake in C6 glioma cells, which endogenously express the neuronal glutamate transporter EAAC1 (pEC₅₀ of 9.89). The stimulatory effect of ET-1 mediated by ET_A receptors corresponds to a 62% increase in the V_max with no modification of the affinity for the substrate. While protein kinase C activity is known to participate in the regulation of EAAC1, the effect of ET-1 on the glutamate uptake was found to be independent of this kinase activation. In contrast, the inactivation of G_o/i type G-protein dependent signaling with pertussis toxin was found to impair ET-1-mediated regulation of EAAC1. An examination of the cell surface expression of EAAC1 by protein biotinylation studies or by confocal analysis of immuno-fluorescence staining demonstrated that ET-1 stimulates EAAC1 translocation to the cell surface. Hence, the disruption of the cytoskeleton with cytochalasin D prevented ET-1-stimulated aspartate uptake. Together, the data presented in the current study suggest that ET-1 participates in the acute regulation of glutamate transport in glioma cells. Considering the documented role of glutamate excitotoxicity in the development of brain tumors, endothelinergic system constitutes a putative target for the pharmacological control of glutamate transmission at the vicinity of glioma cells.

Keywords: Abbreviations; EAAC1; neuronal glutamate transporter; ET-1; endothelin-1; InsP; inositol phosphates; PKC; protein kinase C; PI3-K; phosphatidylinositol 3-kinase; PLC; phospholipase C; PMA; phorbol 12-myristate 13-acetate; PTx; pertussis toxinEAAC1; Glutamate transporter; Endothelin; Pertussis toxin; Glioma cell

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol by Ruthven N.A.H. Lewis; Yuan-Peng Zhang; Ronald N. McElhaney (pp. 203-214).

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and³¹P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (∼11–15 °C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (∼23–25 °C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid–crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing ∼30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the L_c phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the L_c phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen–bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the L_c phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.

Keywords: Abbreviations; PC; phosphatidylcholine; PG; phosphatidylglycerol; DMPC; dimyristoylphosphatidylcholine; DMPG; dimyristoylphosphatidylglycerol; DSC; differential scanning calorimetry; NMR; nuclear magnetic resonance; FTIR; Fourier-transform infrared; Δ; H; _cal; calorimetric enthalpy; L; _α; lamellar liquid/crystalline; P; _β; ′; rippled lamellar gel phase; L; _β; lamellar gel; L; _β; ′; lamellar gel phase with tilted chains; L; _c; lamellar crystalline; T; _m; gel/liquid–crystalline phase transition temperature; T; _p; pretransition temperaturePhosphatidylcholine; Phosphatidylglycerol; Phospholipid bilayer; Model membrane; Phospholipid miscibility; Differential scanning calorimetry; Infrared spectroscopy; Nuclear magnetic resonance spectroscopy

Melatonin strongly interacts with zwitterionic model membranes—evidence from Fourier transform infrared spectroscopy and differential scanning calorimetry by Feride Severcan; Ipek Sahin; Nadide Kazancı (pp. 215-222).

Interactions of melatonin with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) were investigated as a function of temperature and melatonin concentration (1–30 mol%) by using two noninvasive techniques, namely Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The investigation of the C-H, CO, and PO₂⁻ antisymmetric double stretching modes in FTIR spectra and DSC studies reveal that melatonin changes the physical properties of the DPPC bilayers by decreasing the main phase transition temperature, abolishing the pretransition, ordering the system in the gel phase, and increasing the dynamics of the system both in the gel and liquid crystalline phases. It also causes significant decrease in the wavenumber for the CO stretching and PO₂⁻ antisymmetric double bond stretching bands, which indicates strong hydrogen bonding The results imply that melatonin locates in the interfacial region of the membrane. Furthermore, in the DSC curve, more than one signal is observed at high melatonin concentrations (24 and 30 mol%), which indicates melatonin-induced phase separation in DPPC membranes.

Keywords: Melatonin; Dipalmitoyl phosphatidylcholine; Membrane; Liposome; Fourier transform infrared; Differential scanning calorimetry; Phase separation

Electrophysiological and molecular identification of hepatocellular volume-activated K⁺ channels by W.-Z. Lan; H. Abbas; A.-M. Lemay; M.M. Briggs; C.E. Hill (pp. 223-233).

Although K⁺ channels are essential for hepatocellular function, it is not known which channels are involved in the regulatory volume decrease (RVD) in these cells. We have used a combination of electrophysiological and molecular approaches to describe the potential candidates for these channels. The dialysis of short-term cultured rat hepatocytes with a hypotonic solution containing high K⁺ and low Cl⁻ concentration caused the slow activation of an outward, time-independent current under whole-cell configuration of the patch electrode voltage clamp. The reversal potential of this current suggested that K⁺ was the primary charge carrier. The swelling-induced K⁺ current ( I_Kvol) occurred in the absence of Ca²⁺ and was inhibited with 1 μM Ca²⁺ in the pipette solution. The activation of I_Kvol required both Mg²⁺ and ATP and an increasing concentration of Mg–ATP from 0.25 through 0.5 to 0.9 mM activated I_Kvol increasingly faster and to a larger extent. The KCNQ1 inhibitor chromanol 293B reversibly depressed I_Kvol with an IC₅₀ of 26 μM. RT-PCR detected the expression of members of the KCNQ family from KCNQ1 to KCNQ5 and of the accessory proteins KCNE1 to KCNE3 in the rat hepatocytes, but not KCNQ2 and KCNE2 in human liver. Western blotting showed KCNE3 expression in a plasma membrane-enriched fraction from rat hepatocytes. The results suggest that KCNQ1, probably with KCNE2 or KCNE3 as its accessory unit, provides a significant fraction of I_Kvol in rat hepatocytes.

Keywords: cDNA; Human; KCNE; KCNQ; Patch clamp; Rat; RVD

Monovalent cation conductance in Xenopus laevis oocytes expressing hCAT-3 by Wolfgang Gilles; Sebastian D. Vulcu; Jana F. Liewald; Alice Habermeier; Nicole V�kony; Ellen I. Closs; Johanna Rupp; Hermann Nawrath (pp. 234-239).

hCAT-3 (human cationic amino acid transporter type three) was investigated with both the two-electrode voltage clamp method and tracer experiments. Oocytes expressing hCAT-3 displayed less negative membrane potentials and larger voltage-dependent currents than native or water-injected oocytes did. Ion substitution experiments in hCAT-3-expressing oocytes revealed a large conductance for Na⁺ and K⁺. In the presence ofl-Arg, voltage-dependent inward and outward currents were observed. At symmetrical (inside/outside) concentrations ofl-Arg, the conductance of the transporter increased monoexponentially with thel-Arg concentrations; the calculated V_max and K_M values amounted to 8.3 μS and 0.36 mM, respectively. The time constants of influx and efflux of [³H]l-Arg, at symmetrically inside/outsidel-Arg concentrations (1 mM), amounted to 79 and 77 min, respectively. The flux data and electrophysiological experiments suggest that the transport ofl-Arg through hCAT-3 is symmetric, when the steady state ofl-Arg flux has been reached. It is concluded that hCAT-3 is a passive transport system that conducts monovalent cations includingl-Arg. The particular role of hCAT-3 in the diverse tissues remains to be elucidated.

Keywords: Cationic amino acid transporter; Human cationic amino acid transporter type three; l-; Arginine; Na; ⁺; K; ⁺

Synthesis and initial characterization of FGFR3 transmembrane domain: consequences of sequence modifications by Takeo Iwamoto; Min You; Edwin Li; Jamie Spangler; John M. Tomich; Kalina Hristova (pp. 240-247).

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and defects in their dimerization lead to unregulated signaling and disease. RTK transmembrane (TM) domains are proposed to play an important role in the process, underscored by the finding that single amino acids mutations in the TM domains can induce pathological phenotypes. Therefore, many important questions pertaining to the mode of signal transduction and the mechanism of pathology induction could be answered by studying the chemical–physical basis behind RTK TM domain dimerization and the interactions of RTK TM domains with lipids in model bilayer systems. As a first step towards this goal, here we report the synthesis of the TM domain of fibroblast growth factor receptor 3 (FGFR3), an RTK that is crucial for skeletal development. We have used solid phase peptide synthesis to produce two peptides: one corresponding to the membrane embedded segment and the naturally occurring flanking residues at the N- and C-termini (TM_wt), and a second one in which the flanking residues have been substituted with diLysines at the termini (TM_KK). We have demonstrated that the hydrophobic FGFR3 TM domain can be synthesized for biophysical studies with high yield. The protocol presented in the paper can be applied to the synthesis of other RTK TM domains. As expected, the Lys flanks decrease the hydrophobicity of the TM domain, such that TM_KK elutes much earlier than TM_wt during reverse phase HPLC purification. The Lysines have no effect on peptide solubility in SDS and on peptide secondary structure, but they abolish peptide dimerization on SDS gels. These results suggest that caution should be exercised when modifying RTK TM domains to render them more manageable for biophysical studies.

Keywords: Abbreviations; TM; transmembrane; RTK; receptor tyrosine kinase; FGFR3; fibroblast growth factor receptor 3; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; HFIP; hexafluoroisopropanolReceptor tyrosine kinase; FGFR3; Solid phase peptide synthesis; Terminal lysine

Nucleotide dissociation from NBD1 promotes solute transport by MRP1 by Runying Yang; Ali McBride; Yue-xian Hou; Aaron Goldberg; Xiu-bao Chang (pp. 248-261).

MRP1 transports glutathione-S-conjugated solutes in an ATP-dependent manner by utilizing its two NBDs to bind and hydrolyze ATP. We have found that ATP binding to NBD1 plays a regulatory role whereas ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. However, whether ATP hydrolysis at NBD1 is required for the transport was not clear. We now report that ATP hydrolysis at NBD1 may not be essential for transport, but that the dissociation of the NBD1-bound nucleotide facilitates ATP-dependent LTC4 transport. These conclusions are supported by the following results. The substitution of the putative catalytic E1455 with a non-acidic residue in NBD2 greatly decreases the ATPase activity of NBD2 and the ATP-dependent LTC4 transport, indicating that E1455 participates in ATP hydrolysis. The mutation of the corresponding D793 residue in NBD1 to a different acidic residue has little effect on ATP-dependent LTC4 transport. The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Along with their higher transport activities, their Michaelis constant K_ms (ATP) are also higher than that of wild-type. Coincident with their higher K_ms (ATP), their K_ds derived from ATP binding are also higher than that of wild-type, implying that the rate of dissociation of the bound nucleotide from the mutated NBD1 is faster than that of wild-type. Therefore, regardless of whether the bound ATP at NBD1 is hydrolyzed or not, the release of the bound nucleotide from NBD1 may bring the molecule back to its original conformation and facilitate the protein to start a new cycle of ATP-dependent solute transport.

Keywords: Abbreviations; P-gp; P-glycoprotein; MRP1; multidrug resistance-associated protein 1; NBD; nucleotide binding domain; LTC4; leukotriene C4; SDS; sodium dodecyl sulfate; EDTA; ethylenediaminetetraacetic acid; 8-N; ₃; ATP; 8-azidoadenosine 5′-triphosphate; Sf21; Spodoptera frugiperda; 21Multidrug resistance-associated protein 1; Nucleotide binding domain; ATP binding; Dissociation of the bound ATP; ATP hydrolysis; ATP-dependent LTC4 transport

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BBA - Biomembranes (v.1668, #2)