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BBA - Biomembranes (v.1788, #11)

Editorial Board (pp. i).

Sphingolipid/cholesterol regulation of neurotransmitter receptor conformation and function by Jacques Fantini; Francisco J. Barrantes (pp. 2345-2361).

Like all other monomeric or multimeric transmembrane proteins, receptors for neurotransmitters are surrounded by a shell of lipids which form an interfacial boundary between the protein and the bulk membrane. Among these lipids, cholesterol and sphingolipids have attracted much attention because of their well-known propensity to segregate into ordered platform domains commonly referred to as lipid rafts. In this review we present a critical analysis of the molecular mechanisms involved in the interaction of cholesterol/sphingolipids with neurotransmitter receptors, in particular acetylcholine and serotonin receptors, chosen as representative members of ligand-gated ion channels and G protein-coupled receptors. Cholesterol and sphingolipids interact with these receptors through typical binding sites located in both the transmembrane helices and the extracellular loops. By altering the conformation of the receptors (“chaperone-like” effect), these lipids can regulate neurotransmitter binding, signal transducing functions, and, in the case of multimeric receptors, subunit assembly and subsequent receptor trafficking to the cell surface. Several sphingolipids (especially gangliosides) also exhibit low/moderate affinity for neurotransmitters. We suggest that such lipids could facilitate (i) the attachment of neurotransmitters to the post-synaptic membrane and in some cases (ii) their subsequent delivery to specific protein receptors. Overall, various experimental approaches provide converging evidence that the biological functions of neurotransmitters and their receptors are highly dependent upon sphingolipids and cholesterol, which are active partners of synaptic transmission. Several decades of research have been necessary to untangle the skein of a complex network of molecular interactions between neurotransmitters, their receptors, cholesterol and sphingolipids. This sophisticated crosstalk between all four distinctive partners may allow a fine biochemical tuning of synaptic transmission.

Keywords: Lipid domain; Sphingomyelin; Ganglioside; Glycosphingolipid; Cholesterol; Nicotinic acetylcholine receptor; Serotonin receptor; Raft

Enhancement of transdermal drug delivery via synergistic action of chemicals by Pankaj Karande; Samir Mitragotri (pp. 2362-2373).

Transdermal drug delivery is an attractive alternative to conventional techniques for administration of systemic therapeutics. One challenge in designing transdermal drug delivery systems is to overcome the natural transport barrier of the skin. Chemicals offer tremendous potential in overcoming the skin barrier to enhance transport of drug molecules. Individual chemicals are however limited in their efficacy in disrupting the skin barrier at low concentrations and usually cause skin irritation at high concentrations. Multicomponent mixtures of chemicals, however, have been shown to provide high skin permeabilization potency as compared to individual chemicals without necessarily causing irritation. Here we review systems employing synergistic mixtures of chemicals that offer superior skin permeation enhancement. These synergistic systems include solvent mixtures, microemulsions, eutectic mixtures, complex self-assembled vesicles and inclusion complexes. Methods for design and discovery of such synergistic systems are also discussed.

Keywords: Transdermal; Synergy; High throughput; Enhancer; Chemical

Structure–function studies on the voltage-gated sodium channel by Hervé Duclohier (pp. 2374-2379).

Recent research on structure–function relationships aspects of voltage-gated sodium channels (VGSCs) are reviewed. Data issued from the literature are summarized and compared, including results from our own studies. The latter deal with the effects of drug binding, deglycosylation and the role of hydrophobic residues in the voltage sensors. Methods mainly consist of circular dichroism (CD) to asses the channel's secondary structure and conductance measurements after reconstitution into planar lipid bilayers. Molecular modelling was also used to tentatively explain experimental data. Since 30% of the channel's mass are glycoconjugates, the effects of removing them were first investigated. Then, the effects of the neurotoxin Batrachotoxin and the anticonvulsant Lamotrigine were studied. Both drugs induced a significant increase in the channel's helical content and a molecular model shows that lamotrigine interacts with residues previously identified as forming the binding sites in the pore. Finally, the role of hydrophobic residues with long sidechains in the voltage sensors (S4s) was investigated. Recent research on related studies on VGSCs are discussed.

Keywords: Voltage sensor; Activation-inactivation coupling; Heterologous expression; Electrophysiology; Secondary structure; Molecular modelling

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies by Marija Raguz; Justyna Widomska; James Dillon; Elizabeth R. Gaillard; Witold K. Subczynski (pp. 2380-2388).

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation–recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454–1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid–cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 °C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.

Keywords: Lens lipids; Lens cortex; Lens nucleus; Cholesterol; Cholesterol crystalline domain; Membrane; Spin label; EPR

Structure assembly of Bcl-x_L through α5–α5 and α6–α6 interhelix interactions in lipid membranes by Yu Feng; Dongxiang Liu; Xu Shen; Kaixian Chen; Hualiang Jiang (pp. 2389-2395).

Lipid bilayer membrane is the main site where Bcl-x_L executes its anti-apoptotic function. Here we used site-directed mutagenesis and cysteine-directed cross-linking to trap the structure of Bcl-x_L upon membrane insertion. Cys151 on α5-helix and Asn185 on α6-helix of two neighboring Bcl-x_L are found in close positions, respectively. The FRET based binding assay indicated that the BH3-peptide binding pocket in Bcl-x_L is disrupted after its membrane insertion. Co-immunoprecipitation experiments showed that the membrane-bound Bcl-x_L sequestered tBid by direct interaction at physiological pH. If Bcl-x_L behaves similarly at low pH as it does at physiological pH, the membrane-bound Bcl-x_L should bind to tBid through protein regions other than the BH3 domain of tBid and the hydrophobic pocket of Bcl-x_L. Previously, a crystallography study demonstrated that Bcl-x_L formed homodimers through domain swapping in water, where Cys151 and Asn185 of two monomeric subunits are far apart from each other and the BH3-peptide binding pocket is intact. Our results indicated that Bcl-x_L dimer trapped by cross-linking in lipids is distinct from the domain swapped dimer, suggesting that Bcl-x_L transits through a structural change from the water-soluble state to the membrane-bound state and there are multiple possibilities for structural reorganization of Bcl-x_L protein.

Keywords: Abbreviation; BH; Bcl-2 homology; DTT; dithiothreitol; FRET; fluorescence resonance energy transfer; LUV; large unilamellar vesicles; PAGE; polyacrylamide gel electrophoresisBcl-x; _L; Pore formation; Chemical cross-linking; Apoptosis; Membrane protein

Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver by Ikumi Chisaki; Masaki Kobayashi; Shirou Itagaki; Takeshi Hirano; Ken Iseki (pp. 2396-2403).

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. In an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317, but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXRα knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile.

Keywords: Liver X receptor; ABC transporter; Regulation

The role of the Ca²⁺ binding ligand Asn879 in the function of the plasma membrane Ca²⁺ pump by Débora E. Rinaldi; Hugo P. Adamo (pp. 2404-2410).

Asn879 in the transmembrane segment M6 of the plasma membrane Ca²⁺ pump (PMCA human isoform 4xb) has been proposed to coordinate Ca²⁺ at the transport site through its carboxylate. This idea agrees with the fact that this Asn is conserved in other Ca²⁺-ATPases but is replaced by Asp, Glu, and other residues in closely related 2P-type ATPases of different ionic specificity. Previous mutagenesis studies have shown that the substitution of Ala for Asn abolishes the activity of the enzyme (Adebayo et al., 1995; Guerini et al., 1996). We have constructed a mutant PMCA in which the Asn879 was substituted by Asp. The mutant protein was expressed in Saccharomyces cerevisiae, solubilized and purified by calmodulin affinity chromatography. The Asn879Asp PMCA mutant exhibited about 30% of the wild type Ca²⁺-dependent ATPase activity and only a minor reduction of the apparent affinity for Ca²⁺. The decrease in the Ca²⁺-ATPase of the mutant enzyme was in parallel with the reduction in the amount of phosphoenzyme formed from Ca²⁺ plus ATP. Noteworthy, the mutation nearly eliminated the ability of the enzyme to hydrolyze pNPP which is maximal in the absence of Ca²⁺ revealing a major effect of the mutation on the Ca²⁺-independent reactions of the transport cycle. At a pH low enough to protonate the Asp carboxylate the pNPPase activity of Asn879Asp increased, suggesting that the binding of protons to Asn879 is essential for the activities catalyzed by E₂-like forms of the enzyme.

Keywords: Abbreviations; PMCA; plasma membrane calcium pump; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PC; L-α-phosphatidylcholine; BE; lipidic extract from bovine brain containing acidic lipids; C; ₁₂; E; ₁₀; polyoxyethylene-10-lauryletherPlasma membrane Ca; ²⁺; -ATPase; PMCA; Ca; ²⁺; pump; Ca; ²⁺; binding site; Asparagine; Mutagenesis; P-ATPase

Structure–function study of cathelicidin-derived bovine antimicrobial peptide BMAP-28: Design of its cell-selective analogs by amino acid substitutions in the heptad repeat sequences by Aqeel Ahmad; Neeta Asthana; Sarfuddin Azmi; Raghvendra M. Srivastava; Brijesh K. Pandey; Vikas Yadav; Jimut Kanti Ghosh (pp. 2411-2420).

Although BMAP-28 is a potent cathelicidin-derived bovine antimicrobial peptide, its cytotoxic activity against the human and other mammalian cells is of concern for converting it into a novel antimicrobial drug. We have identified a short leucine and isoleucine zipper sequences at the N- and C-terminals of BMAP-28, respectively. To understand the possible role of these structural elements in BMAP-28, a number of alanine-substituted analogs were designed, synthesized and characterized along with the wild-type peptide. The substitution of amino acids at single or multiple ‘a’ position(s) of these structural motifs by alanine showed significant effects on the cytotoxic activity of the molecule on the human red blood cells (hRBCs) and 3T3 cells without showing much effects on their MIC values against the selected bacteria. BMAP-28 and all its analogs depolarized the Escherichia coli cells with almost equal efficacy. In contrast, the alanine-substituted analogs of BMAP-28 depolarized hRBCs much less efficiently than the parent molecule. Results further showed that BMAP-28 assembled appreciably onto the live E. coli and hRBC. However, the selected less toxic analogs of BMAP-28 although assembled as good as the parent molecule onto the live E. coli cells, their assembly onto the live mammalian hRBCs was much weaker as compared to that of the wild-type molecule. Looking at the remarkable similarity with the data presented in our previous work on melittin, it appears that probably the heptad repeat sequence possesses a general role in maintaining the cytotoxicity of the antimicrobial peptides against the mammalian cells and assembly therein.

Keywords: Abbreviations; FITC; fluorescin isothiocyanate; hRBC; human red blood cell; PBS; phosphate buffered saline (pH 7.4); Rho; tetra-methyl rhodamine; PI; propidium iodide; CFU; colony forming unitsAntimicrobial peptide; Antimicrobial and toxic activity; Peptide membrane interaction; Leucine zipper motif; Localization of antimicrobial peptides onto bacterial and mammalian cells

Oleic- and docosahexaenoic acid-containing phosphatidylethanolamines differentially phase separate from sphingomyelin by Saame Raza Shaikh; Daniel S. LoCascio; Smita P. Soni; Stephen R. Wassall; William Stillwell (pp. 2421-2426).

A central tenet of the lipid raft model is the existence of non-raft domains. In support of this view, we have established in model membranes that a phosphatidylethanolamine (PE)-containing docosahexaenoic acid (DHA) forms organizationally distinct non-raft domains in the presence of sphingomyelin (SM) and cholesterol (Chol). We have shown that formation of DHA-rich domains is driven by unfavorable molecular interactions between the rigid Chol molecule and the highly flexible DHA acyl chain. However, the molecular interactions between SM and the DHA-containing PE, which could also contribute to the formation of DHA-rich non-raft domains, have not been sufficiently investigated. To address this issue, we use differential scanning calorimetry (DSC) to study the phase behavior of mixtures of SM with either 1-palmitoyl-2-docosahexaenoyl- sn-glycero-3-phosphoethanolamine (16:0-22:6PE) or 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (16:0-18:1PE), an oleic acid (OA)-containing control, over a wide range of concentrations. Deconvolution of binary DSC scans shows that both 16:0-22:6PE and 16:0-18:1PE phase separate from SM. Analysis of transition temperatures and partial phase diagrams, constructed from the DSC scans for the first time, shows that 16:0-22:6PE displays greater non-ideal mixing with SM compared to 16:0-18:1PE. Our findings support a model in which DHA- and OA-containing PEs differentially phase separate from SM over a wide range of molar ratios to initiate the formation of non-raft domains, which is greatly enhanced by DHA, but not OA, in the presence of cholesterol.

Keywords: Non-raft domain; Docosahexaenoic acid; Differential scanning calorimetry

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery by Cally M. Scherber; Janet L. Schottel; Alptekin Aksan (pp. 2427-2435).

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature ( T_m), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%–40% RH) resulted in an increase in T_m from 8.4±1.7 °C (in undried control samples) to 16.5±1.3 °C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T_m, which began approximately 1 day after initial rehydration and plateaued at 12.5±1.8 °C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T_m and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.

Keywords: Membrane phase change; FTIR; Desiccation; VBNC; E. coli

Lipoprotein interactions with chromatic membranes as a novel marker for oxidative stress-related diseases by Nirit Hanin-Avraham; Bianca Fuhrman; Agnieszka Mech-Dorosz; Sofiya Kolusheva; Angel Porgador; Michael Aviram; Raz Jelinek (pp. 2436-2443).

Changes in the abundance and properties of blood lipoproteins are generally considered major causes for varied pathological conditions and diseases. Using novel chromatic biomimetic vesicle and cell assays, we present here for the first time evidence for significant changes in lipoproteins' interactions with artificial membranes. Specifically, we demonstrate significant differences in membrane binding between lipoproteins (both low-density lipoprotein [LDL] and high-density lipoprotein [HDL]) harvested from diabetic patients vs. healthy controls as well as between oxidized and native lipoproteins. The chromatic assays, complemented by biophysical techniques and electron microscopy, point to significant reduction of surface membrane binding of the lipoproteins as a consequence of diabetes or oxidation. Overall, our results indicate that the substantial modulation of membrane interactions revealed by the chromatic assays may be used as a new and potentially powerful marker for screening and prediction of diseases associated with oxidative stress.

Keywords: Abbreviations; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPE; 1,2-dimyristoyl-; sn; -glycero-3-phospho-ethanolamine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)]; DPH; diphenylhexatriene; FRET; fluorescence resonance energy transfer; HDL; high-density lipoprotein; LDL; low-density lipoprotein; PDA; polydiacetylene; NBD-PE; N; -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)1,2 dihexadecanoyl-; sn-; glycero-3 phospho-ethanolamine, triethylammonium salt; Rhodamine-DHPE; Rhodamine Red™-X 1,2-dihexadecanoyl-; sn; -glycero-3-phospho-ethanolamine, triethylammonium salt

Insights into the effect of detergents on the full-length rhomboid protease from Pseudomonas aeruginosa and its cytosolic domain by Allison R. Sherratt; Michael V. Braganza; Elizabeth Nguyen; Thierry Ducat; Natalie K. Goto (pp. 2444-2453).

Rhomboids comprise a family of intramembrane serine proteases that catalyze the cleavage of transmembrane segments within the lipid membrane to achieve a wide range of biological functions. A subset of bacterial rhomboids possesses an N-terminal cytosolic domain that appears to enhance proteolytic activity via an unknown mechanism. Structural analysis of a full-length rhomboid would provide new insights into this mechanism, an objective that solution NMR has the potential to realize. For this purpose we purified the rhomboid from Pseudomonas aeruginosa in a range of membrane-mimetic media, evaluated its functional status in vitro and investigated the NMR spectroscopic properties of these samples. In general, NMR signals could only be observed from the cytosolic domain, and only in detergents that did not support rhomboid activity. In contrast, media that supported rhomboid function did not show these resonances, suggesting an association between the cytosolic domain and the protein–detergent complex. Investigations into the ability of the isolated cytosolic domain to bind detergent micelles revealed a denaturing interaction, whereas no interaction occurred with micelles that supported rhomboid activity. The cytosolic domain also did not show any tendency to interact with lipid bilayers found in small bicelles or vesicles made from Escherichia coli phospholipid extracts. Based on these data we propose that the cytosolic domain does not interact with the lipid membrane, but instead enhances rhomboid activity through interactions with some other part of the rhomboid, such as the catalytic core domain.

Keywords: Abbreviations; EGFR; epidermal growth factor receptor; NMR; nuclear magnetic resonance; CytD; cytosolic domain; TMD; transmembrane domain; TM; transmembrane; Glp; glycerol-3-phosphate; ec; GlpG; E. coli; GlpG; pa; GlpG; P. aeruginosa; GlpG; Spitz; _TM; Spitz TM segment; Bla; β-lactamase; MBP; maltose binding protein; DDM; dodecyl maltoside; DPC; dodecylphosphocholine; LMPC; 1-myristoyl-2-hydroxy-; sn; -glycero-3-phosphocholine; LPPG; 1-palmitoyl-2-hydroxy-; sn; -glycero-3-[phospho-rac-(1-glycerol)]; NG; n; -nonyl-β-; d; -glucoside; HDPC; hexadecylphosphocholine; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DHPC; 1,2-dihexanoyl-; sn; -glycero-3-phosphocholine; CHAPS; 3-[(3-cholamidopropyl)-dimethylammonio]-1-propansulfonate; MWCO; molecular weight cut off; HSQC; heteronuclear single quantum coherence; SDS–PAGE; sodium dodecyl sulfate–polyacrylamide gel electrophoresisIntramembrane proteolysis; Rhomboid protease; GlpG; Cytosolic domain; NMR; Membrane protein

Differential ability of cholesterol-enriched and gel phase domains to resist benzyl alcohol-induced fluidization in multilamellar lipid vesicles by Terhi Maula; Bodil Westerlund; J. Peter Slotte (pp. 2454-2461).

Benzyl alcohol (BA) has a well-known fluidizing effect on both artificial and cellular membranes. BA is also likely to modulate the activities of certain membrane proteins by decreasing the membrane order. This phenomenon is presumably related to the ability of BA to interrupt interactions between membrane proteins and the surrounding lipids by fluidizing the lipid bilayer. The components of biological membranes are laterally diversified into transient assemblies of varying content and order, and many proteins are suggested to be activated or inactivated by their localization in or out of membrane domains displaying different physical phases. We studied the ability of BA to fluidize artificial bilayer membranes representing liquid-disordered, cholesterol-enriched and gel phases. Multilamellar vesicles were studied by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid, which display different phase partitioning. Domains of different degree of order and thermal stability showed varying abilities to resist fluidization by BA. In bilayers composed of mixtures of an unsaturated phosphatidylcholine, a saturated high melting temperature lipid (sphingomyelin or phosphatidylcholine) and cholesterol, BA fluidized and lowered the melting temperature of the ordered and gel phase domains. In general, cholesterol-enriched domains were more resistant to BA than pure gel phase domains. In contrast, bilayers containing high melting temperature gel phase domains containing a ceramide or a galactosylceramide proved to be the most effective in resisting fluidization. The results of our study suggest that the ability of BA to affect the fluidity and lateral organization of the membranes was dependent on the characteristic features of the membrane compositions studied and related to the intermolecular cohesion in the domains.

Keywords: Abbreviations; BA; benzyl alcohol; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPH; 1,6-diphenyl-1,3,5-hexatriene; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; PCer; d; -; erythro-N-; palmitoyl-ceramide; PGalCer; d; -; erythro; -; N; -palmitoyl-galactosylceramide; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; d; -; erythro; -N; -palmitoyl-sphingomyelin; T; _m; gel-to-liquid phase transition temperature; tPA; trans; -parinaric acidBenzyl alcohol; Membrane fluidity; Steady-state fluorescence anisotropy; trans; -parinaric acid; 1,6-diphenyl-1,3,5-hexatriene

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1 by Peter Teriete; Khang Thai; Jungyuen Choi; Francesca M. Marassi (pp. 2462-2470).

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na⁺/K⁺ ion binding site of the Na,K-ATPase α subunit.

Keywords: FXYD; Phospholemman; Na,K-ATPase; Phosphorylation, Structure; NMR; Micelle

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BBA - Biomembranes (v.1788, #11)