Modulation of nicotinic acetylcholine receptor conformational state by free fatty acids and steroids

Steroids and free fatty acids (FFA) are noncompetitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is purportedly located at the lipid-AChR interface, but their exact mechanism of action is still unknown. Here we studied the effect of structurally different FFA and steroids on the conformational equilibrium of the AChR in Torpedo californica receptor-rich membranes. We took advantage of the higher affinity of the fluorescent AChR open channel blocker, crystal violet, for the desensitized state than for the resting state. Increasing concentrations of steroids and FFA decreased the K(D) of crystal violet in the absence of agonist; however, only cis-unsaturated FFA caused an increase in K(D) in the presence of agonist. This latter effect was also observed with treatments that caused the opposite effects on membrane polarity, such as phospholipase A(2) treatment or temperature increase (decreasing or increasing membrane polarity, respectively). Quenching by spin-labeled fatty acids of pyrene-labeled AChR reconstituted into model membranes, with the label located at the gammaM4 transmembrane segment, disclosed the occurrence of conformational changes induced by steroids and cis-unsaturated FFA. The present work is a step forward in understanding the mechanism of action of this type of molecules, suggesting that the direct contact between exogenous lipids and the AChR transmembrane segments removes the AChR from its resting state and that membrane polarity modulates the AChR activation equilibrium by an independent mechanism.     

Tryptophan-scanning mutagenesis in the alphaM3 transmembrane domain of the muscle-type acetylcholine receptor. A spring model revealed

Membrane proteins constitute a large fraction of all proteins, yet very little is known about their structure and conformational transitions. A fundamental question that remains obscure is how protein domains that are in direct contact with the membrane lipids move during the conformational change of the membrane protein. Important structural and functional information of several lipid-exposed transmembrane domains of the acetylcholine receptor (AChR) and other ion channel membrane proteins have been provided by the tryptophan-scanning mutagenesis. Here, we use the tryptophan-scanning mutagenesis to monitor the conformational change of the alphaM3 domain of the muscle-type AChR. The perturbation produced by the systematic tryptophan substitution along the alphaM3 domain were characterized through two-electrode voltage clamp and 125I-labeled alpha-bungarotoxin binding. The periodicity profiles of the changes in AChR expression (closed state) and ACh EC50 (open-channel state) disclose two different helical structures; a thinner-elongated helix for the closed state and a thicker-shrunken helix for the open-channel state. The existence of two different helical structures suggest that the conformational transition of the alphaM3 domain between both states resembles a spring motion and reveals that the lipid-AChR interface plays a key role in the propagation of the conformational wave evoked by agonist binding. In addition, the present study also provides evidence about functional and structural differences between the alphaM3 domains of the Torpedo and muscle-type receptors AChR.     

Fluorescence and molecular dynamics studies of the acetylcholine receptor gammaM4 transmembrane peptide in reconstituted systems

A combination of fluorescence spectroscopy and molecular dynamics (MD) is applied to assess the conformational dynamics of a peptide making up the outermost ring of the nicotinic acetylcholine receptor (AChR) transmembrane region and the effect of membrane thickness and cholesterol on the hydrophobic matching of this peptide. The fluorescence studies exploit the intrinsic fluorescence of the only tryptophan residue in a synthetic peptide corresponding to the fourth transmembrane domain of the AChR gamma subunit (gammaM4-Trp(6)) reconstituted in lipid bilayers of varying thickness, and combine this information with quenching studies using depth-sensitive phosphatidylcholine spin-labeled probes and acrylamide, polarization of fluorescence, and generalized polarization of Laurdan. A direct correlation was found between bilayer width and the depth of insertion of Trp(6). We further extend our recent MD study of the conformational dynamics of the AChR channel to focus on the crosstalk between M4 and the lipid-belt region. The isolated gammaM4 peptide is shown to possess considerable orientational flexibility while maintaining a linear alpha-helical structure, and to vary its tilt depending on bilayer width and cholesterol (Chol) content. MD studies also show that gammaM4 also establishes contacts with the other TM peptides on its inner face, stabilizing a shorter TM length that is still highly sensitive to the lipid environment. In the native membrane the topology of the M4 ring is likely to exhibit a similar behavior, dynamically modifying its tilt to match the hydrophobic thickness of the bilayer.     

Altered lipid profile,oxidative status and hepatitis B virus interactions in human hepatocellular carcinoma

Altered membrane integrity in hepatocellular carcinoma (HCC) tissue was indicated by an elevation in cholesterol and significant decrease in phosphatidylcholine (PC). The resultant decreased phosphatidylcholine/phosphatidylethanolamine (PC/PE) and increased cholesterol/phospholipid ratios are associated with decreased fluidity in the carcinoma tissue. The lower PC was associated with a decrease in the quantitative levels of the saturated (C16:0, C18:0), ω6 (C18:2, C20:4) and ω3 (C22:5, C22:6) fatty acids (FAs), resulting in reduced long-chain polyunsaturated fatty acids (LCPUFAs), total PUFA and an increase in ω6/ω3 FA ratio. In PE, the saturated and ω3 (C22:5, C22:6) FAs were reduced while the total ω6 FA level was not affected, leading to an increased ω6/ω3 FA ratio. Increased levels of C18:1ω9, C20:2ω6 and reduction of 22:6ω3 in PC and PE suggest a dysfunctional delta-6 desaturase. The reduced PC/PE ratio resulted in a decreased C20:4ω6 (PC/PE) ratio, implying a shift towards synthesis of the 2-series eicosanoids. Lipid peroxidation was reduced in both hepatitis B negative (HBV⁻) and positive (HBV⁺) HCC tissues. Glutathione (GSH) was decreased in HCC while HBV had no effect, suggesting an impairment of the GSH redox cycle. In contrast HBV infection enhanced GSH in the surrounding tissue possibly to counter oxidative stress as indicated by the increased level of conjugated dienes. Apart from the reduced LCPUFA, the low level of lipid peroxidation in the carcinoma tissue was associated with increased superoxide dismutase and glutathione peroxidase activity. The disruption of the redox balance, resulting in increased cellular antioxidant capacity, could create an environment for resistance to oxidative stress in the carcinoma tissue. Alterations in membrane cholesterol, phospholipids, FA parameters, C20:4ω6 membrane distribution and low lipid peroxidation are likely to be important determinants underlying the selective growth advantage of HCC cells.     

Conformation-sensitive steroid and fatty acid sites in the transmembrane domain of the nicotinic acetylcholine receptor

The mechanism by which some hydrophobic molecules such as steroids and free fatty acids (FFA) act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) is still not known. In the present work, we employ F?rster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound Torpedo californica AChR and the fluorescent probe Laurdan using the decrease in FRET efficiency (E) caused by steroids and FFA to identify potential sites of these hydrophobic molecules. Structurally different steroids produced similar changes (DeltaE) in FRET, and competition studies between them demonstrate that they occupy the same site(s). They also share their binding site(s) with FFA. Furthermore, the FRET conditions define the location of the sites at the lipid-protein interface. Endogenous production of FFA by controlled phospholipase A2 enzymatic digestion of membrane phospholipids yielded DeltaE values similar to those obtained by addition of exogenous ligand. This finding, together with the preservation of the sites in membranes subjected to controlled proteolysis of the extracellular AChR moiety with membrane-impermeable proteinase K, further refines the topology of the sites at the AChR transmembrane domain. Agonist-induced desensitization resulted in the masking of the sites observed in the absence of agonist, thus demonstrating the conformational sensitivity of FFA and steroid sites in the AChR.     

Microfilaments and actin-associated proteins at sites of membrane-substrate attachment within acetylcholine receptor clusters

Rat myotubes in tissue culture form broad areas of close contact with the substrate. These areas often display two distinct, interdigitating sets of membrane domains. One, the "contact domain", is close to the substrate; the other, termed the "AChR domain", is further from the substrate and is rich in acetylcholine receptors (AChR). We have used fluorescence techniques to study the organization of the cytoskeleton in these areas. Substrate-apposed membrane of the myotubes was exposed either by shearing or by permeabilizing the cells with a neutral detergent. Phalloidin derivatives and affinity-purified polyclonal or monoclonal antibodies specific for cytoskeletal proteins were then applied to the samples. Sheared samples were observed by epifluorescence microscopy; detergent-permeabilized samples were observed by total internal reflection fluorescence microscopy. We found that, like antivinculin, fluorescent phalloidin derivatives and antibodies to alpha-actinin, filamin, and talin preferentially labeled the contact domains. This suggests that bundles of microfilaments associate with the membrane at sites of myotube-substrate attachment. In contrast, a 43K protein, closely associated with AChR, was present only at AChR domains. A monoclonal antibody to actin labeled both AChR and contact domains, suggesting that actin is enriched over both regions. Our results suggest that, like the plasma membrane of AChR clusters, the underlying membrane skeleton is organized into at least two distinct domains.     

Fluorescence and molecular dynamics studies of the acetylcholine receptor γM4 transmembrane peptide in reconstituted systems

A combination of fluorescence spectroscopy and molecular dynamics (MD) is applied to assess the conformational dynamics of a peptide making up the outermost ring of the nicotinic acetylcholine receptor (AChR) transmembrane region and the effect of membrane thickness and cholesterol on the hydrophobic matching of this peptide. The fluorescence studies exploit the intrinsic fluorescence of the only tryptophan residue in a synthetic peptide corresponding to the fourth transmembrane domain of the AChR γ subunit (γM4-Trp⁶) reconstituted in lipid bilayers of varying thickness, and combine this information with quenching studies using depth-sensitive phosphatidylcholine spin-labeled probes and acrylamide, polarization of fluorescence, and generalized polarization of Laurdan. A direct correlation was found between bilayer width and the depth of insertion of Trp⁶. We further extend our recent MD study of the conformational dynamics of the AChR channel to focus on the crosstalk between M4 and the lipid-belt region. The isolated γM4 peptide is shown to possess considerable orientational flexibility while maintaining a linear α-helical structure, and to vary its tilt depending on bilayer width and cholesterol (Chol) content. MD studies also show that γM4 also establishes contacts with the other TM peptides on its inner face, stabilizing a shorter TM length that is still highly sensitive to the lipid environment. In the native membrane the topology of the M4 ring is likely to exhibit a similar behavior, dynamically modifying its tilt to match the hydrophobic thickness of the bilayer.     

Lipid matters: nicotinic acetylcholine receptor-lipid interactions (Review)

Ligand-gated ion channels mediate fast intercellular communication in response to endogenous neurotransmitters. The nicotinic acetylcholine receptor (AChR) is the archetype molecule in the superfamily of these membrane proteins. Early electron spin resonance studies led to the discovery of a lipid fraction in direct contact with the AChR, with rotational dynamics 50-fold slower than those of the bulk lipids. This AChR-vicinal lipid region has since been postulated to be a possible site of lipid modulation of receptor function. The polarity and molecular dynamics of solvent dipoles-mainly water-of AChR-vicinal lipids in the membrane have been studied with Laurdan extrinsic fluorescence, and Forster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET enabled one to discriminate between the bulk lipid and the AChR-vicinal lipid. The latter is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipids and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. After delineating the topography of the AChR membrane-embedded domains with fluorescence methods, sites for steroids are being explored with site-directed mutagenesis and patch-clamp recording. Pyrene-labelled Cys residues in alphaM1, alphaM4, gammaM1 and gammaM4 transmembrane regions were found to lie in a shallow position. For M4 segments, this is in agreement with a canonical linear alpha-helix; for M1, it is necessary to postulate a substantial amount of non-helical structure, and/or of kinks, to rationalize the shallow location of Cys residues. Mutations of Thr422, a residue close to the extracellular-facing membrane hemilayer in alphaM4, affect the steroid modulation of AChR function, suggesting its involvement in steroid-AChR interactions.     

Substrate-dependent conformational dynamics of the Escherichia coli membrane insertase YidC

The binding of Pf3 coat protein to the membrane insertase YidC from Escherichia coli induces a conformational change in the tertiary structure of the insertase, resulting in a quenching of the intrinsic tryptophan (Trp) fluorescence. Tryptophan mutants of YidC were generated to examine such conformational movements in detail with time-resolved and steady-state fluorescence spectroscopy. Ten of the 11 Trp residues within YidC were substituted to phenylalanines generating single Trp mutants either at position 354, 454, or 508. In addition, a double mutant with Trp residues at 332 and 334 was studied. Purified YidC mutants were reconstituted into DOPC/DOPG vesicles and titrated with a Trp-free mutant of Pf3 coat, enabling a detailed conformational study of the periplasmic P1, P2, and P3 domains of YidC before and after binding of substrate. Time-resolved fluorescence anisotropy revealed that the mobility of the residues W332/W334 and W508 was considerably increased after binding of Pf3 coat to the insertase. Furthermore, analysis of the fluorescence emission spectra and the decay times showed that all Trp residues are embedded in an equivalent environment that is a membrane/water interface.     

Site specificity of agonist-induced opening and desensitization of the Torpedo californica nicotinic acetylcholine receptor

Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were measured using sequential-mixing stopped-flow fluorescence methods to determine the contribution of each individual site to agonist-induced opening and desensitization. Timed dansyl-C6-choline (DC6C) binding followed by its dissociation upon mixing with high, competing agonist concentrations revealed four kinetic components: an initial, fast fluorescence decay, followed by a transient increase, and then two characteristic decays that reflect dissociation from the desensitized agonist sites. The transient increase resulted from DC6C binding to the open-channel based on its prevention by proadifen, a noncompetitive antagonist. Further characterization of DC6C channel binding by the inhibition of [3H]phencyclidine binding and by equilibrium measurements of DC6C fluorescence yielded KD values of 2-4 microM for the desensitized AChR and approximately 600 microM for the closed state. At this site, DC6C displayed a strongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer from tryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned to DC6C dissociation from the alphadelta site of the AChR in its closed conformation, on the basis of inhibition with the site-selective antagonists d-tubocurarine and alpha-conotoxin MI. Fast decay amplitude data indicated an apparent affinity of 0.9 microM for the closed-state alphadelta site; the closed-state alphagamma-site affinity is inferred to be near 100 microM. These values and the known affinities for the desensitized conformation show that the alphagamma site drives AChR desensitization to a approximately 40-fold greater extent than the alphadelta site, undergoes energetically larger conformational changes, and is the primary determinant of agonist potency.     

Treatments that extract the 43K protein from acetylcholine receptor clusters modify the conformation of cytoplasmic domains of all subunits of the receptor.

The nicotinic acetylcholine receptor (AChR) of Torpedo electric organ and vertebrate skeletal muscle is closely associated with a Mr 43,000 protein (43K). In this study, we have examined the effects on the AChR of treatments which remove the 43K protein. We used semiquantitative fluorescence techniques to measure the binding of antibodies to clustered AChR in cultured rat myotubes. We found that labeling by antibodies to the cytoplasmic portions of each of the four receptor polypeptides increased significantly upon extraction of the 43K protein. Labeling by an antibody to an extracellular epitope of the alpha subunits was not affected by removal of the 43K protein, suggesting that changes were restricted to the cytoplasmic domains of the AChR. Increases in labeling by antibodies were more limited following protease treatment, which removes most cytoskeletal structures but leaves the 43K protein bound to the membrane. Competition between an antibody to the beta subunit and an antibody to the gamma and delta subunits suggests that the cytoplasmic portion of the AChR still retains a degree of native structure in the absence of the 43K protein. Our results suggest that, although some of these changes may be due to simply exposing additional epitopes on the AChR, the cytoplasmic portions of all the subunits of the AChR undergo significant conformational changes upon extraction of the 43K protein.     

Lipid matters: nicotinic acetylcholine receptor-lipid interactions (Review)

Ligand-gated ion channels mediate fast intercellular communication in response to endogenous neurotransmitters. The nicotinic acetylcholine receptor (AChR) is the archetype molecule in the superfamily of these membrane proteins. Early electron spin resonance studies led to the discovery of a lipid fraction in direct contact with the AChR, with rotational dynamics 50-fold slower than those of the bulk lipids. This AChR-vicinal lipid region has since been postulated to be a possible site of lipid modulation of receptor function. The polarity and molecular dynamics of solvent dipoles - mainly water - of AChR-vicinal lipids in the membrane have been studied with Laurdan extrinsic fluorescence, and Förster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET enabled one to discriminate between the bulk lipid and the AChR-vicinal lipid. The latter is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipids and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. After delineating the topography of the AChR membrane-embedded domains with fluorescence methods, sites for steroids are being explored with site-directed mutagenesis and patch-clamp recording. Pyrene-labelled Cys residues in &#102 M1, &#102 M4, &#110 M1 and &#110 M4 transmembrane regions were found to lie in a shallow position. For M4 segments, this is in agreement with a canonical linear &#102 -helix; for M1, it is necessary to postulate a substantial amount of non-helical structure, and/or of kinks, to rationalize the shallow location of Cys residues. Mutations of Thr 422, a residue close to the extracellular-facing membrane hemilayer in &#102 M4, affect the steroid modulation of AChR function, suggesting its involvement in steroid-AChR interactions.     

Live-cell molecular analysis of Akt activation reveals roles for activation loop phosphorylation

Activation of the serine/threonine protein kinase Akt/PKB is a multi-step process involving membrane recruitment, phosphorylation, and membrane detachment. To investigate this process in the cellular context, we employed a live-cell fluorescence imaging approach to examine conformational changes of Akt and its membrane association. A fluorescence resonance energy transfer-based reporter of Akt action (ReAktion) reveals a conformational change that is critically dependent on the existence of a phosphorylatable threonine 308 in the activation loop, because mutations to either aspartate or alanine abolished the change. Furthermore, a mutant carrying a phosphorylation mimic at this position showed diminished membrane association, suggesting that this phosphorylation plays an important role of promoting the dissociation of activated Akt from the membrane. In addition, the membrane-associating pleckstrin homology domain was found to associate with the catalytic domain when Thr308 is phosphorylated, suggesting such an interdomain interaction as a mechanism by which phosphorylation within the catalytic domain can affect membrane association. These studies uncover new regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.     

Fatty acid content of the dorsal muscle—an indicator of fat quality in freshwater fish

In 56 samples of freshwater fish, most were low in fat, ≤ 5% of dry weight (D.W.), and the sum of all fatty acids (ΣFA) was about 2% of D.W. Trout, whitefish, and grayling had the highest content of the long-chained FA. of ω3 type, EPA and DHA (1·7–2·6% of D.W.). Two large, low-fat pikes with ΣFA of about12–3% of D.W. and a medium-fat whitefish had the highest ω3/ω6 ratios, 8–9, whereas the fattiest fishes, eels from two lakes and the Baltic (ΣFA =17–26% of D.W.) had lower ω3/ω6 ratios, 1·1–1·8 (ω3 and ω6 FA are two important series of FA). The results indicate that ΣA controls the content of saturated FA (SAFA) and monounsaturated FA (MUFA), whereas the polyunsaturated FA (PUFA) was independent of ΣFA after a break point of about 10%ΣFA of D.W. The P/S ratio (PUFA/SAFA) and the PUFA/ΣFA ratio decreased with increased ΣFA, whereas the ω3/ω6 ratio showed no clear correlation to ΣFA. The difference in fatty acid patterns lay between low-fat and high-fat fishes, rather than between marine and freshwater fishes. The variation, both within and between species of the separate FA is small in fish with similar ΣFA content. Also, low-fat and medium-fat fishes tend to be more dietarily favourable than high-fat fishes, when considering the latest criteria for high nutritional value to humans. Abbreviations used in the text: FA, fatty acids; ΣFA, sum of all FA; AA, arachidonic acid (20 : 4ω6); EPA, eicosapentaenoic acid (20 : 5ω3); DHA, docosahexaenoic acid (22 : 6ω3); SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; D.W., dry weight; F.W. fresh weight; CV, coefficient of variation; ω3 FA, series of PUFA with the first double bond located at carbon number 3; ω6 FA, series of PUFA with the first double bond located at carbon number 6. The fatty acids are described by three numbers, x:ywz, where x=number of carbon atoms, y=number of double bonds, and z=position of the first double bond counted from the methyl end of the molecule.     

Tryptophan residues at subunit interfaces used as fluorescence probes to investigate homotropic and heterotropic regulation of aspartate transcarbamylase

The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications. The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes. These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation. To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-L-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively. The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation.     

Higher de novo synthesized fatty acids and lower ω3- and ω6-long-chain polyunsaturated fatty acids in umbilical vessels of women with preeclampsia and high fish intakes

Umbilical veins (UV) and arteries (UA) of preeclamptic women in Curaçao harbor lower long-chain polyunsaturated fatty acids (LCP). The present aim was to test these findings in Mwanza (Tanzania), whose inhabitants have high LCPω3 and LCPω6 intakes from Lake Victoria fish. Women with preeclampsia (n=28) in Mwanza had lower PUFA and higher 20:0 in UV and UA, compared with normotensive/non-proteinuric controls (n=31). Their UV 22:6ω3, 22:4ω6, LCPω6, ω6, and LCPω3+ω6 were lower, while saturated FA, potentially de novo synthesized FA (Σde novo) and (Σde novo)/(LCPω3+ω6) ratio were higher. Their UA had higher 16:1ω7, ω7, 18:0, and 16:1ω7/16:0. Umbilical vessels in Mwanza had higher 22:6ω3, LCPω3, ω3, and 16:0, and lower 22:5ω6, 20:2ω6, 18:1ω9, and ω9, compared to those in Curaçao. Preeclampsia in both Mwanza and Curaçao is characterized by lower LCP and higher Σde novo. An explanation of this might be placental dysfunction, while the similarity of umbilical vessel FA-abnormalities in preeclamptic and diabetic pregnancies suggests insulin resistance as a common denominator.     

Clustering of nicotinic acetylcholine receptors: From the neuromuscular junction to interneuronal synapses

Fast and accurate synaptic transmission requires high-density accumulation of neurotransmitter receptors in the postsynaptic membrane. During development of the neuromuscular junction, clustering of acetylcholine receptors (AChR) is one of the first signs of postsynaptic specialization and is induced by nerve-released agrin. Recent studies have revealed that different mechanisms regulate assembly vs stabilization of AChR clusters and of the postsynaptic apparatus. MuSK, a receptor tyrosine kinase and component of the agrin receptor, and rapsyn, an AChR-associated anchoring protein, play crucial roles in the postsynaptic assembly. Once formed, AChR clusters and the postsynaptic membrane are stabilized by components of the dystrophin/utrophin glycoprotein complex, some of which also direct aspects of synaptic maturation such as formation of postjunctional folds. Nicotinic receptors are also expressed across the peripheral and central nervous system (PNS/CNS). These receptors are localized not only at the pre- but also at the postsynaptic sites where they carry out major synaptic transmission. In neurons, they are found as clusters at synaptic or extrasynaptic sites, suggesting that different mechanisms might underlie this specific localization of nicotinic receptors. This review summarizes the current knowledge about formation and stabilization of the postsynaptic apparatus at the neuromuscular junction and extends this to explore the synaptic structures of interneuronal cholinergic synapses.     

Lipoxygenases mediate the effect of essential fatty acid in skin barrier formation: a proposed role in releasing omega-hydroxyceramide for construction of the corneocyte lipid envelope

A barrier to water loss is vital to maintaining life on dry land. Formation of the mammalian skin barrier requires both the essential fatty acid linoleate and the two lipoxygenases 12R-lipoxygenase (12R-LOX) and epidermal lipoxygenase-3 (eLOX3), although their roles are poorly understood. Linoleate occurs in O-linoleoyl-ω-hydroxyceramide, which, after hydrolysis of the linoleate moiety, is covalently attached to protein via the free ω-hydroxyl of the ceramide, forming the corneocyte lipid envelope, a scaffold between lipid and protein that helps seal the barrier. Here we show using HPLC-UV, LC-MS, GC-MS, and (1)H NMR that O-linoleoyl-ω-hydroxyceramide is oxygenated in a regio- and stereospecific fashion by the consecutive actions of 12R-LOX and eLOX3 and that these products occur naturally in pig and mouse epidermis. 12R-LOX forms 9R-hydroperoxy-linoleoyl-ω-hydroxyceramide, further converted by eLOX3 to specific epoxyalcohol (9R,10R-trans-epoxy-11E-13R-hydroxy) and 9-keto-10E,12Z esters of the ceramide; an epoxy-ketone derivative (9R,10R-trans-epoxy-11E-13-keto) is the most prominent oxidized ceramide in mouse skin. These products are absent in 12R-LOX-deficient mice, which crucially display a near total absence of protein-bound ω-hydroxyceramides and of the corneocyte lipid envelope and die shortly after birth from transepidermal water loss. We conclude that oxygenation of O-linoleoyl-ω-hydroxyceramide is required to facilitate the ester hydrolysis and allow bonding of the ω-hydroxyceramide to protein, providing a coherent explanation for the roles of multiple components in epidermal barrier function. Our study uncovers a hitherto unknown biochemical pathway in which the enzymic oxygenation of ceramides is involved in building a crucial structure of the epidermal barrier.     

ω-Hydroxyundec-9-enoic acid induces apoptosis through ROS-mediated endoplasmic reticulum stress in non-small cell lung cancer cells

ω-Hydroxyundec-9-enoic acid (ω-HUA), a hydroxyl unsaturated fatty acid derivative, is involved in the antifungal activity of wild rice (Oryza officinalis). Here, we investigated the anti-cancer activity of ω-HUA on a non-small cell lung cancer (NSCLC) cell line. ω-HUA increased apoptosis and induced cleavages of caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP). ω-HUA treatment significantly induced endoplasmic reticulum (ER) stress response. Suppression of CHOP expression and inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly attenuated the ω-HUA treatment-induced activation of caspase-6, caspase-9, and PARP, and subsequent apoptotic cell death, indicating a role for ER stress in ω-HUA-induced apoptosis. In addition, cells subjected to ω-HUA exhibited significantly increased quantity of reactive oxygen species (ROS), and the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited ω-HUA-induced apoptotic cell death and ER stress signals, indicating a role for ROS in ER stress-mediated apoptosis in ω-HUA-treated cells. Taken together, these results suggest that sequential ROS generation and ER stress activation are critical in ω-HUA treatment-induced apoptosis and that ω-HUA represents a promising candidate for NSCLC treatment.     

Cytoplasmic components of acetylcholine receptor clusters of cultured rat myotubes: the 58-kD protein

A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.