Instituto de Investigaciones Bioquímicas de Bahía Blanca, CONICET-UNS

Cholesterol influences ion-channel function, distribution and clustering in the membrane, endocytosis, and exocytic sorting of the nicotinic acetylcholine receptor (AChR). We report the occurrence of a cholesterol recognition motif, here coined "CARC", in the transmembrane regions of AChR subunits that bear extensive contact with the surrounding lipid, and are thus optimally suited to convey cholesterol-mediated signaling from the latter. Three cholesterol molecules could be docked on the transmembrane segments of each AChR subunit, rendering a total of 15 cholesterol molecules per AChR molecule. The CARC motifs contribute each with an energy of interaction between 35 and 52 kJ.mol(-1), adding up to a total of about 200 kJ.mol(-1) per receptor molecule, i.e. ∼40% of the lipid solvation free energy/ AChR molecule. The CARC motif is remarkably conserved along the phylogenetic scale, from prokaryotes to human, suggesting that it could be responsible for some of the above structural/functional properties of the AChR.

Biological membranes show lateral and transverse asymmetric lipid distribution. Cholesterol (Chol) localizes in both hemilayers, but in the external one it is mostly condensed in lipid-ordered microdomains (raft domains), together with saturated phosphatidyl lipids and sphingolipids (including sphingomyelin and glycosphingolipids). Membrane asymmetries induce special membrane biophysical properties and behave as signals for several physiological and/or pathological processes. Alzheimer's disease (AD) is associated with a perturbation in different membrane properties. Amyloid-β (Aβ) plaques and neurofibrillary tangles of tau protein together with neuroinflammation and neurodegeneration are the most characteristic cellular changes observed in this disease. The extracellular presence of Aβ peptides forming senile plaques, together with soluble oligomeric species of Aβ, are considered the major cause of the synaptic dysfunction of AD. The association between Aβ peptide and membrane lipids has been extensively studied. It has been postulated that Chol content and Chol distribution condition Aβ production and posterior accumulation in membranes and, hence, cell dysfunction. Several lines of evidence suggest that Aβ partitions in the cell membrane accumulate mostly in raft domains, the site where the cleavage of the precursor AβPP by β- and γ- secretase is also thought to occur. The main consequence of the pathogenesis of AD is the disruption of the cholinergic pathways in the cerebral cortex and in the basal forebrain. In parallel, the nicotinic acetylcholine receptor has been extensively linked to membrane properties. Since its transmembrane domain exhibits extensive contacts with the surrounding lipids, the acetylcholine receptor function is conditioned by its lipid microenvironment. The nicotinic acetylcholine receptor is present in high-density clusters in the cell membrane where it localizes mainly in lipid-ordered domains. Perturbations of sphingomyelin or cholesterol composition alter acetylcholine receptor location. Therefore, Aβ processing, Aβ partitioning, and acetylcholine receptor location and function can be manipulated by changes in membrane lipid biophysics. Understanding these mechanisms should provide insights into new therapeutic strategies for prevention and/or treatment of AD. Here, we discuss the implications of lipid-protein interactions at the cell membrane level in AD.

The PI3K/Akt pathway is a key component in synaptic plasticity and neuronal survival. The aim of this work was to investigate the participation of the PI3K/Akt pathway and its outcome on different molecular targets such as glycogen synthase kinase 3β (GSK3β) and Forkhead box-O (FoxO) transcription factors during mild oxidative stress triggered by iron overload. The exposure of mouse hippocampal neurons (HT22) to different concentrations of Fe(2+) (25-200 μm) for 24 h led us to define a mild oxidative injury status (50 μm Fe(2+)) in which cell morphology showed changes typical of neuronal damage with increased lipid peroxidation and cellular oxidant levels but no alteration of cellular viability. There was a simultaneous increase in both Akt and GSK3β phosphorylation. Levels of phospho-FoxO3a (inactive form) increased in the cytosolic fraction of cells treated with iron in a PI3K-dependent manner. Moreover, PI3K and Akt translocated to the nucleus in response to oxidative stress. Iron-overloaded cells harboring a constitutively active form of Akt showed decreased oxidants levels. Indeed, GSH synthesis under oxidative stress conditions was regulated by activated Akt. Our results show that activation of the PI3K/Akt pathway during iron-induced neurotoxicity regulates multiple targets such as GSK3β, FoxO transcriptional activity, and glutathione metabolism, thus modulating the neuronal response to oxidative stress.

This short overview addresses the effects of cholesterol (Chol) on the stability, supramolecular organization, and function of the murine muscle-type nicotinic acetylcholine receptor (AChR). The biophysical properties of Chol-AChR interaction are discussed first, as they constitute, in our view, the very source of the modulation exerted by Chol on receptor structure and function. In the absence of innervation, AChRs occur in the form of aggregates that remain stable at the cell-surface membrane of mammalian model cells over a period of hours. Acute Chol depletion drastically reduces (approximately 50%) the number of receptor aggregates by accelerating the rate of endocytosis in a receptor-expressing clonal cell line. Chol depletion also results in ion channel gain-of-function of the remaining cell-surface AChRs, whereas Chol enrichment has the opposite effect. Wide-field and confocal microscopy show AChR clusters as diffraction-limited puncta of approximately 200 nm diameter. Stimulated emission depletion fluorescence microscopy resolves these puncta into nanoclusters with an average diameter of approximately 55 nm. Exploiting the enhanced resolution, the effect of acute Chol depletion is shown to alter the short- and long-range organization of AChR nanoclusters. In the short range, the 50% AChRs remaining at the cell surface form larger nanoclusters. On larger scales (0.5-3.5 microm) Ripley's K-test on stimulated emission depletion images reveal changes in nanocluster distribution, attributable to the Chol-related abolition of cytoskeletal physical barriers normally preventing the lateral diffusion of AChR nanoclusters. We discuss these observations in the light of the so-called 'raft' hypothesis and surmise that Chol content at the plasmalemma homeostatically modulates cell-surface organization and stability of receptor nanodomains and fine tunes receptor channel function to temporarily compensate for acute AChR loss from the cell surface.

In artificial membrane bilayers, saturated long acyl chain-containing phospholipids and cholesterol (Chol) interact to form more ordered domains than those in phospholipids with unsaturated or short fatty acyl chains. We have extended the fluorescence techniques of London et al. [Xu, X., and London, E. (2000) Biochemistry 39, 843-849; Xu, X., Bittman, R., Duportail, G., Heissler, D., Vilchezes, C., and London, E. (2001) J. Biol. Chem. 276, 33540-33546] to study the propensity of several steroids to form or disrupt such ordered lipid domains. Temperature-dependent fluorescence quenching and steady-state polarization of the extrinsic fluorescent probe diphenylhexatriene (DPH) in model membranes composed of dipalmitoylphosphatidylcholine (or sphingomyelin), a nitroxide spin-labeled phosphatidylcholine (12-SLPC), and a given steroid were combined to study the influence of the latter on (a) ordered lipid domain formation, (b) stabilization, and (c) the extension of the ordered lipid assemblies. The results of the two totally independent methods, fluorescence quenching by 12-SLPC and fluorescence polarization of DPH, show that all steroids examined, except for Chol and 25-hydroycholesterol, behave as lipid domain-disrupting compounds. Additionally, we found a positive correlation between the hydrophobicity of steroids and their ordered lipid domain-promoting activity. Comparison of the chemical structures disclosed some distinctive traits of ordered lipid domain-promoting steroids: (i) the presence of an isooctyl side chain bond at C17; (ii) the absence of carbons attached to C23 (i.e., C24-C27) in any of the other (domain-disrupting) steroids; (iii) the presence of a small polar group at position C3; and (iv) the absence of polar groups in the fused rings, with the exception of substitutions at position C3 in the A ring.

There is an increasing body of evidence to support the notion that the function of the nicotinic acetylcholine receptor (AChR) is influenced by its lipid microenvironment [see Barrantes, F. J. (1993) FASEB J. 7, 1460-1467]. We have recently made use of the so-called generalized polarization (GP) of the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) to learn about the physical state of the lipids in Torpedo marmorata AChR native membrane [Antollini, S. S., Soto, M. A., Bonini de Romanelli, I., Gutiérrez Merino, C., Sotomayor, P., and Barrantes, F. J. (1996) Biophys. J. 70, 1275-1284] and cells expressing endogenous or heterologous AChR [Zanello, L. P., Aztiria, E., Antollini, S., and Barrantes, F. J. (1996) Biophys. J. 70, 2155-2164]. In the present work, Laurdan GP was measured in T. marmorata native AChR membrane by direct excitation or under energy transfer conditions in the presence of exogenous lipids. GP was found to diminish in these two regions upon addition of oleic acid and dioleoylphosphatidylcholine and not to vary significantly upon addition of cholesterol hemisuccinate, indicating an increase in the polarity of the single, ordered-liquid lipid phase in the two former cases. Complementary information about the bulk lipid order was obtained from measurements of fluorescence anisotropy of DPH and two of its derivatives. The membrane order diminished in the presence of oleic acid and dioleoylphosphatidylcholine. The location of Laurdan was determined using the parallax method. Laurdan lies at approximately 10 A from the center of the bilayer, i.e., at depth of approximately 5 A from the lipid-water interface. Exogenous lipids modified the energy transfer efficiency from the intrinsic fluorescence to Laurdan. This strategy is introduced as a new analytic tool that discloses for the first time the occurrence of discrete and independent sites for phospholipids and sterols, respectively, both accessible to fatty acids, and presumably located at a shallow depth close to the phospholipid polar head region in the native AChR membrane.

PURPOSE: Eye movements follow a reproducible pattern during normal reading. Each eye movement ends up in a fixation point, which allows the brain to process the incoming information and to program the following saccade. Alzheimer disease (AD) produces eye movement abnormalities and disturbances in reading. In this work, we investigated whether eye movement alterations during reading might be already present at very early stages of the disease. METHODS: Twenty female and male adult patients with the diagnosis of probable AD and 20 age-matched individuals with no evidence of cognitive decline participated in the study. Participants were seated in front of a 20-inch LCD monitor and single sentences were presented on it. Eye movements were recorded with an eye tracker, with a sampling rate of 1000 Hz and an eye position resolution of 20 arc seconds. RESULTS: Analysis of eye movements during reading revealed that patients with early AD decreased the amount of words with only one fixation, increased their total number of first- and second-pass fixations, the amount of saccade regressions and the number of words skipped, compared with healthy individuals (controls). They also reduced the size of outgoing saccades, simultaneously increasing fixation duration. CONCLUSIONS: The present study shows that patients with mild AD evidenced marked alterations in eye movement behavior during reading, even at early stages of the disease. Hence, evaluation of eye movement behavior during reading might provide a useful tool for a more precise early diagnosis of AD and for dynamical monitoring of the pathology.

Alzheimer's disease (AD) is one of the most devastating diseases of the central nervous system (CNS). It is characterized by two neuropathological findings: amyloid plaques and neurofibrillary tangles. AD is also accompanied by an extensive functional deficit in the cholinergic system, involving the neuronal-type nicotinic acetylcholine receptor (AChR). Furthermore there is increasing evidence showing a misregulation of cholesterol metabolism in the development of the disease. Since cholesterol affects AChR protein at multiple levels, the cognitive impairment and other neurological correlates of AD might be partly associated with an abnormal crosstalk between the receptor protein and the sterol in this synaptopathy.

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.

We have previously demonstrated that Lactobacillus reuteri CRL1098 soluble factors were able to reduce TNF-α production by human peripheral blood mononuclear cells. The aims of this study were to determine whether L. reuteri CRL1098 soluble factors were able to modulate in vitro the inflammatory response triggered by LPS in murine macrophages, to gain insight into the molecular mechanisms involved in the immunoregulatory effect, and to evaluate in vivo its capacity to exert anti-inflammatory actions in acute lung injury induced by LPS in mice. In vitro assays demonstrated that L. reuteri CRL1098 soluble factors significantly reduced the production of pro-inflammatory mediators (NO, COX-2, and Hsp70) and pro-inflammatory cytokines (TNF-α, and IL-6) caused by the stimulation of macrophages with LPS. NF-kB and PI3K inhibition by L. reuteri CRL1098 soluble factors contributed to these inhibitory effects. Inhibition of PI3K/Akt pathway and the diminished expression of CD14 could be involved in the immunoregulatory effect. In addition, our in vivo data proved that the LPS-induced secretion of the pro-inflammatory cytokines, inflammatory cells recruitment to the airways and inflammatory lung tissue damage were reduced in L. reuteri CRL1098 soluble factors treated mice, providing a new way to reduce excessive pulmonary inflammation.

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.

Free fatty acids (FFA) are essential components of the cell, where they play a key role in lipid and carbohydrate metabolism, and most particularly in cell membranes, where they are central actors in shaping the physicochemical properties of the lipid bilayer and the cellular adaptation to the environment. FFA are continuously being produced and degraded, and a feedback regulatory function has been attributed to their turnover. The massive increase observed under some pathological conditions, especially in brain, has been interpreted as a protective mechanism possibly operative on ion channels, which in some cases is of stimulatory nature and in other cases inhibitory. Here we discuss the correlation between the structure of FFA and their ability to modulate protein function, evaluating the influence of saturation/unsaturation, number of double bonds, and cis versus trans isomerism. We further focus on the mechanisms of FFA modulation operating on voltage-gated and ligand-gated ion channel function, contrasting the still conflicting evidence on direct versus indirect mechanisms of action.

The nicotinic acetylcholine receptor (nAChR) is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD) and steered rotation molecular dynamics (SRMD) simulations have been conducted on the cryo-electron microscopy (cryo-EM) structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh) binding pockets induced an inward and upward motion of the outer beta-sheet composed of beta9 and beta10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC), transmembrane (TM), and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA). To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O <--> C) motion. The present MD simulations explore the structural dynamics of the receptor under its gating process and provide a new insight into the gating mechanism of nAChR at the atomic level.

Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 μm at –70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the α subunit that differ between vertebrates and nematodes. The replacement of the conserved αGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that αGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs. Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 μm at –70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the α subunit that differ between vertebrates and nematodes. The replacement of the conserved αGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that αGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs. At the neuromuscular junction, acetylcholine (ACh) 1The abbreviations used are: AChR, nicotinic acetylcholine receptor; ACh, acetylcholine; Popen, channel open probability; HEK cells, human embryonic kidney cells. mediates fast neurotransmission by activating nicotinic receptors (AChRs). AChRs in nematode muscle are targets for anthelmintic chemotherapy. Levamisole and pyrantel are two widely used anthelmintic drugs. By binding to the AChR they lead to a depolarization of the somatic muscle of nematodes. The efficacy of these drugs is based on their ability to act as full agonists of AChRs in nematodes (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). Contractility and membrane potential measurements have shown that the nematode axial muscle is 10–100 times more sensitive to the acute action of pyrantel and levamisole than the rat muscle (2Atchison W.D. Geary T.G. Manning B. VandeWaa E.A. Thompson D.P. Toxicol. Appl. Pharmacol. 1992; 112: 133-143Crossref PubMed Scopus (22) Google Scholar). The molecular bases of this selectivity have not been yet elucidated. The kinetics of activation of nematode AChRs by levamisole has been studied in several preparations from parasite muscle (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar, 3Robertson S.J. Martin R.J. Br. J. Pharmacol. 1993; 108: 170-178Crossref PubMed Scopus (80) Google Scholar), but its action on mammalian muscle AChRs has not been described to date. The effects of levamisole on human neuronal α3β2 and α3β4 AChRs have been studied recently (4Levandoski M.M. Piket B. Chang J. Eur. J. Pharmacol. 2003; 471: 9-20Crossref PubMed Scopus (35) Google Scholar) with the voltage clamp method. It was shown that levamisole behaves as a weak partial agonist, an allosteric modulator, and an open channel blocker of neuronal AChRs (4Levandoski M.M. Piket B. Chang J. Eur. J. Pharmacol. 2003; 471: 9-20Crossref PubMed Scopus (35) Google Scholar). ACh is responsible for neuromuscular transmission in nematodes (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). In Caenorhabditis elegans muscle, levamisole-activated AChRs are composed of the unc-38 subunit, which encodes an α subunit, and lev-1 and unc-29, which encode non-α subunits (5Fleming J.T. Squire M.D. Barnes T.M. Tornoe C. Matsuda K. Ahnn J. Fire A. Sulston J.E. Barnard E.A. Sattelle D.B. Lewis J.A. J. Neurosci. 1997; 17: 5843-5857Crossref PubMed Google Scholar). Expression studies in Xenopus oocytes have shown that both unc-38 and unc-29 subunits are necessary for AChR function (5Fleming J.T. Squire M.D. Barnes T.M. Tornoe C. Matsuda K. Ahnn J. Fire A. Sulston J.E. Barnard E.A. Sattelle D.B. Lewis J.A. J. Neurosci. 1997; 17: 5843-5857Crossref PubMed Google Scholar). Both subunits are required for the expression of levamisole-sensitive receptors in body wall muscles of these nematodes (6Richmond J.E. Jorgensen E.M. Nat. Neurosci. 1999; 2: 791-797Crossref PubMed Scopus (356) Google Scholar). Other nematode α subunits have been cloned from the parasitic nematodes Trichostrongylus colubriformis (7Wiley L.J. Weiss A.S. Sangster N.C. Li Q. Gene (Amst.). 1996; 182: 97-100Crossref PubMed Scopus (22) Google Scholar), Haemonchus conturtus (8Hoekstra R. Visser A. Wiley L.J. Weiss A.S. Sangster N.C. Roos M.H. Mol. Biochem. Parasitol. 1997; 84: 79-187Crossref Scopus (46) Google Scholar), and Ascaris suum (9Le Novère N. Changeux J.P. Nucleic Acid Res. 1999; 27: 340-342Crossref PubMed Scopus (83) Google Scholar), showing a 91.6, 91, and 76% similarity with unc-38, respectively. The main structural features of the AChR subunits are strikingly conserved in phylogeny from higher organisms to the nematode. However, residues differentially conserved between mammalian and nematode AChRs may lead to a differential pharmacological action of anthelmintics at AChRs. In this study, we explore for the first time the interaction of levamisole with mammalian muscle AChRs at the single channel and macroscopic current levels. Our results reveal that levamisole shows an extremely low efficacy for channel activation. At high levamisole concentrations, channel blockade also contributes to maintain a low probability of channel opening. In contrast, levamisole has been shown to act as a potent agonist of different nematode muscle AChRs (3Robertson S.J. Martin R.J. Br. J. Pharmacol. 1993; 108: 170-178Crossref PubMed Scopus (80) Google Scholar, 10Lewis J.A. Wu C.H. Levine J.H. Berg H. Neuroscience. 1980; 5: 967-989Crossref PubMed Scopus (191) Google Scholar, 11Harrow I.D. Gration K.F. Pestic. Sci. 1985; 16: 662-672Crossref Scopus (99) Google Scholar, 12Martin R.J. Pennington A.J. Duittoz A.H. Robertson S. Kusel J.R. Parasitology. 1991; 102: 41-58Crossref PubMed Scopus (37) Google Scholar). Thus, this anthelmintic compound therapeutically exploits differences by selectively activating the AChR of the parasite and not that of the host. To identify residues involved in this different selectivity, we combined site-directed mutagenesis at residues differentially conserved between muscle α subunits from nematodes and vertebrates, and we evaluated the changes in levamisole activation. Our results reveal that the glutamic acid at position 153, which is highly conserved in all nematode α subunits cloned to date, may be involved in the potent activation of nematode AChRs by levamisole. The elucidation of the molecular basis of anthelmintic activation of AChRs will greatly contribute to the development of more selective therapies against parasites and to the understanding of how parasites develop resistance to the anthelmintics. In addition, it pinpoints determinants of function. Site-directed Mutagenesis and Expression of AChR—HEK293 cells with α or and at a subunit of for as described C. N. S. PubMed Scopus Google Scholar, C. I. Mol. Pharmacol. PubMed Scopus Google Scholar). was also for to of cells subunits the site-directed mutagenesis and all used for clamp and in the A. B. PubMed Scopus Google Scholar) at C. N. S. PubMed Scopus Google Scholar). The and and levamisole or both drugs to the channel an clamp at with the to the of a the and by the the at a of and time a and a and to the of by the as a of and by than a this was as the of of the time and the in the time showing openings clusters on the basis of their of open probability open and A. N. K. J. 1997; PubMed Scopus Google Scholar, C. J. PubMed Scopus Google Scholar, C. J. PubMed Scopus (46) Google Scholar). of the AChR two which as high and low of mode first to a kinetic for the we used clusters that the high mode activated by ACh and the kinetic was to the activity of a single AChR A. N. K. J. 1997; PubMed Scopus Google Scholar, C. J. PubMed Scopus Google Scholar, C. J. PubMed Scopus (46) Google Scholar). The open and from the clusters to kinetic an of The time was of open and from the and time and on the time as described by A. J. 1996; PubMed Scopus Google Scholar). the probability the open and The was to the low mode of the AChR and to AChRs activated by in which or clusters the of of single channel activity by low agonist concentrations, clusters be and block is on the basis of the activation two agonists to the receptor in the with and and with and AChRs by two agonist open with and with at low agonist about the open and the A. N. K. J. 1997; PubMed Scopus Google Scholar). of and be from the of the briefer of the time its and the as recordings, the and and The was in this and position at the of a as described J.P. C. Mol. Pharmacol. PubMed Scopus Google Scholar, C. 2003; PubMed Scopus Google Scholar). The for a of the the of of ACh, or both drugs to the at at and on the was performed the The current was for current by a single and are the peak and the current and is the time that the current to with at the the current of its Channel by is a full agonist of the nematode muscle AChR (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). In the study, we evaluated this anthelmintic also acts on mammalian muscle AChRs. To this we first single from cells muscle AChRs shown in this levamisole is of activating mammalian AChRs. However, channel openings are significantly briefer than those activated by the ACh. time of μm levamisole-activated AChRs be by a main of The of the main open is briefer than that at μm ACh C. N. S. PubMed Scopus Google Scholar, C. I. Mol. Pharmacol. PubMed Scopus Google Scholar). levamisole from to μm not the with full as ACh. At ACh higher than AChRs open in clusters of activation C. J. PubMed Scopus Google Scholar) activation with the of a single receptor from the to the and by to the At μm ACh, the probability of channel a is C. J. PubMed Scopus Google In contrast, when AChRs are activated by at as high as clusters are not results that levamisole mammalian AChRs with and than ACh. levamisole from to μm to a of open time of AChRs activated by μm levamisole be by a single with a open time of a in the open time that in to its of activating mammalian levamisole may act as an open channel blocker by the efficacy of levamisole in activating mammalian we macroscopic from with levamisole. shows from a single to of μm ACh and μm levamisole. In the current the peak and with a time of about to J.P. C. Mol. Pharmacol. PubMed Scopus Google Scholar). The peak current is about 3% when the is to μm levamisole. in single with levamisole. results the low efficacy of the to activate mammalian AChRs. that the single channel that levamisole may also block AChRs we studied its action as a channel blocker in the and of ACh. levamisole to briefer the open time of AChR activated by μm ACh or by levamisole used the to the action of levamisole as an open channel blocker as shown in and In with a between the of the open time and levamisole is The for the of the in by the of the is and for or levamisole-activated channels. Thus, AChRs activated by ACh or levamisole are by levamisole at a similar The apparent from the with the is for levamisole-activated AChRs and for channels. with the of by kinetic C. J. PubMed Scopus Google Scholar, C. J. PubMed Scopus (46) Google of levamisole AChR in the and of μm ACh levamisole at different concentrations. The open times from the open time The are by the open time α is the of levamisole for channel and α is the apparent channel are shown as of AChR in the of μm ACh levamisole. The time and its from the time are shown as of different for –70 between the time and membrane Levamisole are shown as of activated by ACh and with μm levamisole at –70 and current the of To the blockade by we studied the time of AChRs activated by μm ACh in the of levamisole. In its time to μm a main is on the of in the C. J. PubMed Scopus Google Scholar) The of levamisole significantly changes the time of and a of about is The of this not with levamisole but its increases as a function of its It is to that this to the blockade by levamisole of the in the of this a of is for Thus, the apparent for the is μm at a membrane potential of –70 At levamisole higher than the not as a function of The for the and are and and and for and μm respectively. at higher the channel block from The of the increases with higher membrane that the is The voltage of the effect is by At membrane μm levamisole not the of by ACh, and the be by a single similar to the In contrast, at –70 an fast fast is to open channel The time which to is The peak current is not suggesting that at the of that are levamisole with ACh for channel activation. In the of the blockade that levamisole acts as a open channel blocker at residues that are differentially conserved between mammalian and nematode muscle α subunits To identify these residues are involved in the differential of anthelmintic drugs at parasite and mammalian muscle, we by the residues in cells with the α and non-α and we evaluated channel activity by levamisole. The efficacy of levamisole to activate mammalian AChRs is greatly when the αGly-153 is by a glutamic shown in channel activity in clusters at μm levamisole. In contrast, changes are in the activation by levamisole of AChRs the or the of a of AChRs by levamisole. activated by μm levamisole from HEK cells AChRs and the and are at a of with channel openings as –70 time of AChRs the The are of for of AChR be at higher than The of is by changes in the time The main of the time which to is to briefer as a function of levamisole To the of the of a glutamic acid at we activated by a of levamisole to and the activity of single channel openings in In we the kinetics of activation by the full agonist ACh. in it be that clusters of activated by ACh or levamisole are not that this receptor activates in kinetic The of two different the clusters as to two main an and an At all agonist concentrations, the mode of openings that significantly and clusters that briefer than those of the mode on the we the clusters to mode and as a function of agonist and of AChRs activated by ACh or in a low of ACh or levamisole clusters to the high mode be The of the openings appear as The for these clusters is about at of are differences in the between or levamisole-activated clusters and at low agonist concentrations, levamisole activation of the AChR to be from ACh activation. of the mode are also at higher of both However, the channel as as the at higher of levamisole to channel blockade the agonist is clusters of the mode be In to we for the high the low differences between ACh and levamisole and In the of ACh, low clusters are at higher than The of these clusters increases as a function of ACh and a of about at μm ACh In addition, the open channel and the clusters as ACh is that they to activation levamisole is used as an agonist, low clusters be also at higher than The openings these clusters as a function of as for ACh However, to the open channel the open and the as the is The of the low clusters are significantly for levamisole than for AChRs To the of the mode to levamisole we clusters and the of activation to this between clusters or the of a single at μm of the clusters to the and of all clusters at activation activation of AChRs in the mode may significantly contribute to the selective action of anthelmintics on nematode AChRs. channel activity from cells was in the of agonist in the that mode results from AChR activation. from the of AChRs activated by levamisole a in the in and a in the as with AChRs activated by ACh Thus, the that levamisole is a weak agonist of the AChR to a low efficacy for activation. The also that the for the low mode of activated by levamisole is higher than that of AChRs for and AChRs activated by ACh and in a the we clusters for both and levamisole-activated and we kinetic to the open and time of these In this it is that an from the activation of a single that open and time of the of both and levamisole-activated AChRs we the to the activation and open of the AChR. The is for all ACh as as for levamisole than open blockade is not used the at of ACh and levamisole than μm and respectively. on this the kinetic for the the activation for both ACh and levamisole. from the open and time showing that activation is described by the The are similar for and levamisole-activated AChRs It be concluded that shows a mode which is highly sensitive to the agonist and is activated by levamisole and ACh. has been in a channel K. C. A. PubMed Scopus Google Scholar). studied the activation of this AChR by μm levamisole and it with that of At μm single channel clusters of that levamisole is more to activate than AChRs. However, are differences with to levamisole activation of high clusters are when is activated by μm levamisole. clusters with a open channel of a time of and a of are similar to those of clusters of the AChR at the agonist clusters of the at levamisole higher than but the for clusters is μm for AChRs. it that both the position and the of acid are in AChRs highly sensitive to anthelmintics. Levamisole the in C. and produces of A. suum muscle cells, and activates all nematode muscle AChRs (3Robertson S.J. Martin R.J. Br. J. Pharmacol. 1993; 108: 170-178Crossref PubMed Scopus (80) Google Scholar, 10Lewis J.A. Wu C.H. Levine J.H. Berg H. Neuroscience. 1980; 5: 967-989Crossref PubMed Scopus (191) Google Scholar, J. R. Br. J. Pharmacol. PubMed Scopus Google Scholar). of muscle and membrane potential have shown that the by levamisole is similar to that by ACh in the muscle of H. which is 10–100 times more sensitive to the acute action of levamisole than the rat muscle (2Atchison W.D. Geary T.G. Manning B. VandeWaa E.A. Thompson D.P. Toxicol. Appl. Pharmacol. 1992; 112: 133-143Crossref PubMed Scopus (22) Google Scholar). elucidated mammalian muscles sensitivity to the widely used anthelmintic agent levamisole than parasitic muscle, and we residues involved in differential Our studies at the molecular for the first time that levamisole is a weak agonist of mammalian muscle AChRs and reveal the basis of the low probability of mammalian AChR by an extremely low efficacy for channel activation. In addition, the low efficacy of levamisole is as a function of its to channel By site-directed mutagenesis we that which is conserved in all α subunits of muscle be involved in the high efficacy of levamisole at nematode AChRs. The weak activation of mammalian AChRs by levamisole is as the of a of levamisole concentrations, AChR activated by do not at clusters of activation at ACh higher than In contrast, it has been shown that both ACh and levamisole activate in the in nematode muscle (3Robertson S.J. Martin R.J. Br. J. Pharmacol. 1993; 108: 170-178Crossref PubMed Scopus (80) Google Scholar). The current by with levamisole with that activated by ACh. similar has been described for the partial agonist which produces a peak current of about that activated by the of ACh J.P. 1993; PubMed Scopus Google Scholar). Thus, in to is in the kinetics of levamisole-activated mammalian AChRs greatly differ from those of AChRs activated by the ACh. The increases the efficacy of levamisole as an agonist of mammalian AChRs. different be in this AChR on the basis of their probability of channel opening. kinetics have been in AChRs 1993; PubMed Scopus Google Scholar), and the and of kinetic have been shown to in AChRs K. R. PubMed Scopus Google Scholar, K. J. A. A.P. K. J. PubMed Scopus Google Scholar). The that at low to channel the kinetics of activation of the high mode is almost for ACh or the of levamisole in against The has been in by a K. C. A. PubMed Scopus Google Scholar). channel kinetic of AChRs has a of ACh which the AChR to open ACh K. C. A. PubMed Scopus Google Scholar). it has been that the to in the homologous position of the subunit high affinity binding for agonists with an in N. S. Changeux J.P. J. 2003; PubMed Scopus Google Scholar). of this receptor that the the of activation between the and open channel of the AChR N. S. Changeux J.P. J. 2003; PubMed Scopus Google Scholar). Our results that the to glutamic acid increases the efficacy of the but the is selectively higher for levamisole than for ACh. of levamisole activation between and shows that the of acid at this position is also to the kinetic The bases for the kinetic changes described for the AChR may be on the basis of In and of the receptor in the of agonist, and activation is by of the with binding of agonist to the with the with its the of AChRs is more than that of AChRs K. Mol. PubMed Scopus (22) Google Scholar). the of it that that an in may be of the binding of agonist to the open to of the AChR or to an effect on of the channel in the of that the has been shown to be involved in agonist it is that the may the affinity of and open and that these changes are more for levamisole than for ACh. The of two different binding and and which residues to the ACh binding in α subunits K. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). and a to and of agonist K. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar), a in position not changes in the activation by levamisole of the AChR. is with the of effects R. S. The and and R. Google Scholar) for of at and of an in apparent binding affinity for ACh and S. S. S.J. Changeux J.P. J. Neurosci. PubMed Google Scholar). However, the effects by at position S. S. S.J. Changeux J.P. J. Neurosci. PubMed Google Scholar). In to its agonist levamisole acts as a more potent blocker than ACh. of an open channel blockade are as a in the open a in the of the block and of the all blocker J.H. J. Scopus Google Scholar). are at levamisole than that this acts as an open channel blocker of mammalian AChRs. At higher concentrations, the from that of open channel from the open channel block have been also described for at high J.P. Mol. Pharmacol. 1992; Google Scholar, C. 1999; PubMed Scopus Google Scholar). Our be by the that the receptor may with or the blocker in its J.A. 1991; PubMed Scopus Google Scholar, J. Scopus Google Scholar). be to the of two or more in the J.P. R. J. 1997; PubMed Scopus Google Scholar, J.H. Br. J. Pharmacol. PubMed Scopus Google Scholar). In with studies of the action of the anthelmintic at the muscle AChR from Ascaris have a channel blockade that be by the of two the channel Martin R.J. Br. J. Pharmacol. 1996; PubMed Scopus Google Scholar). studies on Ascaris muscle have shown that the of levamisole for channel block is μm at (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). is similar to the for mammalian AChRs in the activation by levamisole is strikingly different between mammalian and nematode muscle the open channel blockade to be that blockade is not involved in the differential selectivity of anthelmintics on both of Levamisole and pyrantel are to the of action they have different is a levamisole is an By single channel recordings, we have shown C. PubMed Scopus Google Scholar) that pyrantel acts as a low agonist and a high affinity open channel blocker of the mammalian muscle AChRs. Our results that levamisole is potent than pyrantel to activate as as to block the mammalian AChR C. PubMed Scopus Google Scholar). The affinity for levamisole is higher than that of pyrantel μm at –70 to the weak agonist activity of pyrantel at mammalian it is that its effects are by its Our which that the ability of pyrantel is higher than are in with the that pyrantel has more effects than levamisole (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). resistance to the anthelmintics pyrantel and levamisole is an in nematode that in the mammalian AChR be to the nematode AChR, and this in the nematode AChR, it be to at are involved in the development of resistance of the parasite against anthelmintics. for and

We have modelled the transmembrane region of the alpha 7 nicotinic acetylcholine receptor as a mixed alpha-helical/beta-sheet structure. The model was mainly based on the crystal structure of a pore-forming toxin, heat-labile enterotoxin. This is a pentameric protein having a central pore or channel composed of five alpha-helices, one from each of the 5 B subunits that form this pentamer. The remainder of this structure is beta-sheet, loops and a short alpha-helix, not included in the model. The model uses this channel as a template to build the transmembrane region, from M1 to the middle of M3. The remainder of M3 and M4 were built de novo as alpha-helices. Great consideration was given to labelling data available for the transmembrane region. In general terms, the shape of the model agrees very well with that obtained independently by electron microscopic analysis and the secondary structure predicted by the model is in accord with that estimated independently by Fourier transform infrared spectroscopy. The M2 helical region of the model is only slightly kinked, contrary to what is inferred from electron microscopic analysis, but has the same overall shape and form. On the membrane face of the model, the presence of deep pockets may provide the structural basis for the distinction between annular and non-annular lipid binding sites. Also, the transmembrane region is clearly asymmetric in the direction perpendicular to the membrane, and this may have strong influence on the surrounding lipid composition of each leaflet of the cytoplasmic membrane.

To study the effects produced by free fatty acids (FFA) on the biophysical properties of Torpedo marmoratanicotinic acetylcholine receptor-rich native membranes and to investigate the topology of their binding site(s), fluorescence measurements were carried out using the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino) naphthalene) and ADIFAB, an Acrylodan-derivatized intestinal fatty acid-binding protein. The generalized polarization (GP) of the former probe was used to learn about the physical state of the membrane upon FFA binding. Saturated FFA induced a slight increase in GP, whereascis-unsaturated fatty acids decreased GP. Double bond isomerism could also be distinguished; oleic acid (18:1cis) induced a net disordering effect, whereas elaidic acid (18:1trans) produced no changes in GP. The changes in the efficiency of the Förster energy transfer from the protein to Laurdan brought about by addition of FFA, together with the distances involved in this process, indicate that all FFA studied share a common site at the lipid-protein interface. However, despite being located at the same site, each class of FFA differs in its effect on the physical properties of the membrane. These data lead us to suggest that it is the direct action of FFA at the lipid-protein interface, displacing essential lipids from their sites rather than changes in bulk properties such as membrane fluidity that accounts for the effect of FFA on the acetylcholine receptor membrane. To study the effects produced by free fatty acids (FFA) on the biophysical properties of Torpedo marmoratanicotinic acetylcholine receptor-rich native membranes and to investigate the topology of their binding site(s), fluorescence measurements were carried out using the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino) naphthalene) and ADIFAB, an Acrylodan-derivatized intestinal fatty acid-binding protein. The generalized polarization (GP) of the former probe was used to learn about the physical state of the membrane upon FFA binding. Saturated FFA induced a slight increase in GP, whereascis-unsaturated fatty acids decreased GP. Double bond isomerism could also be distinguished; oleic acid (18:1cis) induced a net disordering effect, whereas elaidic acid (18:1trans) produced no changes in GP. The changes in the efficiency of the Förster energy transfer from the protein to Laurdan brought about by addition of FFA, together with the distances involved in this process, indicate that all FFA studied share a common site at the lipid-protein interface. However, despite being located at the same site, each class of FFA differs in its effect on the physical properties of the membrane. These data lead us to suggest that it is the direct action of FFA at the lipid-protein interface, displacing essential lipids from their sites rather than changes in bulk properties such as membrane fluidity that accounts for the effect of FFA on the acetylcholine receptor membrane. nicotinic acetylcholine receptor actual hydrophobicity coefficient free fatty acid(s) Förster resonance energy transfer generalized polarization The nicotinic acetylcholine receptor (AChR)1 is an integral membrane protein deeply embedded in the postsynaptic region of muscle, electrocytes, and nerve cells. Experimental evidence from various groups including ours substantiates the notion that the function of this rapid ligand-gated channel is influenced by its lipid microenvironment (see reviews in Refs. 1Barrantes F.J. FASEB J. 1993; 7: 1460-1467Google Scholar, 2Barrantes F.J. Watts A. Protein-Lipid Interactions: New Comprehensive Biochemistry. 26. Elsevier Science Publishers B.V., Amsterdam1993: 231-257Google Scholar, 3Barrantes F.J. Antollini S.S. Bouzat C. Garbus I. Massol R.H. Kidney Int. 2000; 57: 1382-1389Google Scholar). Although the occurrence of specific interactions between the transmembrane region of the AChR and adjacent lipid molecules in the membrane has been reported (4Giraudat J. Montecucco C. Bisson R. Changeux J.P. Biochemistry. 1985; 24: 3121-3127Google Scholar, 5Blanton M.P. Cohen J.B. Biochemistry. 1992; 31: 3738-3750Google Scholar, 6Blanton M.P. Cohen J.B. Biochemistry. 1994; 33: 2859-2872Google Scholar), the exact nature of these interactions has not been clearly established. The first shell of lipids around the AChR, the so-called annular lipid (7Marsh D. Barrantes F.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4329-4333Google Scholar, 8Marsh D. Watts A. Barrantes F.J. Biochim. Biophys. Acta. 1981; 645: 97-101Google Scholar), exhibits distinct characteristics, such as a higher degree of order and a lower mobility than the bulk lipids. This specialized region of the membrane has received particular attention as the likely candidate domain where modulation of AChR function by lipids occurs (7Marsh D. Barrantes F.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4329-4333Google Scholar, 9Bhushan A. McNamee M.G. Biochim. Biophys. Acta. 1990; 1027: 93-101Google Scholar). The presence of both cholesterol and negatively charged phospholipids (10Criado M. Eibl H. Barrantes F.J. Biochemistry. 1982; 21: 3622-3629Google Scholar, 11Criado M. Eibl H. Barrantes F.J. J. Biol. Chem. 1984; 259: 9188-9198Google Scholar, 12Fong T.M. McNamee M.G. Biochemistry. 1987; 26: 3871-3880Google Scholar, 13McCarthy M.P. Moore M.A. J. Biol. Chem. 1992; 267: 7655-7663Google Scholar, 14Bhushan A. McNamee M.G. Biophys J. 1993; 64: 716-723Google Scholar, 15Ryan S.E. Demers C.N. Chew J.P. Baenziger J.E. J. Biol. Chem. 1996; 271: 24590-24597Google Scholar, 16Rankin S.E. Addona G.H. Kloczewiak M.A. Bugge B. Miller K.W. Biophys. J. 1997; 73: 2446-2455Google Scholar) has been shown to be necessary for proper AChR-mediated ion translocation in vitro. Various hypotheses were postulated to explain this functional dependence, because these lipids may modify the biophysical properties of the lipid annulus and/or the bulk lipid bilayer (12Fong T.M. McNamee M.G. Biochemistry. 1987; 26: 3871-3880Google Scholar, 18Sunshine C. McNamee M.G. Biochim. Biophys. Acta. 1992; 1108: 240-246Google Scholar, 19Sunshine C. McNamee M.G. Biochim. Biophys. Acta. 1994; 1191: 59-64Google Scholar). A second possibility is the occurrence of specific sites for certain lipids at the lipid-facing surface of the AChR, substantiated by the work of several groups (20Jones O.T. McNamee M.G. Biochemistry. 1988; 27: 2364-2874Google Scholar, 21Narayanaswami V. McNamee M.G. Biochemistry. 1993; 32: 12420-12427Google Scholar, 22Dreger M. Krauss M. Herrmann A. Hucho F. Biochemistry. 1997; 36: 839-847Google Scholar, 23Corbin J. Wang H.H. Blanton M.P. Biochim. Biophys. Acta. 1998; 1414: 65-74Google Scholar, 24Addona G.H. Sandermann H.Jr. Kloczewiak M.A. Husain S.S. Miller K.W. Biochim. Biophys. Acta. 1998; 1370: 299-309Google Scholar, 25Antollini S.S. Barrantes F.J. Biochemistry. 1998; 37: 16653-16662Google Scholar). Upon interacting with these sites, lipids would stabilize the secondary structure of the AChR transmembrane segments (12Fong T.M. McNamee M.G. Biochemistry. 1987; 26: 3871-3880Google Scholar, 26Fernandez-Ballester G. Castresana J. Fernandez A.M. Arrondo J.L. Ferragut J.A. Gonzalez-Ros J.M. Biochem. Soc. Trans. 1994; 22: 776-780Google Scholar, 27Baenziger J.E. Darsaut T.E. Morris M.L. Biochemistry. 1999; 38: 4905-4911Google Scholar). Recently, Baenziger et al. (28Baenziger J.E. Morris M.L. Darsaut T.E. Ryan S.E. J. Biol. Chem. 2000; 275: 777-784Google Scholar) proposed a model of how lipid composition modulates the function of the AChR, suggesting that membrane fluidity or some other bulk property of the membrane modulates the equilibrium between the resting and desensitized states of AChR. They also suggested that in addition to this indirect effect, the AChR has a specific requirement for anionic lipids (such as phosphatidic acid) binding to a specific site on the AChR or exerting a less specific charge effect on AChR conformation. Previous studies from several laboratories demonstrate that free fatty acids (FFA) inhibit the ion flux mediated by the AChR in vitro (29Andreasen T.J. McNamee M.G. Biochemistry. 1980; 19: 4719-4726Google Scholar, 30Villar M.T. Artigues A. Ferragut J.A. Gonzalez-Ros J.M. Biochim. Biophys. Acta. 1988; 938: 35-43Google Scholar) or in vivo (31Bouzat C.B. Barrantes F.J. Receptors and Channels. 1993; 1: 251-258Google Scholar). Analysis of single-channel electrophysiological data argues for a mechanism compatible with noncompetitive inhibition of the AChR. From these studies the conclusion was drawn that the effect of FFA is related to their hydrophobic character, but the exact mechanism of FFA action is still not clear. To further investigate the possibility that these compounds exert their effect on AChR at the lipid-protein interface and to investigate the nature of these effects on the biophysical property of the AChR-rich membrane, we carried out fluorescence studies using the fluorescent probes Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) and ADIFAB, an Acrylodan-derivatized intestinal fatty acid-binding protein. Laurdan possesses an exquisite sensitivity to the phase state of the membrane. The physical origin of Laurdan spectral properties resides in its capacity to sense the polarity and the molecular dynamics of dipoles in its environment because of the effect of dipolar relaxation processes (32De Parasassi T. Stasio G. d'Ubaldo A. Gratton E. Biophys. J. 1990; 57: 1179-1186Google Scholar, 33De Parasassi T. Stasio G. Ravagnan G. Rusch R.M. Gratton E. Biophys. J. 1991; 60: 179-189Google Scholar). The principal dipoles sensed by Laurdan in the membrane are water molecules. When no relaxation occurs, high Laurdan GP values result, indicative of low water content in the hydrophobic/hydrophilic interface region of the membrane. Thus, GP values depend on the extent of water penetration allowed by the local membrane packing and hence provide a direct report on the AChR membrane environment. In the present work, we have exploited the advantageous spectroscopic properties of Laurdan to study the effect of FFA with different structure in the native membrane in which the AChR protein is embedded. Complementary studies with ADIFAB were also performed to determine the partition coefficient of the different fatty acids in the membrane. This information enabled us to compare the effects caused by fatty acids at the same effective concentration in the membrane. We found that the carbon chain length of fatty acids, as well as the number of double bonds and their stereochemical configuration, are important determinants of the unique effects of the different FFA species on the physical properties of the AChR-rich membrane. Furthermore, changes in the efficiency of the energy transfer from the protein to Laurdan, brought about by the addition of exogenous FFA, revealed the presence of sites for FFA at the lipid-protein interface in the native AChR membrane. Preliminary data have been presented in abstract form (34Antollini S.S. Barrantes F.J. Biophys. J. 1999; 76 (abstr.): 370Google Scholar). Torpedo marmorata specimens were obtained from the Mediterranean coast off Alicante, Spain. They were killed by pithing, and the electric organs were dissected and stored at −70 °C until further use. Laurdan and ADIFAB were purchased from Molecular Probes (Eugene, OR). All other drugs were obtained from Sigma. Membrane fragments rich in AChR were prepared from the electric tissue of T. marmorata as described previously (35Barrantes F.J. Hucho F. Neuroreceptors. W. de Gruyter, Berlin, New York1982: 315-328Google Scholar). Specific activities in the order of 2.0–2.8 nmol α-bungarotoxin sites/mg protein were obtained. The orientation of AChR in vesicles was measured as described by Hartig and Raftery (36Hartig P.R. Raftery M.A. Biochemistry. 1979; 18: 1146-1150Google Scholar) by determining the total toxin-binding sites in the presence of Triton X-100 and the right side out toxin-binding sites in the absence of detergent (36Hartig P.R. Raftery M.A. Biochemistry. 1979; 18: 1146-1150Google Scholar) as in previous work from our laboratory (37Gutiérrez-Merino C. Bonini de Romanelli I.C. Pietrasanta L.I. Barrantes F.J. Biochemistry. 1995; 34: 4846-4855Google Scholar). For fluorescent measurements, AChR-rich membranes were suspended in buffer A (150 mm NaCl, 0.25 mmMgCl2, and 20 mm HEPES buffer, pH 7.4) at a final concentration of 50 μg protein/ml (0.2 μm). The optical density of the membrane suspension was kept below 0.1 to minimize light scattering. Sodium salts of FFA were dissolved in buffer A with a bath sonicator. In same cases we first prepared a sodium salt FFA stock solution in 4 mm NaOH, and aliquots were diluted to final concentrations with buffer A. FFA were dissolved in ethanol (in all cases the amount of ethanol added to the samples was kept below 0.5%). All fluorimetric measurements were performed in an SLM model 4800 fluorimeter using a light from a obtained with a and and were for The was at 20 °C with a water bath Laurdan was added to AChR-rich membrane samples from an ethanol solution to a final probe concentration of The amount of was kept below The samples were in the for at GP (32De Parasassi T. Stasio G. d'Ubaldo A. Gratton E. Biophys. J. 1990; 57: 1179-1186Google Scholar, 33De Parasassi T. Stasio G. Ravagnan G. Rusch R.M. Gratton E. Biophys. J. 1991; 60: 179-189Google Scholar) was as where are the at the of the phase and the phase GP values were obtained from obtained with an of The energy transfer efficiency in to all other processes of the on the of the between and to Scholar), is by the where is the is a for each as the at which is be as where and are the fluorescence of in the presence and absence of the and and are the in and to the protein which is When was measured in the presence of exogenous FFA, a further was to for of the fluorescence of by other mechanism induced by 4 where is the of by the of the fluorescence by the values were using in the presence of FFA, with or Laurdan, was with the where and are the concentrations of fatty acid in the membrane phase and in the and and are the of the and membrane is as where is the added FFA the values were obtained using ADIFAB, which to FFA binding with a in fluorescence from in the to in the a be from the of the fluorescence at to that at A. A.M. Biochemistry. 1993; 32: Scholar), to the where is the measured of to is this with no FFA is the ADIFAB is with FFA, and and are the ADIFAB with and concentrations of FFA, the of the of FFA and values were found to be and A.M. J. Biol. Chem. 1992; 267: Scholar). These values are for all is the for FFA ADIFAB from a obtained by of ADIFAB with FFA, to a form of were obtained using the where A and was The for each addition was to its effective concentration the membrane using the where is the lipid and is the lipid concentration in the which for each addition of A of was because AChR-rich membranes are in a phase at 20 °C S.S. Bonini de Romanelli C. Barrantes F.J. Biophys. J. 1996; Scholar). were by was as To compare the effects caused by the presence of different exogenous FFA on the native AChR-rich membrane from T. we first measured their partition are several for partition of lipids in but the of using ADIFAB is that it is not necessary to free and fatty as is the using fatty acids, a to ADIFAB, an intestinal fatty acid-binding protein A. A.M. Biochemistry. 1993; 32: Scholar, A.M. J. Biol. Chem. 1992; 267: Scholar), is a fluorescent for the of FFA concentration in the to 20 The of FFA by ADIFAB is on a in the of the fluorescent to the binding of the protein the is by a fatty ADIFAB a spectral upon FFA the of FFA concentrations from the of the fluorescence of and measured at about and In the shown in the in at and the increase at of the ADIFAB is upon of AChR-rich membrane marmorata with and we obtained the values of the and partition coefficient for the different fatty acids and ADIFAB in solution and in the presence of Torpedo AChR-rich The values allowed us to the fatty acids different hydrophobic fatty acids, such as and less hydrophobic fatty acids, such as and and fatty acids, such as and and fatty Torpedo AChR-rich membrane partition in a The values of the fatty acids in native AChR-rich membrane were used to the concentration added in the an effective concentration in the membrane for each fatty acid (see To study the of the physical properties of the membrane induced by the presence of FFA, we exploited the fluorescence probe exquisite sensitivity to the phase state of the membrane. We have previously used the so-called GP of Laurdan as a to the physical state native AChR-rich membrane S.S. Barrantes F.J. Biochemistry. 1998; 37: 16653-16662Google Scholar, S.S. Bonini de Romanelli C. Barrantes F.J. Biophys. J. 1996; Scholar). In the present work we the of the polarity of both the AChR and bulk lipid by fatty Laurdan GP values were obtained by direct or using the membrane fluorescence as at and Laurdan molecules as In a previous work we this and that the GP values obtained higher GP values than obtained by direct of the that the microenvironment of the AChR has lower polarity than the bulk lipid S.S. Bonini de Romanelli C. Barrantes F.J. Biophys. J. 1996; Scholar). The changes in GP values caused by different FFA could be using the effective concentration of each fatty acid the membrane, using their values We found that addition of FFA native membrane GP to different in a on the structure of the fatty fatty acids decreased GP whereas caused a were between different fatty and the being In a fatty each double bond a of in the chain R. R. of Membrane Berlin, Scholar). Thus, a fatty acid with several double bonds changes in the of its molecular in an increase in the of the chain the found in a fatty The in the GP values induced by FFA an increase in polarity the Laurdan This in is a of the disordering of the bilayer caused by the of fatty acids, which the content of water molecules in the membrane. In to FFA, FFA a increase in GP and a slight in polarity and hence in the amount of water in the membrane. In other fatty acids a slight effect in the membrane, because of their The effect of fatty acid on GP was studied A was in the effect caused on Laurdan GP by acid) and acid) oleic acid decreased GP elaidic acid caused no the in molecular structure of these fatty acids is a for this because as bonds a in the whereas bonds no in chain In a previous work we were to between distinct sites for phospholipids and both to fatty acids, at the lipid-protein interface in Torpedo native membrane, by the of between the membrane fluorescence and Laurdan upon addition of different exogenous lipids S.S. Barrantes F.J. Biochemistry. 1998; 37: 16653-16662Google Scholar). we measured changes between AChR and Laurdan, brought about by the addition of FFA of different chain length and degree of The addition of FFA decreased the efficiency of in a The by all fatty acids was This that all fatty acids, of their physical characteristics, to sites at the lipid-protein interface in the native membrane. To different FFA to sites, we an of using the We added first a FFA, the of we added a second FFA, with or different characteristics, to the different FFA to different sites, further a second FFA is whereas no of the FFA for the same and such of for was added Laurdan GP When a second FFA was changes in GP on its caused a in GP, whereas and values to the same extent as by The between GP values obtained addition of different fatty acids were in all cases in the of the fatty acids and we The that Laurdan GP was is a that the second FFA well the membrane and in the The of with the first fatty acid or in the presence of a second fatty of its physical In the present work, we used fluorescence to study the of the physical properties of native membrane by the addition of fatty acids with different We first the partition coefficient of different FFA using an fatty acid-binding This enabled us to the effective concentration of the FFA in the membrane. of Laurdan GP information on the physical state of the bulk and region of the AChR-rich membrane in the presence of concentrations of in efficiency induced by fatty acids the of fatty interactions distances of the probe The FFA partition using the fluorescent properties of ADIFAB, in between different fatty acids Although FFA are hydrophobic their properties have a common their carbon and the double bonds in the R. R. of Membrane Berlin, Scholar). We have to these effects in the same by using an that we have the actual hydrophobicity coefficient of of in double of of the We this coefficient for all FFA studied and it with their partition coefficient this it is that the of different FFA to partition in the membrane the to this FFA be different high fatty acids, such as and fatty acids of such as and and fatty acids, such as and All fatty acids decreased the efficiency of the energy between the membrane fluorescence and In a previous work S.S. Barrantes F.J. Biochemistry. 1998; 37: 16653-16662Google Scholar), we this as an increase in the between and from the of Laurdan molecules from the AChR lipid microenvironment caused by the added lipid We were also to distinct sites for and oleic we the sites to which oleic acid are the same as to which other fatty acids The that a in was for all FFA is a that this is the evidence in of the is that the addition of a second fatty acid not a further of the caused by the first fatty The of the present work is that all fatty acids a fatty acid such as Laurdan to the same extent different fatty acids the physical properties of native Torpedo AChR-rich membranes in a that on their structure Thus, fatty acids the membrane, whereas fatty acids a of the changes in GP it to FFA where are the GP values obtained at the same fatty acid effective concentration in the membrane and addition of the fatty and are the and GP values obtained for a and and were measured at 20 °C in and which are in the and the changes induced by FFA from values of FFA to disordering values of FFA effects and the Furthermore, whereas acid) induced a net disordering effect, acid) produced no in GP values This us to that FFA carbon chain length as well as the number of double bonds and their stereochemical are important determinants of the unique effects of the different FFA on the physical properties of AChR-rich membrane. In in fatty acids exert effects on AChR channel (31Bouzat C.B. Barrantes F.J. Receptors and Channels. 1993; 1: 251-258Google Scholar). These effects are also in membrane from the and are not mediated by that such as and Thus, fatty acids to the action of the AChR channel as the action of several and other 1991; Scholar). In and in AChR in FFA or with double bond effect on whereas FFA with or double bonds produced effects but less than acid S. B. J. 1995; Scholar). The FFA has also been reported to inhibit in S. S. J. 1997; Scholar). The mechanism by which or other fatty acids inhibit nicotinic is To for these effects on AChR binding of fatty acid to a at the interface and/or of the local receptor microenvironment have been different fatty acids all to AChR function in a in of channel channel at the of (31Bouzat C.B. Barrantes F.J. Receptors and Channels. 1993; 1: 251-258Google sites at the lipid-protein interface, in the same sites as cholesterol and phospholipids S.S. Barrantes F.J. Biochemistry. 1998; 37: 16653-16662Google and changes in the fluidity of the AChR lipid microenvironment that clearly depend on their Thus, an of fatty acid action mediated fluidity changes is to because different fatty acids changes in membrane fluidity of different and some in no at We suggest that it is the direct action of FFA in displacing essential lipids from their sites at the lipid-protein interface and not changes in bulk properties such as membrane fluidity that is for the effect of FFA on AChR

The nicotinic acetylcholine receptor (AChR) is the prototype ligand-gated ion channel, and its function is dependent on its lipid environment. In order to study the involvement of sphingolipids (SL) in AChR trafficking, we used pharmacological approaches to dissect the SL biosynthetic pathway in CHO-K1/A5 cells heterologously expressing the muscle-type AChR. When SL biosynthesis was impaired, the cell surface targeting of AChR diminished with a concomitant increase in the intracellular receptor pool. The SL-inhibiting drugs increased unassembled AChR forms, which were retained at the endoplasmic reticulum (ER). These effects on AChR biogenesis and trafficking could be reversed by the addition of exogenous SL, such as sphingomyelin. On the basis of these effects we propose a 'chaperone-like' SL intervention at early stages of the AChR biosynthetic pathway, affecting both the efficiency of the assembly process and subsequent receptor trafficking to the cell surface.

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.

We investigated the molecular mechanisms and the binding site location for the fluorophor crystal violet (CrV), a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR). To this end, radiolabeled competition binding, fluorescence spectroscopy, Schild-type analysis, patch-clamp recordings, and molecular dynamics approaches were used. The results indicate that (i) CrV interacts with the desensitized Torpedo AChR with higher affinity than with the resting state at several temperatures (5-37 degrees C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [(3)H]thienylcyclohexylpiperidine binding to the desensitized or resting AChR is mediated by a steric mechanism; (iii) tetracaine inhibits CrV binding to the resting AChR, probably by a steric mechanism; (iv) barbiturates modulate CrV binding to the resting AChR by an allosteric mechanism; (v) CrV itself induces AChR desensitization; (vi) CrV decreases the peak of macroscopic currents by acting on the resting AChR but without affecting the desensitization rate from the open state; and (vii) two tertiary amino groups from CrV may bind to the alpha1-Glu(262) residues (located at position 20') in the resting state. We conclude that the CrV binding site overlaps the PCP locus in the resting and desensitized state. The noncompetitive action of CrV may be explained by an allosteric mechanism in which the binding of CrV to the extracellular mouth of the resting receptor leads to an inhibition of channel opening. Binding of CrV probably increases desensitization of the resting channel and stabilizes the desensitized state.

Patients with Alzheimer's disease (AD) develop progressive language, visuoperceptual, attentional, and oculomotor changes that can have an impact on their reading comprehension. However, few studies have examined reading behavior in AD, and none have examined the contribution of predictive cueing in reading performance. For this purpose we analyzed the eye movement behavior of 35 healthy readers (Controls) and 35 patients with probable AD during reading of regular and high-predictable sentences. The cloze predictability of words N - 1, and N + 1 exerted an influence on the reader's gaze duration. The predictabilities of preceding words in high-predictable sentences served as task-appropriate cues that were used by Control readers. In contrast, these effects were not present in AD patients. In Controls, changes in predictability significantly affected fixation duration along the sentence; noteworthy, these changes did not affect fixation durations in AD patients. Hence, only in healthy readers did predictability of upcoming words influence fixation durations via memory retrieval. Our results suggest that Controls used stored information of familiar texts for enhancing their reading performance and imply that contextual-word predictability, whose processing is proposed to require memory retrieval, only affected reading behavior in healthy subjects. In AD patients, this loss reveals impairments in brain areas such as those corresponding to working memory and memory retrieval. These findings might be relevant for expanding the options for the early detection and monitoring in the early stages of AD. Furthermore, evaluation of eye movements during reading could provide a new tool for measuring drug impact on patients' behavior.