Environmental Enrichment in Murine Models and Its Translation to Human Factors Improving Conditions in Alzheimer Disease.

With the aging of the world population, there has been a notable increase in the incidence of Alzheimer disease (AD), the most prevalent neurodegenerative disease affecting the elderly. Several studies have reported a delay in the onset of AD symptoms and age-related cognitive dysfunction upon changes to a healthier lifestyle. These positive adjustments find support in the cognitive reserve hypothesis, which holds that the ability to defer disease inception and protect cognitive performance is related to healthier lifestyle habits such as cognitive and physical activity, social engagement, and sensorial stimulation. These lifestyle habits can be compounded under the umbrella of the environmental enrichment (EE) paradigm. The mechanisms underlying EE's capacity to modulate disease expression remain unclear. Since ethical and methodological considerations rule out direct analysis of such changes in the human brain, researchers have resorted to animal models to carry out in-depth characterizations of post-EE structural and functional brain modifications using a variety of behavioral, electrophysiological, genetic, biochemical, and biophysical approaches. Moreover, given the shorter lifespan of animals compared to humans, it is possible to address the effects of aging in control and AD models. In this review we analyze and classify EE data from studies using AD murine models and compare the setup variables employed. We also delve into various aspects of neuroplasticity, under the posit that this property is the key mechanistic process underlying the benefits of EE in both animal and human subjects.

Corticosterone affects the differentiation of a neuronal cerebral cortex-derived cell line through modulation of the nicotinic acetylcholine receptor.

Chronic exposure to stress hormones has an impact on brain structures relevant to cognition. Nicotinic acetylcholine receptors (AChRs) are involved in numerous cognitive processes including learning and memory formation. In order to better understand the molecular mechanisms of chronic stress-triggered mental disease, the effect of corticosterone (CORT) on the biology of AChRs was studied in the neuronal cell line CNh. We found that chronic treatment with CORT reduced the expression levels of the α7-type neuronal AChR and, to a lesser extent, of α4-AChR. CORT also delayed the acquisition of the mature cell phenotype in CNh cells. Chronic nicotine treatment affected the differentiation of CNh cells and exerted a synergistic effect with CORT, suggesting that AChR could participate in signaling pathways that control the cell cycle. Overexpression of α7-AChR-GFP abolished the CORT effects on the cell cycle and the specific α7-AChR inhibitor, methyllycaconitine, mimicked the proliferative action exerted by CORT. Whole-cell voltage-clamp recordings showed a significant decrease in nicotine-evoked currents in CORT-treated cells. Taken together, these observations indicate that AChRs, and the α7-AChR in particular, could act as modulators of the differentiation of CNh cells and that CORT could impair the acquisition of a mature phenotype by affecting the function of this AChR subtype.

Ceramides modulate cell-surface acetylcholine receptor levels.

The effects of ceramides (Cer) on the trafficking of the nicotinic acetylcholine receptor (AChR) to the plasma membrane were studied in CHO-K1/A5 cells, a clonal cell line that heterologously expresses the adult murine form of the receptor. When cells were incubated with short- (C6-Cer) or long- (brain-Cer) chain Cer at low concentrations, an increase in the number of cell-surface AChRs was observed concomitant with a decrease in intracellular receptor levels. The alteration in AChR distribution by low Cer treatment does not appear to be a general mechanism since the surface expression of the green fluorescent protein derivative of the vesicular stomatitis virus protein (VSVG-GFP) was not affected. High Cer concentrations caused the opposite effects, decreasing the number of cell-surface AChRs, which exhibited higher affinity for [125I]-alpha-bungarotoxin, and increasing the intracellular pool, which colocalized with trans-Golgi/TGN specific markers. The generation of endogenous Cer by sphingomyelinase treatment also decreased cell-surface AChR levels. These effects do not involve protein kinase C zeta or protein phosphatase 2A activation. Taken together, the results indicate that Cer modulate trafficking of AChRs to and stability at the cell surface.

Sphingolipids are necessary for nicotinic acetylcholine receptor export in the early secretory pathway.

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.

Cholesterol depletion activates rapid internalization of submicron-sized acetylcholine receptor domains at the cell membrane.

Novel effects of cholesterol (Chol) on nicotinic acetylcholine receptor (AChR) cell-surface stability, internalization and function are reported. AChRs are shown to occur in the form of submicron-sized (240-280 nm) domains that remain stable at the cell-surface membrane of CHO-K1/A5 cells over a period of hours. Acute (30 min, 37 degrees C) exposure to methyl-beta-cyclodextrin (CDx), commonly used as a diagnostic tool of endocytic mechanisms, is shown here to enhance AChR internalization kinetics in the receptor-expressing clonal cell line. This treatment drastically reduced ( approximately 50%) the number of receptor domains by accelerating the rate of endocytosis (t(1/2) decreased from 1.5-0.5 h). In addition, Chol depletion produced ion channel gain-of-function of the remaining cell-surface AChR, whereas Chol enrichment had the opposite effect. Fluorescence measurements under conditions of direct excitation of the probe Laurdan and of Förster-type resonance energy transfer (FRET) using the intrinsic protein fluorescence as donor both indicated an increase in membrane fluidity in the bulk membrane and in the immediate environment of the AChR protein upon Chol depletion. Homeostatic control of Chol content at the plasmalemma may thus modulate cell-surface organization and stability of receptor domains, and fine tune receptor channel function to temporarily compensate for acute AChR loss from the cell surface.

Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy.

Acetylcholine receptor (AChR) supramolecular aggregates that have hitherto only been accessible to examination by electron microscopy were imaged with stimulated emission depletion (STED) fluorescence microscopy, providing resolution beyond limits of diffraction of classical wide-field or confocal microscopes. We examined a Chinese hamster ovary cell liner CHO-K1/A5, that stably expresses adult murine AChR. Whereas confocal microscopy displays AChR clusters as diffraction-limited dots of approximately 200 nm diameter, STED microscopy yields nanoclusters with a peak size distribution of approximately 55 nm. Utilizing this resolution, we show that cholesterol depletion by acute (30 min, 37 degrees C) exposure to methyl-beta-cyclodextrin alters the short and long range organization of AChR nanoclusters on the cell surface. In the short range, AChRs form larger nanoclusters, possibly related to the alteration of cholesterol-dependent protein-protein associations. Ripley's K-test on STED images reveals changes in nanocluster distribution on larger scales (0.5-3.5 microm), which possibly are related to the abolition of cytoskeletal physical barriers preventing the lateral diffusion of AChR nanoclusters.

Charged amino acid motifs flanking each extreme of the alphaM4 transmembrane domain are involved in assembly and cell-surface targeting of the muscle nicotinic acetylcholine receptor.

The alphaM4 transmembrane domain of the nicotinic acetylcholine receptor (AChR) is flanked by two basic amino acids (His(408) and Arg(429)) located at its cytoplasmic- and extracellular-facing extremes, respectively, at the level of the phospholipid polar head regions of the postsynaptic membrane. A series of single and double alphaM4 mutants (His(408)Ala, Arg(429)Ala, Arg(429)Glu, His(408)Ala/Arg(429)Ala, and His(408)Ala/Arg(429)Glu) of the adult muscle-type AChR were produced and coexpressed with wild-type beta, delta, and epsilon subunits as stable clones in a mammalian heterologous expression system (CHO-K1 cells). The mutants were studied by alpha-bungarotoxin ([(125)I]alpha-BTX) binding, fluorescence microscopy, and equilibrium sucrose gradient centrifugation. Cell-surface [(125)I]alpha-BTX binding diminished approximately 40% in His(408)Ala and as much as 95% in the Arg(429)Ala mutant. Reversing the amino acid charge (e.g., Arg(429)Glu) abolished cell-surface expression of AChR. Fluorescence microscopy disclosed that AChR was retained at the endoplasmic reticulum, with an enhanced occurrence of unassembled AChR species in the mutant clones. Centrifugation analysis confirmed the lack of fully assembled AChR pentamers in all mutants with the exception of His(408)Ala. We conclude that His(408) and Arg(429) in alphaM4 are involved in assembly and cell-surface targeting of muscle AChR. Arg(429) plays a more decisive role in these two processes, suggesting an asymmetric weight of the charged motifs at each extreme of the alpha subunit M4 transmembrane segment. (c) 2006 Wiley-Liss, Inc.

Structural and dynamic studies of the gamma-M4 trans-membrane domain of the nicotinic acetylcholine receptor.

A structural characterization of a synthetic peptide corresponding to the fourth transmembrane domain (M4-TMD) of the gamma-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been undertaken. Solid-state NMR and CD spectroscopy studies indicate that upon reconstitution into lipid vesicles or magnetically aligned lipid bilayers, the synthetic M4-TMD adopts a linear alpha-helical conformation with the helix aligned within 15 degrees of the membrane normal. Furthermore, analysis of the motional averaging of anisotropic interactions present in the solid-state NMR spectra of the reconstituted peptide, indicate that the dynamics of the peptide within the bilayer are highly sensitive to the phase adopted by the lipid bilayer, providing an insight into how the interaction of lipids with this domain may play a important role in the modulation of this receptor by its lipid environment.

Structural basis for lipid modulation of nicotinic acetylcholine receptor function.

The nicotinic acetylcholine receptor (AChR) is the archetype molecule in the superfamily of ligand-gated ion channels (LGIC). Members of this superfamily mediate fast intercellular communication in response to endogenous neurotransmitters. This review is focused on the structural and functional crosstalk between the AChR and lipids in the membrane microenvironment, and the modulation exerted by the latter on ligand binding and ion translocation. Experimental approaches using Laurdan extrinsic fluorescence and Förster-type resonance energy transfer (FRET) that led to the characterization of the polarity and molecular dynamics of the liquid-ordered phase AChR-vicinal lipids and the bulk membrane lipids, and the asymmetry of the AChR-rich membrane are reviewed first. The topological relationship between protein and lipid moieties and the changes in physical properties induced by exogenous lipids are discussed next. This background information lays the basis for understanding the occurrence of lipid sites in the AChR transmembrane region, and the selectivity of the protein-lipid interactions. Changes in FRET efficiency induced by fatty acids, phospholipid and cholesterol (Chol), led to the identification of discrete sites for these lipids on the AChR protein, and electron-spin resonance (ESR) spectroscopy has recently facilitated determination of the stoichiometry and selectivity for the AChR of the shell lipid. The influence of lipids on AChR function is discussed next. Combined single-channel and site-directed mutagenesis data fostered the recognition of lipid-sensitive residues in the transmembrane region, dissecting their contribution to ligand binding and channel gating, opening and closing. Experimental evidence supports the notion that the interface between the protein moiety and the adjacent lipid shell is the locus of a variety of pharmacologically relevant processes, including the action of steroids and other lipids.

Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor.

The effects of metabolic inhibition of cholesterol biosynthesis on the trafficking of the nicotinic acetylcholine receptor (AChR) to the cell membrane were studied in living CHO-K1/A5, a Chinese hamster ovary clonal line that heterologously expresses adult alpha2betadeltaepsilon mouse AChR. To this end, we submitted CHO-K1/A5 cells to long-term cholesterol deprivation, elicited by Mevinolin, a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase and applied a combination of biochemical, pharmacological and fluorescence microscopy techniques to follow the fate of the AChR. When CHO-K1/A5 cells were grown for 48 h in lipid-deficient medium supplemented with 0.5 microM Mevinolin, total cholesterol was significantly reduced (40%). Concomitantly, the maximum number of binding sites (Bmax) of the cell-surface AChR for the competitive antagonist alpha-bungarotoxin was reduced from 647+/-30 to 352+/-34 fmol/mg protein, i.e. by 46%. The apparent dissociation constant (Kdapp) for alpha-bungarotoxin of the AChRs remaining at the cell surface was not modified by cholesterol depletion. Similarly, the half-concentration inhibiting the specific binding of the radioligand (IC50) for another competitive antagonist, d-tubocurarine, did not differ from that in control cells. The decrease in cell-surface AChR was paralleled by an increase in intracellular AChR levels, which rose from 44+/-2.1% in control cells to 74+/-3.3% in Mevinolin-treated cells. When analyzed by wide-field fluorescence microscopy, the fluorescence signal arising from alpha-bungarotoxin labeled cell-surface AChRs was reduced by approximately 70% in Mevinolin-treated cells. The distribution of intracellular AChR also changed: Alexa594-alpha-bungarotoxin-labeled AChR exhibited a highly compartmentalized pattern, concentrating at the perinuclear and Golgi-like regions. Temperature-arrest of protein trafficking magnified this effect, emphasizing the Golgi localization of the AChR. Colocalization studies using the transiently expressed fluorescent trans-Golgi/trans-Golgi network marker pEYFP/human beta1,4-galactosyltransferase and the trans-Golgi network marker syntaxin 6 provided additional support for the Golgi localization of intracellular AChRs. The low AChR cell-surface expression and the increase in intracellular AChR pools in cholesterol-depleted cells raise the possibility that cholesterol participates in the trafficking of the receptor protein to the plasmalemma and its stability at this surface location.

Lipid-protein interactions and effect of local anesthetics in acetylcholine receptor-rich membranes from Torpedo marmorata electric organ.

The selectivity of lipid-protein interaction for spin-labeled phospholipids and gangliosides in nicotinic acetylcholine receptor-rich membranes from Torpedo marmorata has been studied by ESR spectroscopy. The association constants of the spin-labeled lipids (relative to phosphatidylcholine) at pH 8.0 are in the order cardiolipin (5.1) approximately equal to stearic acid (4.9) approximately equal to phosphatidylinositol (4.7) > phosphatidylserine (2.7) > phosphatidylglycerol (1.7) > G(D1b) approximately equal to G(M1) approximately equal to G(M2) approximately equal to G(M3) approximately equal to phosphatidylcholine (1.0) > phosphatidylethanolamine (0.5). No selectivity for mono- or disialogangliosides is found over that for phosphatidylcholine. Aminated local anesthetics were found to compete with spin-labeled phosphatidylinositol, but to a much lesser extent with spin-labeled stearic acid, for sites on the intramembranous surface of the protein. The relative association constant of phosphatidylinositol was reduced in the presence of the different local anesthetics to the following extents: tetracaine (55%) > procaine (35%) approximately benzocaine (30%). For stearic acid, only tetracaine gave an appreciable reduction (30%) in association constant. These displacements represent an intrinsic difference in affinity of the local anesthetics for the lipid-protein interface because the membrane partition coefficients are in the order benzocaine >> tetracaine approximately procaine.

Search for alpha4 and alpha7 nicotinic acetylcholine receptor markers in a pedigree of benign familial infantile convulsions (BFIC).

In this study we investigate the possible involvement of the recently reported locus for benign familial infantile convulsions (BFIC) in human chromosome 19 and that of the neuronal acetylcholine receptor alpha4 (CHRNA4) and alpha7 (CHRNA7) subunits in a family with at least twelve clinically diagnosed cases of BFIC. Six polymorphic microsatellite markers covering the BFIC locus on chromosomal region 19q, one marker for CHRNA4 (chromosome 20) and two for CHRNA7 (chromosome 15) were used for the screening. The two-point lod score analysis showed no evidence of BFIC phenotype on chromosome 19. Similarly, when markers for chromosome 20 (CHRNA4 intron1, Amplimer: CHRNA4. PCR.1) and chromosome 15 (D15S165 and D15S1010) were used, score analysis showed no indication of linkage. The most likely interpretation of these results is that BFIC is a genetically heterogeneous form of epilepsy.

Steroids differentially inhibit the nicotinic acetylcholine receptor.

The effect of various natural and synthetic steroids on the function of the nicotinic acetylcholine receptor (AChR) was studied at the single-channel level. AChR channel kinetics was affected by some substitutions in the cyclopentaneperhydrophenantrene ring. Functionally relevant substitutions shortened channel open state duration, an effect that varied for different steroids. The presence of a polar group at C11 contributed to the inhibitory potency of the steroid. Among mono-hydroxylated steroids such as 11- and 17-OH progesterone, the highest potency was displayed by the former showing a level similar to that of the reference compound, hydrocortisone. When the effects were analyzed in terms of the octanol-water partition coefficient, a linear relationship was unexpectedly found between the hydrophilicity of the steroids and their inhibitory potency.

Topography of nicotinic acetylcholine receptor membrane-embedded domains.

The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized with N-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in alphaM1, alphaM4, gammaM1, and gammaM4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear alpha-helical structure, but not so for M1, for which "classical" models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.

The neuronal nicotinic acetylcholine receptor in some hereditary epilepsies.

Recent advances in human genetics and in the neurobiology of neurotransmitter receptors and channels have led to the discovery of specific genes associated with hereditary epileptic phenotypes. All the genes identified to date code for ligand- and voltage-gated ion channels. Some clinically rare idiopathic epilepsies are associated with mutations in genes coding for different neuronal nicotinic acetylcholine receptor (AChR) subunits. Distinct alpha subunits are found in the brain and in the peripheral nervous system, and structural, non-alpha subunits like beta2 and beta4 confer different properties to neuronal receptors. Thus, the final properties of the oligomeric AChR depend on the different combinations of alpha and beta subunits. Most mutations found so far occur in the alpha4 chain, the most abundant subunit in the central nervous system. Specifically, the identification of mutations in the alpha4 subunit of neuronal AChR in human benign familial neonatal convulsions (BFNC) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) raise the possibility that the observed gene defects are linked (causatively) with these two diseases or, alternatively, that AChR alpha4 mutants increase the probability of epileptic discharges. We discuss testable hypotheses for unraveling the pathophysiology of these two disorders associated with AChR mutations.

Nongenomic effects of steroids on the nicotinic acetylcholine receptor.

A fast signaling mode of natural and synthetic steroids is exerted on some ion channels and cell-surface receptors. This activity contrasts with their classic mode of action, via intracellular receptors. Early studies from our laboratory demonstrated that spin-labeled androstanol and cholestane interact with the nicotinic acetylcholine receptor (AChR) and that lipid mobility at the lipid belt surrounding the AChR is reduced relative to that of the bulk membrane lipid. The occurrence of discrete and independent sites for phospholipids and sterols, both accessible to fatty acids, was subsequently disclosed in the native membrane. Synthetic and natural glucocorticoids were found to act as noncompetitive inhibitors of AChR function. The influence of different substituent groups in the cyclepentane perhydrophenanthrene ring on the channel-shortening potency of various steroids has also been assayed in muscle-type AChR, and we found a certain selectivity of this effect. Some organochlorine pesticides are xenoestrogens, that is, environmental agents capable of disrupting endocrine system signaling. We determined their effects on the AChR membrane using novel fluorescence techniques.

Effect of organochlorine insecticides on nicotinic acetylcholine receptor-rich membranes.

The so-called generalized polarization (GP) of the fluorescent probe Laurdan and the steady-state fluorescence anisotropy of the probe diphenylhexatriene (DPH) and its phenylpropionic derivative (PA-DPH) were used to study the effects of several organochlorine insecticides of the chlorophenylethane, chlorinated cyclohexane and chlorinated cyclodiene families on the Torpedo nicotinic acetylcholine receptor (AChR)-rich native membrane. All insecticides, with the exception of Lindane, augmented Laurdan GP both in the native membrane and in model lipid systems. Most organochlorine compounds produced a concentration-dependent decrease of DPH and PA-DPH anisotropy in the AChR-rich membrane. These compounds exhibited a dual behavior vis-à-vis the native AChR-rich membrane, exerting disordering effects at the bilayer core while ordering and/or excluding water molecules from the lipid-protein interface region, as sensed by DPH anisotropy and Laurdan GP, respectively. Furthermore, all insecticides decreased the efficiency of fluorescence resonance energy transfer between the intrinsic protein and Laurdan, albeit to different extents. On the basis of all these observations, the existence of potential target sites for insecticides in the protein-lipid interface region is postulated.

Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems.

Different mammalian and insect somatic host cell systems were tested in their ability to express, fold, and assemble alpha7-type neuronal acetylcholine receptor (AChR) both at the transcriptional and translational level. For this purpose we employed clonal cell lines derived from the neural crest, such as PC12 cells from a rat adrenal pheochromocytoma, and GH3 cells isolated from a rat pituitary tumor, as well as non-neuronal cells such as NIH-3T3 fibroblasts from embryonic NIH Swiss mouse and Sf9 cells from ovary tissue of the Spodoptera frugiperda butterfly. Total RNA, isolated from either transfected or non-transfected PC12, GH3 or 3T3 cells, or recombinant AcNPV-infected and mock-infected Sf9 cells was analyzed by Northern blot. PC12 cells, which endogenously express alpha7 AChR, and all its heterologous alpha7-transfectant clones, exhibited variable but generally high amounts of a single transcript. GH3 and NIH-3T3 transfectant clones and recombinant AcNPV-infected Sf9 cells expressed variable levels of alpha7-mRNA, with a single transcript that co-migrated with the 28S rat rRNA. Only the neural crest-derived cell lines appeared to functionally express the alpha7 AChR, as measured by their [125I]alpha-bungarotoxin binding ability. The results suggest that heterologous expression of alpha7 is regulated not at the transcriptional, but at the postranslational level and that not all host cell systems appear to express the cellular factors needed for the correct postranslational modifications leading to mature and functional alpha7 AChR. Furthermore, the results suggest that tightly controlled expression mechanisms have evolved in parallel with this ancient cholinergic sequence.

[Study of consultation in an Internal Medicine Department]. / Estudio de la interconsulta en un Servicio de Medicina Interna.

Consultation in Internal Medicine is a common task of the attending labor of the internist in a general hospital and is a relevant link for intrahospital cohesion. In the present work the characteristics of 738 consultations, which represented all consultation request forms received at our Department during a nine-month period, were studied. A 55.4 percentage of consultation forms were requested urgently and 93.6% of them were answered on the same day. Most consultation parts received came from General Surgery and traumatology, with a predominance of cardiologic, pneumologic, infectious and multiple causes. The assessment of the urgency made by the internist coincided with the urgent request only in 38.3% of cases. A clinical follow-up was made for 33.3% of patients and 2% of them were transferred to the Internal Medicine Department. The consultation was repeated for 22.5% of the total of patients. Only in 23.1% of cases an oral link with the referring physician occurred. To remark the relevance of the consultation for the optimization of care quality to our patients and its influence on the inter-relationship between specialists in different fields of medicine.

Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor.

The properties of the nicotinic acetylcholine receptor (AChR) are modulated by its lipid microenvironment. Studies of such modulation are hampered by the cell's homeostatic mechanisms that impede sustained modification of membrane lipid composition. We have devised a novel strategy to circumvent this problem and study the effect of changes in plasma membrane lipid composition on the functional properties of AChR. This approach is based on the stable transfection of AChR subunit cDNAs into cells defective in a specific lipid metabolic pathway. In the present work we illustrate this new strategy with the successful transfection of a temperature-sensitive Chinese hamster ovary (CHO) cell line, SPB-1, with the genes corresponding to the four adult mouse AChR subunits. The new clone, SPB-1/SPH, carries a mutation of the gene coding for serine palmitoyl transferase, the enzyme that catalyses the first step in sphingomyelin (Sph) biosynthesis. This defect causes a decrease of Sph de novo synthesis at non-permissive temperatures. The IC50 for inhibition of alpha-BTX binding with the agonist carbamoylcholine exhibited values of 3.6 and 2.7 microm in the wild-type and Sph-deficient cell lines, respectively. The corresponding IC50 values for the competitive antagonist D-tubocurarine (D-TC) were 2.8 and 3.4 microm, respectively. No differences in single-channel properties were observed between wild-type and mutant cell lines grown at the non-permissive, lipid defect-expressing temperature using the patch-clamp technique. Both cells exhibited two open times with mean values of 0.35 +/- 0.05 and 1.78 +/- 0.2 ms at 12 degrees C. Taken together, these results suggest that the AChR is expressed as the complete heteroligomer. However, only 10-20% of the total AChR synthesized reached the surface membrane in the mutant cell line and exhibited a higher metabolic turnover, with a half-life about 50% shorter than the wild-type cells. When control CHO-K1/A5 cells were treated with fumonisin B1, an inhibitor of sphingosine (sphinganine) N-acetyltransferase (ceramide synthase), a 45.5% decrease in cell surface AChR expression was observed. The results suggest that sphingomyelin deficiency conditions AChR targeting to the plasma membrane.