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.

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.

Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the Mr 43,000 nonreceptor peptide.

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5'-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate. The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments. Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and "holey" native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane. The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.

Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes.

Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000-43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending.

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.

Conversion of acetylcholine receptor dimers to monomers upon depletion of non-receptor peripheral proteins.

The influence of treatments for extracting non-receptor peripheral proteins on the oligomeric states of the acetylcholine receptor has been studied in receptor-rich membranes from Torpedo marmorata. Conventional alkaline treatment of non-alkylated membranes resulted in the extraction of peripheral proteins (30% of total membrane proteins). Concomitantly, partial conversion of the dimer into the monomer was observed in the absence of exogenous reduction. Alkaline extraction at high ionic strength resulted in a marked decrease in protein solubilization, and no conversion of the dimer to the monomer occurred. Alkaline treatment extracted only one half of the peripheral proteins (15% of total protein) from membranes previously alkylated with N-ethylmaleimide or iodoacetamide, or oxidized by sodium periodate. Conversion of dimer to monomer was totally prevented by these treatments. Similar results were obtained by treatment of the membranes with lithium 3,5-diiodosalicylate. The above effects of alkaline extraction on the acetylcholine receptor can be interpreted in the context of two mutually non-exclusive mechanisms: (a) some of the peripheral proteins may directly participate in the thiol-dependent receptor aggregational states. Their extraction destroys this dynamic control. (b) Extraction of peripheral proteins destabilizes the receptor and makes it more susceptible to inter or intramolecular sulfhydryl-disulfide exchange, leading to the endogenous reduction of a proportion of the dimers.

Phospholipid chain immobilization and steroid rotational immobilization in acetylcholine receptor-rich membranes from Torpedo marmorata.

1. The ESR spectra of both phosphatidylcholine and phosphatidylethanolamine spin labels reveal an immobilized lipid component (tau R greater than or equal to 50 ns), in addition to a fluid component (tau R approximately 1 ns), in acetylcholine receptor-rich membranes prepared from Torpedo marmorata electroplax according to the method of Cohen et al. (Cohen, J.B., Weber, M., Huchet, M. and Changeux, J.-P. (1972) FEBS Lett. 26, 43--27). 2. The ESR spectra of the androstanol spin label display a component corresponding to molecules which are immobilized with respect to rotation about the long molecular axis (tau R greater than or equal to 50 ns), in addition to the fluid lipid bilayer component in which the molecules are rotating rapidly about their long axes (tau R approximately 1 ns). This immobilized component is observed throughout the temperature range 2--22 degrees C, at an approximately constant relative intensity of approx. 45% of the total, which is quantitatively the same as previously observed with fatty acid spin labels.

Lipid composition of purified transverse tubule membranes isolated from amphibian skeletal muscle.

The level and proportion of lipids and their fatty acid composition were analyzed in highly purified transverse tubule membranes of amphibian skeletal muscle. Tubule membranes show (a) a higher content of lipids, (b) a higher phospholipid/cholesterol ratio and (c) a different phospholipid composition from other subcellular fractions, such as the light and heavy membranes from sarcoplasmic reticulum, which are similar in lipid profile. Transverse tubule membranes are characterized by a high percentage of phosphatidylserine and sphingomyelin and a low proportion of phosphatidylcholine compared with the other membranes. All three show a high proportion of ethanolamine plasmalogens (50% of the total ethanolamine glycerophospholipid). Transverse tubule membrane lipids contain a high proportion of 20- and 22-carbon polyunsaturated fatty acids, predominantly 20:4, 20:5, 22:5 and 22:6. Arachidonate predominates in phosphatidylinositol, eicosapentaenoate and docosahexaenoate in ethanolamine and serine glycerophospholipids.

Effect of local anaesthetics on steroid-nicotinic acetylcholine receptor interactions in native membranes of Torpedo marmorata electric organ.

Interactions between steroids and the nicotinic acetylcholine receptor (AChR) have been studied in native membrane vesicles from Torpedo marmorata electric organ by electron spin resonance (ESR) and fluorescence techniques. ESR spectra of spin-labelled cholestane (CSL) revealed that this steroid probe was incorporated into the AChR-rich membrane vesicles in regions which were to a certain extent enriched preferentially in the steroid, both in the presence and in the absence of local anaesthetics. Since the nitroxide group present in CSL is also a paramagnetic quencher of the intrinsic protein fluorescence, this property was used to characterize the AChR-steroid interactions. The quenching induced by CSL was sensitive both to AChR concentration and to the action of cholinergic agonists. In competition experiments, the ability of CSL to quench the AChR intrinsic fluorescence was markedly inhibited by benzocaine, tetracaine and QX-222 (a quaternary trimethylammonium derivative of lidocaine), and was totally inhibited by procaine. The effectiveness of local anaesthetics in inhibiting CSL-induced quenching followed the order: procaine much greater than benzocaine approximately greater than tetracaine greater than QX-222. This inhibition effect was shown not to be charge-dependent. The data can be interpreted in terms of a model requiring specific association sites for local anaesthetics on the hydrophobic surface of the AChR which at least partially overlap with those for steroids.

Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ.

Interactions between merocyanine 540 (MC540) and nicotinic acetylcholine receptor (AChR) have been studied by visible absorption spectroscopy using native receptor-rich membranes from Discopyge tschudii electric tissue and liposomes obtained by aqueous dispersion of endogenous lipids extracted from the same tissue. The fact that merocyanine partitions into the membrane when this is in the liquid-crystalline state, exhibiting a characteristic peak at 567 nm, was exploited to obtain quantitative information about the physical state of the AChR-rich membrane. Spectra of MC540 revealed that this molecule was preferentially incorporated into AChR-rich membranes, with an affinity (Kdapp 30 microM) 10-fold higher than that in liposomes (Kdapp 290 microM). Changes were observed in the equilibrium dissociation constant of MC540 at different temperatures: the two-fold higher affinity at 8 degrees C than at 23 degrees C can be rationalized in terms of a higher value of the overall dimerization constant (Kdim) at the lower temperature. The local anaesthetic benzocaine competed for MC540 binding sites with higher potency in AChR-rich native membranes than in liposomes made with endogenous lipids. This competition was found to be AChR concentration-dependent, whereas in liposomes the displacement was constant at different lipid/MC540 molar ratios. Titration experiments yielded an apparent dissociation constant for benzocaine of 0.6 mM and 0.7 mM for liposomes and AChR-rich membranes, respectively. The possible location of the benzocaine binding site is deduced from the competition experiments to be at the lipid annulus surrounding the nicotinic AChR protein.

Structural and functional crosstalk between acetylcholine receptor and its membrane environment.

Nicotinic acetylcholine receptor (AChR) is a transmembrane protein belonging to the superfamily of rapid, ligand-operated channels. Theoretical models based on thermodynamic criteria assign portions of the polypeptide chains to the lipid bilayer region. From an experimental point of view, however, the relationship between the two moieties remains largely unexplored. Current studies from our laboratory are aimed at defining the structural, dynamic, and functional relationship between membrane lipids and AChR. We are particularly interested in establishing the characteristics of and differences between the lipids in each leaflet of the bilayer and the belt or "annular" lipids immediately surrounding AChR and the bulk bilayer lipids. We are also interested in determining the possible implications of lipid modifications on AChR channel properties. Toward these ends, fluorescence and other spectroscopic techniques, together with biochemical analyses and patch-clamp studies, are currently being undertaken. Correlations can be established between structural aspects of phospholipid packing in the immediate perimeter of AChR and other properties of these annular lipids revealed by dynamic spectroscopic and molecular modeling techniques. Lipid compositional analyses of the clonal muscle cell line BC3H-1 and chemical modification studies have been carried out by incubation of intact cells in culture and of membrane patches excised therefrom with liposomes of different lipid composition. These studies have been combined with electrophysiological measurements using the patch-clamp technique, with the aim of determining the possible effects of lipids on the channel properties of muscle-type AChR. A variety of experimental conditions, involving polar head and fatty acyl chain substitution of phospholipids and cholesterol incorporation, are being assayed in the BC3H-1 cells.

Screening structural-functional relationships of neuropharmacologically active organic compounds at the nicotinic acetylcholine receptor.

The mechanisms of action and pharmacological effects on the nicotinic cholinoceptor of a large database of organic compounds were analyzed using a new computational procedure. This procedure is a screening method based on comparison of the molecular structures (shape and charge) of the putative active organic compounds. The resulting predictions can be used as an exploratory tool in the design of experiments aimed at testing the effects of several compounds on a target macromolecule. Unlike a conventional database search for structural similarities, the present method is able to circumscribe objectively the results to the most statistically significant molecules.

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.

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.

Inherited and experimentally induced changes in gating kinetics of muscle nicotinic acetylcholine receptor.

Ligand-gated ion channels (LGIC) allow rapid responses in the nervous system. The nicotonic acetylcholine receptor (AChR) has been the model for structure-function relationship studies on this superfamily. The AChR undergoes the following functional events: 1. Binding of the neurotransmitter. 2. Opening of the ion channel. 3. Conduction of ions across the pore. 4. Desensitization. The equilibrium among these processes can be perturbed by alteration in the primary structure of the AChR or by the presence of pharmacological agents. Changes in the primary sequence leading to modifications in gating kinetics may occur in association with physiological or pathological processes. Such changes can also be genetically engineered to gain insights into structure-function relationships.

High levels of phosphorylation in minor phospholipids of Discopyge tschudii electrocyte membranes.

Phospholipid phosphate group metabolism has been studied in the electrocyte of Discopyge tschudii (Torpedinidae), following for several hours the incorporation and distribution of [(32)P] in living electrocyte stacks and acetylcholine receptor membranes prepared therefrom. Conditions are reported for the incubation of electrocyte columns in vitro with [(32)P]O(4)Na(2)H resulting in active and sustained incorporation of the precursor in minority phospholipids. Phosphatidic acid, phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, representing 2.3, 2.1, 0.3 and 0.2% respectively of the total phospholipids were almost exclusively labelled. Relative specific activities of 13, 7, 56 and 140 respectively were attained. These values were much higher than those of major phospholipids like phosphatidylcholine (0.03) or phosphatidylethanolamine (0.01). Subcellular fractionation of the metabolically active electrocyte stacks yielded various membrane fractions differing in their degree of [(32)P]-labelling, specially in phosphatidic acid and polyphosphoinositides. The fraction richest in nicotinic acetylcholine receptor protein showed the highest levels of [(32)P] incorporation. The results indicate that phosphorylation reactions are actively operative in certain phospholipid classes of the electrocyte and in membranes obtained from these cells after in vitro labelling with [(32)P]. In particular, phosphomonoester groups in polyphosphoinositides and the phosphate group in phosphatidic acid display a high metabolic activity.

Effects of periodate oxidation and glycosidases on structural and functional properties of the acetylcholine receptor and the non-receptor, peripheral ?-polypeptide (M(r) 43,000).

NaIO(4) oxidation, exo- and endo-glycosidase treatments and combinations thereof have been applied to acetylcholine receptor from Torpedo marmorata in its membrane-bound and detergent-solubilised forms. The effects of these chemical and enzymatic treatments are made apparent in the electrophoretic properties of the four receptor subunits (?, ?, ? and ?) and of the non-receptor polypeptides, their thermal and proteolytic susceptibility, and the steady-state and kinetic parameters of receptor-toxin complex formation. The electrophoretic pattern of the membrane polypeptides is found to depend on the redox state of the membranes, presence or absence of the non-receptor peripheral ?-peptide (M(r) 43,000), pH and temperature. Very low NaIO(4) concentrations (50 ?M) suffice to prevent the penetration of the ?-peptide into NaDodSO(4) polyacrylamide gels. This effect could be abolished by N-ethylmaleimide alkylation of free sulphydryl groups, suggesting the involvement of easily oxidizable vicinal thiols in the aggregation of the peptide. Higher reagent concentrations resulted in the altered mobility and subsequent splitting of the receptor subunit carrying the ligand recognition site (?, M(r) 40,000) into a doublet. In contrast, NaIO(4) treatment of the detergent-solubilized receptor aggregated the ?-subunit, presumably via chemical groups hidden in the membrane but exposed in detergent. Only this subunit underwent such NaIO(4)-dependent changes within the concentration range in which (a) an increase of the 13-S dimeric receptor species at the expense of the 9-S monomeric form was observed and (b) half-maximal quenching of the intrinsic fluorescence occurred (?2 mM NaIO(4)). Neuraminidase digestion affected exclusively the ?- and ?-subunits of the receptor, suggesting the presence of substantial amounts of sialic acid residues in these subunits. ?-Glucosidase and endoglycosidase D had no effect on the electrophoretic properties of receptor and non-receptor polypeptides. Neither NaIO(4) nor neuroaminidase treatments had any effect on the thermal sensitivity of the receptor. Similarly, the equilibrium and kinetic properties of receptor-?-neurotoxin complex formation were not modified by such treatment nor was the susceptibility to tryptic digestion. The thermal and proteolytic sensitivities were affected by acid pH (5.2) and ?-glucosidase treatments. The latter enzymatic digestion reduced the ?-toxin binding capacity of the receptor by 35% and increased the equilibrium dissociation constant by 2-fold.

Interactions of fluorescent cholinergic antagonists with the membrane-bound acetylcholine receptor.

The synthesis of pyrenyl-choline derivatives is reported. The interactions of these compounds with the acetylcholine receptor has been studied in developing chick myotubes in culture and in the adult frog neuromuscular junction with electrophysiological techniques. In vivo, the compounds display the pharmacological characteristics of nicotinic antagonists. Similar conclusions can be derived from in vitro experiments using radiolabelled derivatives in equilibrium binding studies with the membrane-bound receptor from Torpedo, steady-state fluorescence titrations, and toxin kinetic inhibition studies. The rapid kinetics of receptor-probe interactions were studied with the temperature-jump method. The fluorescence emission of the probes, excited by energy transfer from the protein chromophores, enabled the observation of two relaxation processes. The concentration dependence of the relaxation times and associated amplitudes can most economically be accounted for in terms of a reaction mechanism consisting of two sequential ligand binding-isomerization steps. The two ligands find themselves in an environment of high electronic polarizability, having a dielectric constant of about 40, after rapidly binding to two sites which differ in their affinities roughly by a factor of 10 (Apparent Kd's 1.5 and 0.2 ?M).

Hydrocortisone and 11-desoxycortisone modify acetylcholine receptor channel gating.

Acute exposure of the nicotinic acetylcholine receptor (AchR) to hydrocortisone (HC) induced a dose-dependent reduction in the channel open time and burst duration and an increase in the closed time, with no changes in channel amplitude. Similar effects were observed with 11-desoxycortisone, thus suggesting that the oxygen atom at position 11 is not required for channel modification. The changes were observed when HC was added to either face of the membrane, but the concentration dependence of the effect differed, indicating a certain sidedness of the corticoid action. The results are consistent with the corticoids acting either at a site on the AChR which can be reached via a membrane pathway or at the lipid annulus immediately surrounding the AChR, i.e. at the lipid-protein interface.

Acetylcholine receptor channel properties are modified by benzyl alcohol.

The effect of the general anaesthetic, benzyl alcohol, on the nicotinic cholinergic receptor (AChR) was evaluated at the single channel level using the patch-clamp technique. Benzyl alcohol decreases both the conductance (about 2-fold with 40 mM benzyl alcohol) and the mean open time (about 2.5-fold) of the AChR channels. When modified channels are activated by high ACh concentrations, groups of brief channel openings are observed. Each group is in turn composed of a higher number of openings than in non-treated receptors. Similar modifications are observed when benzyl alcohol is applied from the cytoplasmic side of the membrane, suggesting that the general anaesthetic interacts with a nonspecific site, possibly the lipid-protein interface.