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.

The anthelmintic pyrantel acts as a low efficacious agonist and an open-channel blocker of mammalian acetylcholine receptors.

Pyrantel is an anthelmintic which acts as an agonist of nicotinic receptors (AChRs) of nematodes and exerts its therapeutic effects by depolarizing their muscle membranes. Here we explore at the single-channel level the action of pyrantel at mammalian muscle AChR. AChR currents are elicited by pyrantel. However, openings do not appear in clearly identifiable clusters over a range of pyrantel concentrations (1-300 microM). The mean open time decreases as a function of concentration, indicating an additional open-channel block. Single-channel recordings in the presence of high ACh concentrations and pyrantel demonstrate that the anthelmintic acts as a high-affinity open-channel blocker. When analyzed in terms of a sequential blocking scheme, the calculated forward rate constant for the blocking process is 8x10(7) M(-1) x s(-1), the apparent dissociation constant is 8 microM at a membrane potential of -70 mV and the process is voltage dependent. Pyrantel displaces alpha-bungarotoxin binding but the concentration dependence of equilibrium binding is shifted towards higher concentrations with respect to that of ACh binding. Thus, by acting at the binding site pyrantel activates mammalian AChRs with low efficacy, and by sterical blockade of the pore, the activated channels are then rapidly inhibited.

The noncompetitive inhibitor quinacrine modifies the desensitization kinetics of muscle acetylcholine receptors.

Quinacrine has been shown to act as a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAChR). However, its mechanism of action is still a matter of controversy. We analyzed in detail the action of quinacrine at both the single-channel and macroscopic current levels. The main effect of quinacrine is a profound concentration-dependent decrease in both the frequency of opening events and the duration of clusters elicited by high acetylcholine concentrations. Quinacrine also significantly increases (40-fold at 30 microM) the decay rate of macroscopic currents elicited by rapid perfusion of acetylcholine to outside-out patches. This decay is still well-described by a single exponential. Quinacrine has very little effect on the peak amplitude of the response, suggesting that it acts mainly on open channels. The recovery from desensitization after removal of acetylcholine is delayed in the presence of quinacrine. Results from both single-channel and macroscopic current recordings indicate that quinacrine increases the rate of nAChR desensitization and stabilizes the desensitized state. Interestingly, in equilibrium agonist-binding assays, quinacrine does not promote the typical high-affinity desensitized state. Thus, quinacrine seems to induce an intermediate state exhibiting the permeability but not the agonist binding properties of desensitization.

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.

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.

Nicotinic receptor fourth transmembrane domain: hydrogen bonding by conserved threonine contributes to channel gating kinetics.

The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated alphaT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of alphaT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of alphaT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.

Amphetamine acts as a channel blocker of the acetylcholine receptor.

Non-competitive inhibitors (NCIs) of the nicotinic receptors (AChR) comprise a wide range of compounds. The chemical scaffold of amphetamine is similar to those of some NCIs. We investigated the effects of amphetamine (1-100 microM) on the muscle AChR by recording single-channel currents. The drug reduces the duration of the open state in a concentration-dependent manner and causes the appearance of brief closings, resembling the action of open-channel blockers. The forward rate constant for the blocking process is of the order of 10(7) M(-1) s(-1) and the blocking process is voltage dependent. The results are consistent with the steric block of the open channel as the primary action of amphetamine. At high drug concentrations the mechanism of inhibition deviates from that of classical open-channel blockers.

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.

Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics.

The firmest candidate among the transmembrane portions of the nicotinic acetylcholine receptor (AChR) to be in contact with the lipid bilayer is the fourth segment, M4. To explore the contribution of alphaM4 amino acid residues of mouse AChR to channel gating, we combined site-directed mutagenesis with single-channel recordings. Two residues in alphaM4, Cys418 and Thr422, were found to significantly affect gating kinetics when replaced by alanine. AChRs containing alphaC418A and alphaT422A subunits form channels characterized by a 3- and 5-fold reduction in the mean open time, respectively, suggesting an increase in the closing rate due to the mutations. The calculated changes in the energy barrier for the channel closing process show unequal and coupled contributions of both positions to channel gating. Single-channel recordings of hybrid wild-type alpha/alphaT422A AChR show that the closing rate depends on the number of alpha subunits mutated. Each substitution of threonine to alanine changes the energy barrier of the closing process by approximately 0.5 kcal/mol. Recordings of channels activated by high agonist concentration suggest that these mutations also impair channel opening. Both Cys418 and Thr422 have been postulated to be in contact with the lipid milieu and are highly conserved among species and subunits. Our results support the involvement of lipid-exposed residues in alphaM4 in AChR channel gating mechanism.

Assigning functions to residues in the acetylcholine receptor channel region (review).

This review is concerned with the functional domains of the nicotinic acetylcholine receptor (AChR) involved in ion permeation. These comprise the ion pore and its gate. The latter allows the channel to be almost exclusively closed in the absence of agonist and favours ion flux in its presence. Early photoaffinity labelling experiments using open-channel blockers and site-directed mutagenesis studies identified M2 of each AChR subunit as the transmembrane domain lining the walls of the ion pore. Several biochemical, electrophysiological, and mutagenesis studies as well as molecular modelling and in vitro studies of ion channel formation with synthetic peptides corroborate these findings. Point mutations combined with electrophysiological techniques have contributed to dissecting the AChR channel region assigning functions to individual amino acid residues, thus revealing structural and functional stratification of the M2 channel domain. Specific residues have been found to be structural determinants of conductance, ion selectivity, gating, and desensitization. The three-dimensional structure of the AChR protein at 9A resolution suggests a possible arrangement of the M2 alpha-helices in the open and closed states, respectively. In spite of the current wealth of knowledge on the AChR ion channel stemming from the combination of experimental approaches discussed in this review, the mechanistic structure by which the interaction with the agonist favours the opening of the cationic channel remains unknown.

Ephedrine blocks wild-type and long-lived mutant acetylcholine receptor channels.

A combination of transient expression of wild-type and a mutant muscle nicotinic receptor (AChR) found in a patient suffering from a congenital myasthenic syndrome and single-channel recordings was used to characterize the action of ephedrine on the AChR. In the presence of ephedrine, both types of AChR channel showed a flickering behaviour and a dose-dependent reduction in the mean open time. The duration of the blocking events was dependent on the membrane potential. The forward rate constant for the blocking process was of the order of 10(7) M-1 s-1 in both types of AChR. The results are consistent with ephedrine acting as a channel blocker in wild-type AChR as well as in long-lived AChR channels present in patients with the slow-channel syndrome.

Modulation of muscle nicotinic acetylcholine receptors by the glucocorticoid hydrocortisone. Possible allosteric mechanism of channel blockade.

Mechanisms of ion channel blockade by noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) have been particularly difficult to elucidate. We have combined here transient expression of embryonic, adult, and a mutated adult muscle AChR associated with a slow channel syndrome (Ohno, K., Hutchinson, D. O., Milone, M., Brengman, J. M., Bouzat, C., Sine, S., and Engel, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 758-762) with single channel recordings to determine subunit specificity and mechanisms of action of the prototype glucocorticoid hydrocortisone (HC). HC affected in a similar manner the gating kinetics of all types of muscle AChR, producing briefer openings with normal amplitudes. We postulate that this steroid acts as a noncompetitive inhibitor of the AChR and that its mechanism of action can be interpreted in terms of blocking models. The forward rate constant for the blocking process was also similar for all channel types, indicating that the structural differences between them are not responsible for the effect. The reduction in the channel open time was not dependent on agonist concentration; it was slightly voltage dependent, suggesting that HC binds to a site located inside the membrane that senses the electric field. Recordings at high acetylcholine concentration in the presence of HC showed a reduced number of openings per activation period and the long closed times typically observed in the desensitization phenomenon. In competition studies with the classical open channel blocker QX-222, HC induced an early termination of the burst, suggesting that the two act at different sites. Taken together the results support the existence of specific sites sensed by the membrane field, different from those of open channel blockers and probably located at the lipid-protein interface. From this site(s), glucocorticoids and other hydrophobic noncompetitive inhibitors could allosterically mediate channel blockade.

Heterologous retinal cultured neurons and cell adhesion molecules induce clustering of acetylcholine receptors and polynucleation in mouse muscle BC3H-1 clonal cell line.

Several features of the clonal cell line BC3H-1 resemble those of embryonic muscle cells at their early stage of development (Patrick et al.: J Biol Chem 252:2143-2153, 1977). Under normal culture conditions, fully differentiated BC3H-1 cells possess a spindle-shaped, muscle-like morphology with a single nucleus. Like embryonic muscle cell counterparts, they neither express the epsilon subunit nor exhibit the clustered organization of the nicotinic acetylcholine receptor (AChR) characteristic of mature myocytes. Instead, AChRs are evenly distributed upon the sarcolemma upon differentiation. Here we report that BC3H-1 cells can be induced to express AChR clusters by co-culturing the cell line with heterologous retinal neurons, which establish contacts with former cells. Clustering was also triggered by pretreating the culture substratum with several cell adhesion molecules. Polynucleation, a phenomenon observed in mature myotubes in vivo, was also observed in BC3H-1 cells under the two sets of experimental conditions. AChR clustering and polynucleation, both characteristic of the mature postsynaptic region, can thus be induced in BC3H-1 cells by at least two quite distinct pathways: neuronal cell contact and/or cell adhesion molecules. It is suggested that heterologous retinal neurons can elicit only an incomplete differentiation of BC3H-1 cells, failing to concentrate the clusters beneath the neuronal endings, and failing to induce expression of the epsilon subunit, characteristic of the mature AChR.

Effect of chemical modification of extracellular histidyl residues on the channel properties of the nicotinic acetylcholine receptor.

We have examined the effect of chemical modification with diethyl pyrocarbonate (DEP) on the properties of acetylcholine (ACh)-activated channels in the cloned muscle-cell line BC3H-1. After protein modification, patch-clamp recordings showed alterations in the kinetics of the nicotinic acetylcholine receptor (AChR) channel. The major effect was observed in the channel mean open time, which was reduced up to about 12-fold at 466 microM DEP. The specificity of the effect was first established through comparison with both untreated cells and cells treated with inactivated DEP. Consistent with an increase in the number of unprotonated histidine residues (pKa = 6.0), this effect increased concomitantly with the pH of the reaction medium, being faster at pH 8 than at pH 6. The changes were dependent on time and DEP concentration, with an apparent EC50 = 114 microM. Modified channels also showed an increase in the number of events per burst of openings together with a decrease in burst durations. The amplitude of the channel-closed time component of about 1 ms increased with respect to the longest-duration-closed component. The number of alpha-bungarotoxin sites was slightly reduced after the modification, without affecting ligand binding affinity. The results suggest that DEP affects extracellular histidine residues involved in the ion translocation function of the AChR, but not its toxin-recognition ability. DEP could, therefore, induce a dissociation between toxin and agonist binding, as is often observed in neuronal AChR.

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.

Effects of long-chain fatty acids on the channel activity of the nicotinic acetylcholine receptor.

We have analyzed the effect of free fatty acids on the function of the acetylcholine receptor (AChR) at the single-channel level, using the patch-clamp technique. Long-chain fatty acids, in the presence of albumin as a carrier, were applied to intact cells or to the cytoplasmic surface of excised membrane patches. In the latter case, AChR channels underwent immediate changes in their behaviour and only very brief opening events were apparent. This could be accounted for by a four-fold reduction in the channel mean open time, with no significant changes occurring in the conductance. An increase in the duration of intermediate closed intervals and a decrease in the burst duration were also observed. The modification appeared not to be critically dependent on the degree of saturation of fatty acyl chains. Addition of free fatty acids in the absence of albumin, as well as treatment of the excised membrane patches with phospholipase A2, resulted in complete inhibition of AChR channel activity. In intact cells, fatty acids could reach and affect AChR channels in the plasmalemma under the patch pipette when added from outside the patch-clamped area. The fatty acid-modified AChR was still able to undergo desensitization. The open channel probability was higher than 0.8 for 100 microM agonist, decreasing to 0.4 in the fatty acid-modified receptors. The results are discussed within the framework of the hypothesis that some lipophilic compounds exert their action on the AChR-lipid interface (the "annulus") (see Barantes, 1993).

Myogenic differentiation of the muscle clonal cell line BC3H-1 is accompanied by changes in its lipid composition.

Phospholipid and neutral lipid composition was studied in the course of myogenic differentiation of the clonal cell line BC3H-1. Total phospholipid content increased during differentiation, predominantly in the major classes of choline and ethanolamine glycerophospholipids. The contents of other lipids, such as triacylglycerols, diminished more than 50% during this period. The content and distribution of fatty acids also underwent marked differentiation-dependent changes. The polyunsaturated (tetrapenta- and hexaenoic) fatty acid species of several phospholipid classes diminished during differentiation, especially those in choline, serine and inositol glycerophospholipids. Most noticeable were the changes in phosphatidylserine; long-chain fatty acids having 20 to 22 carbon atoms and 4 to 6 double bonds decreased from about 30 to about 10 mol%. Although increased levels of saturation in other phospholipid fatty acyl chains appear to accompany the myogenic changes of BC3H-1 cells, some unsaturated fatty acids, such as oleic acid (18:1), increased by as much as 80% during the same period, suggesting the activation of a delta 9 desaturase. Sphingomyelin contained only saturated and monoenoic fatty acids and exhibited a four- to five-fold decrease in its content of monoenoic acyl groups. Diacylglycerols became enriched in arachidonate and docosahexaenoate. The amount of cholesterol and its esters increased slightly during differentiation of BC3H-1 cells. The data show that several metabolic pathways change during myogenic differentiation of the BC3H-1 clonal cell line, particularly de novo biosynthetic pathways, elongation/desaturation reactions, and acyl chain turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Changes in channel properties of acetylcholine receptors during the time course of thiol chemical modifications.

The changes occurring during chemical modification of thiol groups in single acetylcholine receptor (AChR) channels of BC3H-11 cells were examined by the patch-clamp technique in the "cell-attached" configuration. Treatment with either 1 mM or 5 mM dithiothreitol or with 5 mM N-ethylmaleimide (NEM) does not cause significant changes in the conductance and mean open time of the channels. However, reduction with dithiothreitol followed by alkylation with NEM produces modifications of AChRs. Under these conditions, channels activated by 2 microM acetylcholine show decreased open times (about 15-fold shorter for the most-modified AChRs) and a slight reduction in single-channel current. Both changes are dependent on the time of exposure and concentration of NEM. The rate of occurrence of openings, however, changes little during NEM treatment. When reduced and alkylated AChRs are activated by 100 microM acetylcholine, clusters of short openings separated by silent periods of about 1 s are observed. The channel-open probability, determined for openings within a cluster, is decreased by about 10-fold when compared with control receptors. The observations at high agonist concentration indicate that the modified AChR is still able to undergo desensitization.

Muscarinic cholinergic receptor of rat cerebral cortex. Location and characterization of ligand binding site-carrying peptides in synaptosomal membranes and isolated neuronal perikarya.

A careful examination of the location and biochemical properties of the tryptic peptides identified by site-specific labelling of the muscarinic cholinergic receptor (mAChR) of rat cerebral cortex has been carried out. In brain synaptosomal membranes and isolated neuronal perikarya, mAChR labelled with [3H]propylbenzilylcholine mustard (PrBCM) was tryptically cleaved to peptides of Mr 50,000, 30,000. 18,000 and a limiting fragment of Mr 8000. All of these binding site-carrying fragments, characterized in terms of their content of carbohydrates and thiol groups, were quantitatively recovered as membrane-bound peptides. The delipidated [3H]PrBCM-labelled tryptic limiting fragment was found to be highly hydrophobic and insoluble in aqueous media. Experiments performed with proteinase on the tryptic limiting fragment suggest the existence of an ester linkage between the ligand and the peptide. The results strongly support the hydropathicity profile which predicts the location of the muscarinic receptor protein with respect to the membrane bilayer.