Cations Mediate Interactions between the Nicotinic Acetylcholine Receptor and Anionic Lipids

Interactions between the nicotinic acetylcholine receptor (nAChR) and phosphatidic acid (PA) are bidirectional in that membranes containing PA are effective at stabilizing an agonist-responsive nAChR, whereas incorporation of the nAChR into the same membranes leads to a substantial increase in lipid lateral packing density. A previous study suggested that the ability of PA to adopt a dianionic ionization state is key. We monitored the ionization state of PA in both reconstituted and protein-free membranes. In model membranes composed of PA and 3:2 (mol/mol) phosphatidylcholine (PC)/PA, the monoanionic-to-dianionic transition of PA was detected with a pKa of 8.7 and 6.5, respectively. In the reconstituted 3:2 PC/PA membranes, however, PA was stabilized in a monoanionic state at pH values up to 10. Although dianionic PA does not play a role in nAChR function, we found that both the stabilization of monoanionic PA and the concentration of other cations at the bilayer surface can account for changes in bilayer physical properties that are observed upon incorporation of the nAChR into 3:2 PC/PA membranes. A nAChR-induced concentration of cations at the bilayer surface likely mediates interactions between the nAChR and the anionic lipids in its membrane environment.     

Anionic Lipids Modulate the Activity of the Aquaglyceroporin GlpF

The structure and composition of a biological membrane can severely influence the activity of membrane-embedded proteins. Here, we show that the E. coli aquaglyceroporin GlpF has only little activity in lipid bilayers formed from native E. coli lipids. Thus, at first glance, GlpF appears to not be optimized for its natural membrane environment. In fact, we found that GlpF activity was severely affected by negatively charged lipids regardless of the exact chemical nature of the lipid headgroup, whereas GlpF was not sensitive to changes in the lateral membrane pressure. These observations illustrate a potential mechanism by which the activity of an α-helical membrane protein is modulated by the negative charge density around the protein.     

Neuronal Nicotinic Acetylcholine Receptor Modulators Reduce Sugar Intake

Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases α4β2 * and decreases α6β2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption.     

Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival

It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers.     

The Nicotinic Acetylcholine Receptor: Structure and Autoimmune Pathology

Abstract

The nicotinic acetylcholine receptors (AChR) are presently the best-characterized neurotransmitter receptors. They are pentamers of homologous or identical subunits, symmetrically arranged to form a transmembrane cation channel. The AChR subunits form a family of homologous proteins, derived from a common ancestor. An autoimmune response to muscle AChR causes the disease myasthenia gravis. This review summarizes recent developments in the understanding of the AChR structure and its molecular recognition by the immune system in myasthenia.     

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 subunits are found in the brain and in the peripheral nervous system, and structural, non- subunits like 2 and 4 confer different properties to neuronal receptors. Thus, the final properties of the oligomeric AChR depend on the different combinations of and subunits. Most mutations found so far occur in the 4 chain, the most abundant subunit in the central nervous system. Specifically, the identification of mutations in the 4 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 4 mutants increase the probability of epileptic discharges. We discuss testable hypotheses for unraveling the pathophysiology of these two disorders associated with AChR mutations.     

Cloning of a Putative Neuronal Nicotinic Acetylcholine Receptor Subunit

A cDNA clone was isolated from a rat superior cervical ganglion cDNA library with an oligonucleotide that hybridized to muscle-like nicotinic acetylcholine receptor (nAChR) subunit cDNA. The deduced amino acid sequence possesses characteristics expected of a nAChR subunit that does not bind acetylcholine, in addition to distinctive features such as unique cysteine residues and N-linked glycosylation sites.     

Neural Regulation of Properties of the Nicotinic Acetylcholine Receptor

Abstract

During nerve-muscle synapse formation, acetylcholine receptors become localized and modified to allow efficient transfer of information from nerve to muscle. In this paper we summarize our studies on two aspects of receptor modulation—their concentration at synaptic sites and their ability to desensitize in response to prolonged application of agonist. We demonstrate that receptor localization is a complex event which extensively reorganizes the structure of the junctional region. This allows the subsequent influences of contraction to be exerted differently in junctional and extrajunctional regions. We indicate that increases in muscle cell Ca²⁺ appear to mediate some of the effects of muscle contraction and suggest how regulation of Ca²⁺ levels may specify junctional and extrajunctional differences. Finally, we discuss the role of receptor phosphorylation in determining the rate of desensitization.     

Neurotoxins Distinguish Between Different Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations

Neuronal and muscle nicotinic acetylcholine receptor subunit combinations expressed in Xenopus oocytes were tested for sensitivity to various neurotoxins. Extensive blockade of the alpha 3 beta 2 neuronal subunit combination was achieved by 10 nM neuronal bungarotoxin. Partial blockade of the alpha 4 beta 2 neuronal and alpha 1 beta 1 gamma delta muscle subunit combinations was caused by 1,000 nM neuronal bungarotoxin. The alpha 2 beta 2 neuronal subunit combination was insensitive to 1,000 nM neuronal bungarotoxin. Nearly complete blockade of all neuronal subunit combinations resulted from incubation with 2 nM neosurugatoxin, whereas 200 nM neosurugatoxin was required for partial blockade of the alpha 1 beta 1 gamma delta muscle subunit combination. The alpha 2 beta 2 and alpha 3 beta 2 neuronal subunit combinations were partially blocked by 10,000 nM lophotoxin analog-1, whereas complete blockade of the alpha 4 beta 2 neuronal and alpha 1 beta 1 gamma delta muscle subunit combinations resulted from incubation with this concentration of lophotoxin analog-1. The alpha 1 beta 1 gamma delta muscle subunit combination was blocked by the alpha-conotoxins G1A and M1 at concentrations of 100 nM. All of the neuronal subunit combinations were insensitive to 10,000 nM of both alpha-conotoxins. Thus, neosurugatoxin and the alpha-conotoxins distinguish between muscle and neuronal subunit combinations, whereas neuronal bungarotoxin and lophotoxin analog-1 distinguish between different neuronal subunit combinations on the basis of differing alpha subunits.     

Neurosteroids Modulate Nicotinic Receptor Function in Mouse Striatal and Thalamic Synaptosomes

Abstract: Progesterone and its A-ring reduced metabolites are allosteric activators of GABA_A receptors. The studies reported here examined the effects of these steroids on brain nicotinic receptors using an ⁸⁶Rb⁺ efflux assay that likely measures the function of α4β2-type nicotinic receptors and [³H]dopamine release, which may be modulated by an α3-containing nicotinic receptor. Both of the A-ring reduced metabolites of progesterone were noncompetitive inhibitors of both assays, whereas progesterone inhibited only the ⁸⁶Rb⁺ efflux assay. The ⁸⁶Rb⁺ efflux assay was slightly more sensitive than was the dopamine release assay to steroid inhibition. Inhibition developed slowly for both assays ( t _1/2 = 0.4 min) and was reversed even more slowly ( t _1/2 = 10–15 min). Steroid addition did not alter either the rate of association of [³H]nicotine binding to brain membranes, nor was equilibrium binding changed. These findings argue that neurosteroids are allosteric inhibitors of brain nicotinic receptors.     

Myelin-Derived Lipids Modulate Macrophage Activity by Liver X Receptor Activation

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence Studies of the Nicotinic Acetylcholine Receptor in its Membrane Environment

Since the discovery of the fraction of immobilized lipid in contact with the nicotinic acetylcholine receptor (AChR), the lipid-belt region around this protein has become the focus of a variety of biophysical studies aimed at defining its properties. Here we summarize recent spectroscopic studies from our laboratory using Laurdan fluorescence to characterize distinct sites for lipids and to describe their effect on the AChR microenvironment.     

Ion Channel Topography of the Neuronal Nicotinic Acetylcholine Receptor: Pharmacochemical Approaches

Forty-three bisammonium ganglionic blockers were synthesized to study the structure of the ion channel of nicotinic acetylcholine receptor. The conformational parameters of these blockers were studied, and their effects toward the ganglionic transmission in situ on the sympathetic feline superior cervical ganglia and in vitro on the parasympathetic guinea-pig small intestine ganglia were determined. A model of the binding site for the bisammonium ganglionic blockers in the neuronal ion channel was proposed.     

Composition of Lipids in Elasmobranch Electric Organ and Acetylcholine Receptor Membranes

The composition of phospholipids from electric organ and from membranes enriched in acetylcholine receptors (AChRs) is analyzed in three elasmobranch fish (Torpedo marmorata, Torpedo californica, and Discopyge tschudii). Irrespective of their purity, AChR-containing membranes are similar to electric organ in lipid and fatty acid composition. The following characteristics are common to the three species: (a) Choline, ethanolamine, and serine glycerophospholipids account for 80-90% of the phospholipids. (b) Their major fatty acid constituents are monoenes, saturates, and long-chain (n-3) polyenes (especially docosahexaenoate). (c) A large proportion of the ethanolamine glycerophospholipids (30-50%) is made up by plasmenylethanolamine, which contains fewer polyenes than phosphatidylethanolamine per mole of lipid. (d) Polyphosphoinositides represent 20-30% of the inositides of electric organ. (e) Phosphatidylinositol and phosphatidate have large proportions of 20- and 22-carbon polyenes. (f) Diphosphatidylglycerol and triacylglycerols are rich in oleate but also contain long-chain polyenes. (g) Sphingomyelin has monoenes and saturates ranging from 14 to 26 carbons. Species-related variations are observed (a) in the ratios between some phospholipid classes and subclasses and (b) in the relative abundance of the major polyunsaturated acyl chains of phospholipids. Despite these differences, the average unsaturation and length of fatty acids in major phospholipid classes are similar for the three species.     

The Role of Lipids in the Function of the Acetylcholine Receptor

Abstract

The composition of membranes containing acetylcholine receptor was altered in order to examine the possible role of lipids in receptor function. Polyethylene glycol was used to fuse AcChR-rich membranes with an excess of lipid vesicles of defined composition. By this procedure, the cholesterol composition was reduced to as little as 20% of that found in native membranes. Using a TI⁺ flux assay it was possible to measure receptor function in such altered membrane environments. The apparent K_d for carbamylcholine was found to decrease as the cholesterol content was reduced. This result was confirmed by measuring the agonist-induced fluorescence change of the covalently attached probe, 4-[N-(iodoacetoxy)-ethyl-N-methyl] amino-7-nitrobenz-2-oxa-1,3-diazole. When the phospholipid composition was manipulated by membrane fusion, ion flux was found to be optimal when the lipid composition resembled that of native receptor membranes. These results indicate that membrane lipids potentially play a role in the regulation of acetylcholine receptor function.