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SARS-CoV-2 epidemics raises a considerable issue of public health at the planetary scale. There is a pressing urgency to find treatments based upon currently available scientific knowledge. Therefore, we tentatively propose a hypothesis which hopefully might ultimately help save lives. Based on the current scientific literature and on new epidemiological data which reveal that current smoking status appears to be a protective factor against the infection by SARS-CoV-2 [1], we hypothesize that the nicotinic acetylcholine receptor (nAChR) plays a key role in the pathophysiology of Covid-19 infection and might represent a target for the prevention and control of Covid-19 infection. [ABSTRACT FROM AUTHOR]

α6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of α6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. α-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of α-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyxproline increased the IC50 by 2800-fold at α6/α3β2β3 but only by 6-fold at α6/α3β4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepi nephrine release. Release was abolished in α6-null mutant mice. α-Conotoxin BuIA[T5A;P60] partially blocked norepinephrine release in wild-type controls but failed to block release in β4-/- mice. In contrast, BuIA[T5A;P60] failed to block dopamine release in the wild-type striatum known to contain α6β2* nAChRs. BuIA[T5A;P60] is a novel ligand for distinguishing between closely related α6* nAChRs; α6β4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum. [ABSTRACT FROM AUTHOR]

Nicotinic receptors - a family of ligand-gated ion channels that mediate the effects of the neurotransmitter acetylcholine - are among the most well understood allosteric membrane proteins from a structural and functional perspective. There is also considerable interest in modulating nicotinic receptors to treat nervous-system disorders such as Alzheimer's disease, schizophrenia, depression, attention deficit hyperactivity disorder and tobacco addiction. This article describes both recent advances in our understanding of the assembly, activity and conformational transitions of nicotinic receptors, as well as developments in the therapeutic application of nicotinic receptor ligands, with the aim of aiding novel drug discovery by bridging the gap between these two rapidly developing fields. [ABSTRACT FROM AUTHOR]

Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system and modulate neuronal function in most mammalian brain structures. The contribution of defined nAChR subunits to a specific behavior is thus difficult to assess. Mice deleted for β2-containing nAChRs (β22/2) have been shown to be hyperactive in an open-field paradigm, without determining the origin of this hyperactivity. We here develop a quantitative description of mouse behavior in the open field based upon first order Markov and variable length Markov chain analysis focusing on the timeorganized sequence that behaviors are composed of. This description reveals that this hyperactivity is the consequence of the absence of specific inactive states or ''stops''. These stops are associated with a scanning of the environment in wildtype mice (WT), and they affect the way that animals organize their sequence of behaviors when compared with stops without scanning. They characterize a specific ''decision moment'' that is reduced in β22/2 mutant mice, suggesting an important role of β2-nAChRs in the strategy used by animals to explore an environment and collect information in order to organize their behavior. This integrated analysis of the displacement of an animal in a simple environment offers new insights, specifically into the contribution of nAChRs to higher brain functions and more generally into the principles that organize sequences of behaviors in animals. [ABSTRACT FROM AUTHOR]

To understand the structural causes of myasthenic mutations, molecular models of the nicotinic acetycholine receptor were first constructed. Then, the gating transition between resting and open conformation was investigated in silico by normal mode analysis using a physically meaningful description of protein flexibility. This analysis revealed a global quaternary twist motion that opens the ion pore. Second, it was found that most (24/27) of the spontaneous mutations known to cause congenital myasthenia (and autosomal dominant nocturnal frontal lobe epilepsy) are located either at the interface between subunits or, within a given subunit, at the interface between rigid blocks. These interfaces are modified significantly by the twist mode together with significant changes of the tertiary organization of the subunits. These data provide a qualitative interpretation of the molecular phenotype of pathological mutations responsible for congenital myasthenia. [ABSTRACT FROM AUTHOR]

Chronic nicotine exposure results in long-term homeostatic regulation of nicotinic acetylcholine receptors (nAChRs) that play a key role in the adaptative cellular processes leading to addiction. However, the relative contribution of the different nAChR subunits in this process is unclear. Using genetically modified mice and pharmacological manipulations, we provide behavioral, electrophysiological, and pharmacological evidence for a long-term mechanism by which chronic nicotine triggers opposing processes differentially mediated by β2*- vs. α7*nAChRs. These data offer previously undescribed insights into the understanding of nicotine addiction and the treatment of several human pathologies by nicotine-like agents chronically acting on β2*- or α7*nAChRs. [ABSTRACT FROM AUTHOR]

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or ‘Cys-loop’ receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change. [ABSTRACT FROM AUTHOR]

Nicotinic acetylcholine receptors (nAChR) are pentameric ligand-gated ion channels composed of subunits that consist of an extracellular domain that carries the ligand-binding site and a distinct ion-pore domain. Signal transduction results from the allosteric coupling between the two domains: the distance from the binding site to the gate of the pore domain is 50 A. Normal mode analysis with a Cα Gaussian network of a new structural model of the neuronal α7 nAChR showed that the lowest mode involves a global quaternary twist motion that opens the ion pore. A molecular probe analysis, in which the network is modified at each individual amino acid residue, demonstrated that the major effect is to change the frequency, but not the form, of the twist mode. The largest effects were observed for the ligand-binding site and the Cys-loop. Most (24/27) of spontaneous mutations known to cause congenital myasthenia and autosomal dominant nocturnal frontal lobe epilepsy are located either at the interface between subunits or, within a given subunit, at the interface between rigid blocks. These interfaces are modified significantly by the twist mode. The present analysis, thus, supports the quaternary twist model of the nAChR allosteric transition and provides a qualitative interpretation of the effect of the mutations responsible for several receptor pathologies. [ABSTRACT FROM AUTHOR]

Rationale Several studies have suggested that nicotine treatment can modulate the behavioral and neurochemical responses to other psychostimulants, such as cocaine. Objectives The current study examined the hypothesis that nicotinic acetylcholine receptor (nAChR) blockade influences the ability of cocaine to elicit increases in extracellular dopamine levels. Materials and methods Pharmacological studies using nicotinic antagonists as well as genetic inactivation of β2* nAChRs were used to determine the effect of nAChR blockade on dopamine levels in ventral striatum elicited by acute or repeated administrations of cocaine in mice. Results Administration of mecamylamine (a general nicotinic antagonist that is not highly selective for individual nAChR subtypes) or co-administration of methyllycaconitine (a more selective antagonist of α7* nAChRs) with dihydro-β-erythroidine (a more selective antagonist of α2* nAChRs and other heteromeric nAChR subtypes) prevented sensitization of cocaine-elicited increases in extracellular DA levels in the ventral striatum in wild-type mice. In contrast, neither of the more specific antagonists alone was effective in preventing sensitization. Finally, methyl-lycaconitine administration prevents sensitization in β2-/- mice but not in β2+/+ or wild-type mice. Conclusions These data indicate that inhibition of both β7* and β2* nAChRs is necessary to prevent development of sensitization of cocaine-elicited increases in extracellular dopamine levels in the ventral striatum of mice. [ABSTRACT FROM AUTHOR]

The article discusses research on the use of β2 subunit of the nicotinic acetylcholine receptor for nicotine-conditioned place preference in mice. It highlights the importance of determining the nicotinic receptor subtypes involved in nicotine reward to understand how nicotine addiction progresses.

We investigated the role played by $2-containing neuronal nicotinic receptors [nicotinic acetyicholine receptors (nAChRs)I in mediating nicotine's side effects in the fetus and newborn. Pregnant WT and mutant mice lacking the β2 nAChR subunit were implanted with osmotic minipumps that delivered either water or a controlled dose of nicotine. Subsequently, we compared the development of the sympathoadrenal system and breathing and arousal reflexes of offspring shortly after birth, a period of increased vulnerability to nicotine exposure. Newborn WT pups exposed to nicotine exhibited all of the deficits associated with maternal tobacco and nicotine use, and linked to poor neonatal outcome: growth restriction, unstable breathing, and impaired arousal and catecholamine biosynthesis. Remarkably similar deficits were detected in pups lacking β2-containing nAChRs. Loss-of-function of these nAChRs consequently reproduces with astonishing fidelity many of the abnormalities caused by perinatal nicotine exposure. We propose that the underlying mechanisms of nicotine's detrimental side effects on a range of crucial defensive reflexes involve loss of function of nAChR subtypes, possibly via activity-dependent desensitization. [ABSTRACT FROM AUTHOR]

A major progress in 20th century neuroscience has been the identification of the acetylcholine nicotinic receptor, the first neurotransmitter and ligand-gated ion channel to be characterized as a protein. The work leading to this achievement, which spanned the late 1960s and early 1970s, influenced significantly, in my opinion, the evolution of methods and experimental approaches used to investigate neural activity but also our general understanding of brain functions. Circumstances were such that, perhaps, for the first time in history, disciplines as culturally separate as electrophysiology (the dry science) (Fatt, 1964; Katz, 1966), biochemistry (the wet science) (Nachmansohn, 1959), and pharmacology (Dale, 1943) were joined, in a unified and rational manner, with the common goal of identifying the elementary molecular device that transforms, in the nervous system, a chemical signal into an electrical one. [ABSTRACT FROM AUTHOR]

Cholinergic axons and nicotinic receptors are abundant in all layers of the olfactory bulb (OB), the main region of newborn neuron integration in the adult brain. Here, we report that the OB granule cell layer in mice lacking the predominant form of brain high- affinity nicotinic acetylcholine receptors (β2-/- mice) displayed nearly 50% more newborn neurons and significantly fewer apoptotic cells than did β2+/+ mice. Conversely, in vivo chronic nicotine exposure significantly decreased the number of newborn granule cells in β2+/+ but not β2-/- adult mice, confirming that the survival of newborn neurons can be controlled by the activation of β2-containing nicotinic acetyicholine receptors. Unexpectedly, investigating the behavioral consequence of an increased number of granule cells in β2-/- mice revealed that these animals have a less robust short-term olfactory memory than their wild-type counter- parts. Taken together, these results provide evidence that high- affinity nicotinic receptors are involved in the maturation of adult OB local circuits. They also indicate that an increase in the number of granule cells does not necessarily correlate with better olfactory performance and further highlight the importance of cholinergic afferents for olfactory processing. [ABSTRACT FROM AUTHOR]

The cholinergic system is involved in arousal and in rapid eye movement sleep (REMS). To evaluate the contribution of nicotinic acetylcholine receptors (nAChRs) to these functions, we studied with polygraphic recordings the regulation of sleep in mice lacking the β2 subunit gene of the nAChRs, a major component of high-affinity nicotine binding sites in the brain. Nicotine (1-2 mg/kg, i.p.) increased wakefulness in wild-type but not knock-out animals, indicating that β2-containing nAChRs mediate the arousing properties of nicotine. Under normal conditions, the β2-/- mice displayed the same amounts of waking, non-REM sleep (NREMS) and REMS as their wild-type counterparts. However, they exhibited longer REMS episodes and a reduced fragmentation of NREMS by events characterized notably by a transient drop in EEG power and frequently associated with EMG activation, tentatively referred to as micro-arousals. Respiration monitoring showed that these events were accompanied with, but not caused by, breathing irregularities. Sleep deprivation of β2-/- mice resulted in a normal increase in REMS episode duration and NREMS δ power but yielded a reduction of the number of micro-arousals in NREMS. In contrast, in β2-/- mice, a 1 hr immobilization stress failed to produce the normal rebound in REMS in the following 12 hr and, instead, was associated with increased NREMS fragmentation and sustained corticosterone levels. Our results show that the β2-containing nAChRs contribute to the organization of sleep by regulating the transient phasic activity in NREMS, the REMS onset and duration, and the REMS-promoting effect of stress. [ABSTRACT FROM AUTHOR]

Presents a study which determined the subunit composition of functional nicotinic acetylcholine receptors in dopaminergic neurons. Materials and methods; Results; Discussion.

The binding of a fluorescent agonist to the acetycholine receptor from Torpedo electric organ has been studied by time-resolved spectroscopy in three different environments: in native membrane fragments, in the detergent CHAPS, and after complexation by amphipathic polymers (‘amphipols’). Binding kinetics was similar in the membrane and in amphipols, demonstrating that the receptor can display unaltered allosteric transitions outside its natural lipid environment. In contrast, allosteric equilibria were strongly shifted towards the desensitized state in CHAPS. Therefore, the effect of CHAPS likely results from molecular interactions rather than from the loss of bulk physical properties of the membrane environment. [Copyright &y& Elsevier]

Studies the requirement of the nicotinic acetycholine receptor beta 2 subunit for the anatomical and functional development of the visual system. Altered retinofugal projections in adult beta 2 -/- mice; Development of the retinogeniculate projections in beta 2 -/- mice; Pharmacologically elicited alteration of retinogeniculate projections.

AbstractNicotinic acetylcholine receptors (nAChRs) are expressed in the midbrain ascending dopaminergic system, a target of many addictive drugs. Here we assessed the intracellular Ca2+ level by imaging fura-2-loaded cells in substantia nigra pars compacta in mouse brain slices, and we examined the influence on this level of prolonged exposures to nicotine using mice lacking the nAChR β2-subunit. In control cells, superfusion with nicotine (10–100 μm) caused a long-lasting rise of intracellular Ca2+ level which depended on extracellular Ca2+. This nicotinic response was almost completely absent in β2–/– mutant mice, leaving a small residual response to a high concentration (100 μm) of nicotine which was inhibited by the α7-subunit-selective antagonist, methyllycaconitine. Conversely, the α7-subunit-selective agonist choline (10 mm) caused a methyllycaconitine-sensitive increase in intracellular Ca2+ level both in wild-type and β2–/– mutant mice. Nicotine-elicited Ca2+ mobilization was reduced by the Na+ channel blocker tetrodotoxin (TTX) and by T-type Ca2+ channel blocking agents, whereas the choline-elicited Ca2+ increase was insensitive to TTX. Neither nicotine nor choline produced Ca2+ increase following inhibition of the release of Ca2+ from intracellular stores by dantrolene. These results demonstrate that in nigral dopaminergic neurons, nicotine can elicit Ca2+ mobilization via activation of two distinct nAChR subtypes: that of β2-subunit-containing nAChR followed by activation of Na+ channel and T-type Ca2+ channels, and/or activation of α7-subunit-containing nAChR. The Ca2+ influx due to nAChR activation is subsequently amplified by the recruitment of intracellular Ca2+ stores. This Ca2+... [ABSTRACT FROM AUTHOR]

Provides information on nicotinic acetylcholine receptors (nAChR). Genetic encoding of nAChR oligomer; Transmembrane organization and subunit structure of nAChR; Discussion on the physical chemistry of acetylcholine binding; Pharmacological diversity of nAChR binding sites.

We have examined neuroanatomical, biochemical and endocrine parameters and spatial learning in mice lacking the β2 subunit of the nicotinic acetylcholine receptor (nAChR) during ageing. Aged β2­/­ mutant mice showed region-specific alterations in cortical regions, including neocortical hypotrophy, loss of hippocampal pyramidal neurons, astroand microgliosis and elevation of serum corticosterone levels. Whereas adult mutant and control animals performed well in the Morris maze, 22- to 24-month-old β2­/­ mice were significantly impaired in spatial learning. These data show that 2 subunit-containing nAChRs can contribute to both neuronal survival and maintenance of cognitive performance during ageing. β2­/­ mice may thus serve as one possible animal model for some of the cognitive deficits and degenerative processes which take place during physiological ageing and in Alzheimer's disease, particularly those associated with dysfunction of the cholinergic system. [ABSTRACT FROM AUTHOR]

Recombinant DNA techniques have been used to identify the family of molecules that mediate nicotine's effects on the brain. Nicotine binds and activates nicotinic acetylcholine receptors (nAChRs) which are made up of combinations of individual nicotinic subunits. It is important to determine which of the many possible subunit combinations are responsible for the physiological and behavioral effects of nicotine that lead to addiction. Molecular genetic tools such as antisense strategies have been useful in elucidating the electrophysiological properties of nAChRs in different tissues. Use of knock-out mice lacking individual nAChR subunits has also begun to elucidate how nicotine exerts its actions from the molecular level to the behavioral level. Experiments using mice lacking the β2 subunit of the nAChR have shown that binding of nicotine to receptors containing this subunit is the first step in a pathway leading to increased dopamine levels in the mesolimbic dopamine system, and ultimately to the behavioral effects of nicotine in a test of nicotine reinforcement. Mice deficient in various α subunits of the nAChR will identify the partners of β2 mediating the addictive properties of nicotine. In addition, more data needs to be gathered on the electrophysiological properties of different subunit combinations, the effects of nicotine on different neurotransmitter systems and the links between the molecular biology of nicotine receptors, their physiology and the ultimate role of individual receptor subtypes in complex behaviors. Multidisciplinary approaches to nAChR function will be essential to answering these questions. [ABSTRACT FROM AUTHOR]

Summarizes what is currently known of the mechanisms involved in the development of the postsynaptic membrane. Role of the motor nerve and electrical activity in subcellular differentiation during formation of the neuromuscular junction (NMJ); Acetycholine as critical synaptic component in differentiation; Posttranslational mechanisms during end-plate development.

The transmembrane orientation of the polypeptide chains present in acetylcholine receptor-rich membranes from Torpedo marmorata was studied by selective proteolysis. These membranes contain two major polypeptides of Mr 40000 and 43000 and two minor ones of Mr 50000 and 66000. With intact membranes all four proteins were found remarkably resistant to the action of the proteolytic enzymes pronase and trypsin. In contrast, when membranes were opened by sonication in the presence of trypsin, all four proteins were degraded. Stable degradation products of Mr 47000, 38000, 35000 and 32000 were found. Addition of protease after sonication was not effective; sonication alone did not remove the four major proteins from the membrane. It is concluded that: (a) these four chains were cut at sites normally exposed to the inside surface of the membrane and (b) the membrane fragments were sealed in vitro with the same orientation as in vivo Various treatments were found to render the intact receptor-rich membranes susceptible to proteolytic attack: incubation at pH 4 with the protease pepsin led to the selective degradation of the 40000-Mr, 50000-Mr and 66000-Mr chains; the same results were obtained when the membranes were heated at 62°C for 1 min and then digested with trypsin. The 43000-Mr chain remained resistant in both cases. No degradation fragments with Mr greater than 20000 were found. Furthermore, when these membranes were sonicated in the presence of pepsin, the 43000-Mr polypeptide was degraded. It is concluded that (a) the 43000-Mr polypeptide is entirely enclosed by the lipid bilayer, and (b) that the 40000-Mr, 50000-Mr and 66000-Mr polypeptides are transmembrane proteins. Trypsin digestion of the receptor-rich membranes opened by phospholipase A2 (crotoxin) or alkaline treatment, or solubilization by 1% Triton X-100 gave the same series of degradation products as the sonicated membranes. The 47000-Mrr fragment was shown to be derived from the 50000-Mr chain; and the 35000-Mr and 32000-Mr fragments, which were labelled by the affinity reagent 4(N-maleimido)-phenyl-[3H]trimethylammonium, were derived from the 40000-Mr chain. After dodecytsulfate/polyacrylamide gel electrophoresis, fragments of Mr 47000, 38000, 35000 and 32000 were labelled by 125I-concanavalin A, like the native 40000-Mr, 50000-Mr and 66000-Mr chains. The NH2-terminal sequence of the 35000-Mr fragment was identical to that of the 40000-Mr chain. [ABSTRACT FROM AUTHOR]

In receptor-rich membrane fragments from Torpedo, acetylcholine binds, in the presence of 70 μM Tetram, to a homogeneous population of high-affinity sites with Kd = (3.4 ± 0.8) × 10-8 M. Dissolution of these membrane fragments by sodium cholate causes a decrease of affinity associated with the appearance of medium-affinity (Kd ≈ 10-7 M) and low-affinity (Kd ≥ 10-6 M) sites. Dissolution by neutral detergents Triton X-100 or Emulphogene preserves the high affinity of the acetylcholine binding sites. In all the soluble states of the receptor protein, Ca-2 ions and local anaesthetics no longer enhance the affinity for acetylcholine. Elimination of sodium cholate by dilution leads to the reassociation of the receptor protein, the recovery of high-affinity sites and the control by Ca-2 ions and local anaesthetics. Purification by affinity chromatography of the receptor protein in Triton X-100 is accompanied by a conversion of a majority of the acetylcholine sites into their state of low affinity. High-affinity sites can no longer be recovered by detergent dilution from these low-affinity ones. [ABSTRACT FROM AUTHOR]

Binding of heregulin (HRG) to its receptor, ErbB3, results in a dimerization with ErbB2/neu and activation of their intrinsic tyrosine kinases, initiating a cascade of events resulting in the stimulation of acetylcholine receptor (AChR) genes in muscle. Here we have examined the signalling downstream of the HRG receptor. We show that phosphatidylinositol 3′-kinase (PI3K) and SHC bind to the HRG-activated ErbB3 in myotubes. Subsequently, p70S6 kinase (p70S6k), and MAP kinase ERK2 and thereby p90rsk are activated. However, inhibition of PI3K and p70s6k by wortmannin and rapamycin, respectively, failed to antagonize AChR α-subunit gene expression stimulated by HRG, despite the fact that the activities of the kinases were inhibited. In contrast, these inhibitors elevated AChR α-subunit mRNA levels, by themselves, independently of muscle electrical activity. On the other hand, the 17mer anti-sense oligonucleotide, EAS1, caused a specific depletion of ERK2 and eliminated the ability of HRG to stimulate AChR α-subunit gene expression. These results indicate that HRG stimulates expression of AChR genes via ERK2 activation, and provide a physiological example of neurotrophic factor-associated repression of AChR genes by stimulation of p70s6k activity which may contribute to the expression of adult type AChR genes at the neuromuscular junction. [ABSTRACT FROM AUTHOR]

Highly purified plasma membrane fragments rich in acetylcholine (nicotinic) receptor were prepared from Torpedo marmorata electric organ, depleted at alkaline pH of the 43000-Mr protein and solubilized with sodium cholate in the presence or absence of exogeneous lipids. The solubilized receptor (essentially in its 9-S light form) was reinserted into artificial lipid vesicles in the course of detergent removal by two methods yielding reconstituted vesicles with mean diameters of about 40 nm and 100 nm respectively. The influence of the composition of the lipid and aqueous phases on the physical reinsertion of the receptor protein was studied by gel filtration, gradient Centrifugation and electron microscopy. At low ionic strength, the α-toxin binding sites were quantitatively reintegrated into vesicles from soy-bean lipids, but not into egg phosphatidylcholine vesicles Raising the ionic strength during detergent removal decreased receptor incorporation, the permeability properties of the reconstituted vesicles were investigated by a Millipore filtration method using radioactive tracer ions. Passive equilibration of 22Na+ or 134Cs+ ions between internal and external compartments was slow and remained incomplete after several days. Addition of the agonists acetylcholine or carbamylcholine stimulated the exchange of cations which reached a plateau value within less than 15 s, These agonists did not affect the rate of 36Cl- exchange. The permeability response was totally blocked by snake α-toxins, trimethisoquin, histrionicotoxin, and by preincubation with agonists (desensitization) It is concluded that these properties do not depend on the dimerization of the receptor protein into the 13-S form nor on its association with the 43000-Mr protein and therefore appear to be intrinsic to the 9-S form of the receptor. [ABSTRACT FROM AUTHOR]

The acetylcholine (nicotinic) receptor from the electric organ of Electrophorus electricus has been purified approx. 300-fold from Triton X-100 extracts of electroplax membrane fragments. The receptor protein was first adsorbed from crude extracts on an affinity column containing 1,3-bis(tri-ethylammonium ethoxy)-4-iodoacetamidobenzene diiodide, an analogue of flaxedil, linked to Sepharose 2B, then eluted with a solution of flaxedil and concentrated. The receptor protein was further purified by centrifugation on sucrose gradients containing 1% Triton X-100, 1% Emulphogene, or 0.5% sodium cholate. If desired, detergent could be removed after centrifugation in the presence of sodium cholate by gel filtration on Sephadex G-75. At ail stages the receptor was assayed by incubation with α-[³H]toxin from Naja nigricollis followed by isolation of the toxin, receptor complex by filtration on Millipore filters at low detergent concentration. The best preparations bound 1 mol toxin per 150 000 g protein. In the presence of non-denaturing detergents the preparations gave a single major band for both protein and activity upon polyacrylamide disc gel electrophoresis or sucrose gradient centrifugation. The receptor protein appears essentially homogeneous in the electron microscope after negative staining with uranyl acetate. Upon gel electrophoresis in the presence of dodecylsulphate, there were two major bands corresponding to molecular weights of 43 000 and 48 000. The ultraviolet-absorption spectrum and amino-acid composition of the purified receptor protein are typical of those of globular, water-soluble proteins in general and do not reflect the hydrophobic character of the macromolecule. Furthermore, the purified protein interacts with concanavalin A and other plant agglutinins indicating that it contains a carbohydrate moiety. The purified receptor protein carries a single class of binding sites for [³H]decamethonium (Kd = 0.02 µM) at ligand concentrations up to 1 µM and for acetylcholine (Kd = 0.06 µM) at up to 1 µM. No evidence for cooperativity m binding of cholinergic effectors to the receptor protein, either purified or in crude extracts, has been detected and all effectors tested inhibit the binding of [³H]decamethonium in a competitive manner. The affinity of agonists for the solubilized receptor protein is approx. 20-fold greater than for the membrane-bound protein. [ABSTRACT FROM AUTHOR]

Presents research which investigated the contribution of the high-affinity neuronal nicotinic acetylcholine receptor to the effects of nicotine on the mesolimbic dopamine system in mice lacking the beta-2 subunit of this receptor. Role of dopamine in mediating the properties of drugs; Findings; Use of patch-clamp recording; Idea that beta-2-containing neuronal nicotinic acetylcholine receptor is involved in nicotine reinforcement.

Nicotinic acetylcholine receptors are transmembrane oligomeric proteins that mediate interconversions between open and closed channel states under the control of neurotransmitters. Fast in vitro chemical kinetics and in vivo electrophysiological recordings are consistent with the following multi-step scheme. Upon binding of agonists, receptor molecules in the closed but activatable resting state (the Basal state, B) undergo rapid transitions to states of higher affinities with either open channels (the Active state, A) or closed channels (the initial Inactivatable and fully Desensitized states, I and D). In order to represent the functional properties of such receptors, we have developed a kinetic model that links conformational interconversion rates to agonist binding and extends the general principles of the Monod-Wyman-Changeux model of allosteric transitions. The crucial assumption is that the linkage is controlled by the position of the interconversion transition states on a hypothetical linear reaction coordinate. Application of the model to the peripheral nicotinic acetylcholine receptor (nAChR) accounts for the main properties of ligand-gating, including single-channel events, and several new relationships are predicted. Kinetic simulations reveal errors inherent in using the dose-response analysis, but justify its application under defined conditions. The model predicts that (in order to overcome the intrinsic stability of the B state and to produce the appropriate cooperativity) channel activation is driven by an A state with a K[sub d] in the 50 nM range, hence some 140-fold stronger than the apparent affinity of the open state deduced previously. According to the model, recovery from the desensitized states may occur via rapid transit through the A state with minimal channel opening, thus without necessarily undergoing a distinct recovery pathway, as assumed in the standard ‘cyclic’ model. Transitions to the desensitized states by low... [ABSTRACT FROM AUTHOR]

Protein kinase C has previously been implicated in the regulation of chicken acetylcholine receptor (AChR) gene expression. To investigate the molecular basis of this regulation, the promoter of the AChR α-subunit (αAChR) gene was linked to a reporter gene and introduced into cultured chick myotubes by transient transfection. Treatment of myotubes with protein-kinase-C-activating phorbol esters was found to inhibit promoter activity. These inhibitory actions were mediated by promoter sequences between nucleotides -110 and -45, relative to the start point of transcription of the αAChR gene. In particular, phorbol-ester responsiveness could be conferred by a short DNA sequence that contains one of the two MyoD binding sites of the αAChR gene muscle-specific enhancer. 12-O-Tetradecanoylphorbol 13-acetate was found to inhibit rapidly and potently the expression of mRNAs coding for the myogenic regulators CMD1 and myogenin. Moreover, its inhibitory effect on the αAChR gene promoter could be attenuated by cotransfection of a MyoD1 expression vector. These results provide a molecular basis for the previously demonstrated involvement of protein kinase C in the regulation of αAChR biosynthesis. In addition, they lend further support to the notion that myogenic proteins play an important role in the control of αAChR gene expression. [ABSTRACT FROM AUTHOR]

The concept of pharmacological receptor was proposed at the turn of the 20th century but it took almost 70 years before the first receptor for a neurotransmitter was isolated and identified as a protein. This review retraces the history of the difficulties and successes in the identification of the nicotinic acetylcholine receptor, the first neurotransmitter receptor to be identified. [ABSTRACT FROM AUTHOR]

Presents a study which examined the contributions of individual nicotinic acetylcholine receptors (nAChrs) to nicotine-mediated behaviors. Information on nAChrs; Materials and methods; Results.

Nicotine acetylcholine receptors form a family of membrane channel proteins expressed throughout the nervous system. In response to ligand activation nicotine receptors modulate important processes such as neurotransmitter release and synaptic plasticity. We have examined protein interactions comprising the high affinity nicotine receptor beta2 site in the mouse brain by MALDI-TOF MS analysis of receptor complexes derived from immunoprecipitation and pull-down experiments. The validity of the nicotine receptor interacting proteins was examined in knockout mice lines with genetic deletions in the beta2 subunit. [ABSTRACT FROM AUTHOR]

The article presents an abstract related to the study of a novel specific phosphorylation of the receptor-associated 43K rapsyn. 43K rapsyn is a peripheral protein especially related with the nicotinic acetylcholine receptor (AChR) enhanced postsynaptic membrane at the neuromuscular junction and in the electrocyte. Reseachers demonstrated a new type of specific phosphorylation of 43K rapsyn by endogenous kinase(s) present in Torpedo electrocyte AChR-rich membrane.

Abstract: Nicotine addiction, the primary cause of tobacco consumption, is mediated through nicotine binding to brain nicotinic acetylcholine receptor (nAChRs). Upon chronic exposure, nicotine elicits a cascade of events, starting with nAChR activation and desensitization, followed by a long term up-regulation that corresponds to an increase in the number of the high affinity nAChRs, a paradoxical process that occurs in the brain of smokers. Recent investigation of the maturation and trafficking of the major brain α4β2 nAChR demonstrates that up-regulation is initiated in the endoplasmic reticulum soon after protein translation. The data thus far accumulated provide evidence that nicotine elicits up-regulation by promoting maturation of nAChR precursors that would otherwise be degraded. This “maturational enhancer” action of nicotine probably contributes to the long term effect of chronic nicotine, and suggests a novel mechanism of neuronal plasticity through an yet unknown endogenous substance which would modulate the receptor expression under physiological conditions. [Copyright &y& Elsevier]

Abstract: The presence of nicotinic acetylcholine receptors (nicotinic receptors) composed of either α7 or α4 and β2 subunits is revealed in B lymphocytes by means of radioligand binding assay and Cell ELISA. Mouse B lymphocytes contained 12,200±3200 of epibatidine-binding sites and 3130±750 of α-Bungarotoxin-binding sites per cell. Mice lacking nicotinic receptor subunits α4, β2 or α7 had less serum IgG and IgG-producing cells in the spleen, but showed stronger immune response to both protein antigen in vivo and CD40-specific antibody in vitro than wild-type mice. Anti-CD40-stimulated proliferation of B lymphocytes from β2 knockout, but not wild-type mice was inhibited with nicotine. Our results indicate that signalling through nicotinic receptors affects both the pre-immune state and activation of B lymphocytes in the immune response, possibly via CD40-dependent pathway. This could contribute to immune depression found in tobacco smokers. [Copyright &y& Elsevier]

Presents a simulation of a single ligand-ligand events for the nicotinic acetylcholine receptor to provide comparisons with single channel events under pulsed agonist conditions. Biochemical function of ligand-gated channels; Description of single channel stochastic simulations; Simulations comparing equivalent and nonequivalent sites.

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClβ receptor of Caenorhabditis elegans and the vertebrate cationic α7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluClβ receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluClβ receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the PCl/PNa ratios, but the GluClβ receptor's mutants retained high capacity to select Cl- over Na+. These results indicate that, unlike the proposed case of anionic Gly- and γ-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClβ receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClβ receptor's channel, Cl- interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore. [ABSTRACT FROM AUTHOR]