Regulating role of acetylcholine and its antagonists in inward rectified K+ channels from guard cell protoplasts ofVicia faba

The inward rectified potassium current ofVicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K⁺ current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K⁺ current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%. However, if guard cell protoplasts are treated with d-Tub and Atr together, the inward K⁺ current is inhibited by 60% –75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K⁺ channels has no effect on the inward K⁺ current regulated by ACh, suggesting that there are inward K⁺ channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement.     

Regulating role of acetylcholine and its antagonists in inward rectified K~ channels from guard cell protoplasts of Vicia faba

The inward rectified potassium current of Vicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%. However, if guard cell protoplasts are treated with d-Tub and Atr together, the inward K current is inhibited by 60%-75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K channels has no effect on the inward K current regulated by ACh, suggesting that there are inward K channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement.     

Regulating role of acetylcholine and its antagonists in inward rectified K+ channels from guard cell protoplasts of Vicia faba

The inward rectified potassium current of Vicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K+ current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K+ current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%.However,if guard cell protoplasts are treated with d-Tub and Atr together, the inward K+ current is inhibited by 60%-75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K+ channels has no effect on the inward K+ current regulated by ACh, suggesting that there are inward K+ channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement.     

Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7

In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor (nAChR) is a valid target in the treatment of a variety of diseases, including Alzheimer’s disease, anxiety, and nicotine addiction. As with most membrane proteins, information on the three-dimensional (3D) structure of nAChR is limited to data from electron microscopy, at a resolution that makes the application of structure-based design approaches to develop specific ligands difficult. Based on a high-resolution crystal structure of AChBP, homology models of the extracellular domain of the neuronal rat and human nAChR subtypes α4β2 and α7 (the subtypes most abundant in brain) were built, and their stability assessed with molecular dynamics (MD). All models built showed conformational stability over time, confirming the quality of the starting 3D model. Lipophilicity and electrostatic potential studies performed on the rat and human α4β2 and α7 nicotinic models were compared to AChBP, revealing the importance of the hydrophobic aromatic pocket and the critical role of the α-subunit Trp—the homolog of AChBP-Trp 143—for ligand binding. The models presented provide a valuable framework for the structure-based design of specific α4β2 nAChR subtype ligands aimed at improving therapeutic and diagnostic applications. Figure Electrostatic surface potential of the binding site cavity of the neuronal nicotinic acetylcholine receptor (nAChR). Nicotinic models performed with the MOLCAD program: a rat α7, b rat α4β2, c human α7, d human α4β2. All residues labeled are part of the α7 (a,c) or α4 (b,d) subunit with the exception of Phe 117, which belongs to subunit β2 (d). Violet Very negative, blue negative, yellow neutral, red very positive     

α7 mutants mimicking atypical motifs (YxxCC of loop-C, and E to H at −1′ in TM2) in the C. elegans LEV-8 subunit affect nicotinic acetylcholine receptor function

The ACR-8-like group of C. elegans nicotinic acetylcholine receptor (nAChR) subunits contain unusual motifs in the ACh binding site and in the −1′ position of transmembrane region two (TM2). Using site-directed mutagenesis (SDM) we have introduced these motifs into chicken α7 as it has not been possible to express C. elegans nAChR in vitro. Oocytes expressing α7 with the C. elegans binding motif show a reduced affinity and efficacy for both ACh and nicotine. The blocking action of the anthelmintic drug levamisole is reduced. The TM2 motif resulted in a non-functional receptor. We conclude that the TM2 motif profoundly restricts cation movement through the α7 channel but does not confer anion permeability. The altered form of the ACh binding motif is likely to result in a receptor with altered pharmacology, adding potential functional diversity at synapses in the nervous system and neuromuscular junctions of C. elegans.     

Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains

In insects, acetylcholine (ACh) is the main neurotransmitter, and nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee, nAChRs are expressed in diverse structures including the primary olfactory centres of the brain, the antennal lobes (AL) and the mushroom bodies. Whole-cell, voltage-clamp recordings were used to characterize the nAChRs present on cultured AL cells from adult honeybee, Apis mellifera. In 90% of the cells, applications of ACh induced fast inward currents that desensitized slowly. The classical nicotinic agonists nicotine and imidacloprid elicited respectively 45 and 43% of the maximum ACh-induced currents. The ACh-elicited currents were blocked by nicotinic antagonists methyllycaconitine, dihydroxy-β-erythroidine and α-bungarotoxin. The nAChRs on adult AL cells are cation permeable channels. Our data indicate the existence of functional nAChRs on adult AL cells that differ from nAChRs on pupal Kenyon cells from mushroom bodies by their pharmacological profile and ionic permeability, suggesting that these receptors could be implicated in different functions.     

Interactions between tachykinins and diverse,human nicotinic acetylcholine receptor subtypes

Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. Reports of effects of substance P (SP) on nAChR function prompted us to investigate interactions between several tachykinins and human nAChR subtypes using clonal cell lines as simple experimental models. Acute exposure to SP inhibits carbamylcholine- or nicotinestimulated function measured using⁸⁶Rb⁺ efflux assays of human ganglionic (α3β4) nAChR expressed in SH-SY5Y neuroblastoma cells (IC₅₀∼2.3 μM) or of human muscle-type (α1β1γδ) nAChR expressed in TE671/RD clonal cells (IC₅₀∼21 μM). SP also acutely blocks function of rat ganglionic nAChR expressed in PC12 pheochromocytoma cells (IC₅₀∼2.1 μM). Neurokinin A and eledoisin inhibit function (extrapolated IC₅₀ values between 60 and 160 μM) of human muscle-type or ganglionic nAChR, but neurokinin B does not, and neither human nAChR is as sensitive as PC12 cell α3β4-nAChR to eledoisin or neurokinin A inhibition. At concentrations that produce blockade of nAChR function, SP fails to affect binding of [³H]acetylcholine to human muscle-type or ganglionic nAChR. SP-mediated blockade of rat or human ganglionic nAChR function is insurmountable by increasing agonist concentrations. Collectively, these results indicate that tachykinins act noncompetitively to inhibit human nAChR function with potencies that vary across tachykinins and nAChR subtypes. They also indicate that tachykinin actions at nAChR could further contribute to complex cross-talk between nicotinic cholinergic and tachykinin signals in regulation of nervous system activity.     

Construction of SH-EP1-α4β2-hAPP695 Cell Line and Effects of Nicotinic Agonists on β-amyloid in the Cells

(1) Nicotinic acetylcholine receptors in central nervous system are thought to be new targets for Alzheimer’s disease. However, the most involved nicotinic receptor subtype in Alzheimer’s disease is unclear. α4β2 receptor is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human amyloid precursor protein (695) gene into SH-EP1 cells which have been transfected with human nicotinic receptor α4 subunit and β2 subunit gene, to observe effects of α4β2 receptors activation on β-amyloid, expecting to provide a new cell line for drug screening and research purpose. (2) Liposome transfection was used to express human amyloid precursor protein (695) gene in SH-EP1-α4β2 cells. Function of the transfected α4β2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective α4β2 nicotinic receptor agonist) on β-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using real-time PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1-α4β2 cells; Nicotine (1 μM) and epibatidine (0.1 μM) decreased intracellular and secreted β-amyloid in the cells; and activation of α4β2 receptors did not affect amyloid precursor protein (695) mRNA level. (4) These results suggest that the constructed cell line, expressing both amyloid precursor protein (695) gene and human nicotinic receptor α4 subunit and β2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer’s disease. Alteration of Aβ level induced by activation of α4β2 nAChR in our study might occur at a post-translational level.     

Sgβ1, a novel locust (Schistocerca gregaria) non-α nicotinic acetylcholine receptor-like subunit with homology to the Drosophila melanogaster Dβ1 subunit

The cloning, sequencing and functional expression of Sgβ1, a novel locust (Schistocerca gregaria) non-α nicotinic acetylcholine receptor (nAChR) subunit is described. This subunit shows 80% identity with the Drosophila melanogaster Dβ1 and 92% identity with the Locusta migratoria β1, non-α subunits but only 38% identity to Sgα1 (also referred to as αL1), a previously cloned S. gregaria nAChR α-subunit. When expressed in Xenopus laevis oocytes, Sgβ1 does not respond to nicotine. Responses to nicotine are observed, however, in oocytes co-expressing Sgα1 and Sgβ1, but the pharmacology is indistinguishable from that of currents produced by expressing Sgα1 alone. We conclude that either Sgβ1 does not co-assemble with Sgα1, or that it is unable to contribute to the functional properties of the receptor, in the Xenopus oocyte expression system.     

The α7 nicotinic acetylcholine receptor in neuronal plasticity

A growing body of evidence indicates that neuronal nicotinic acetylcholine receptors (nAChRs), in addition to promoting fast cholinergic transmission, may modulate other neuronal activities within the central nervous system (CNS). In particular, the α7 nAChR is highly permeable to Ca²⁺ and may serve a distinct role in regulating neuronal plasticity. By elevating intracellular Ca²⁺ levels in discrete neuronal locations, these ligand-gated ion channels may influence numerous physiological processes in developing and adult CNS. In this article, we review evidence that both pre- and postsynaptic α7 nAChRs modulate transmitter release in the brain and periphery through Ca²⁺-dependent mechanisms. The possible role of α7 nAChRs in regulating neuronal growth and differentiation in developing CNS is also evaluated. We consider an interaction between cholinergic and glutamatergic transmission and propose a hypothesis on the possible coregulation of intracellular Ca²⁺ byN-methyl-d-aspartate (NMDA) receptors and α7 nAChRs. Finally, the clinical significance of alterations in the normal function of α7 nAChRs is discussed as it pertains to prenatal nicotine exposure, schizophrenia, and epilepsy.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.     

Nicotinic acetylcholine receptor α7 and β4 subunits contribute nicotine-induced apoptosis in periodontal ligament stem cells

Nicotine, a major component of cigarette smoking, is the important risk factor for the development of periodontal disease. However, the mechanisms that underlie the cytotoxicity of nicotine in human periodontal ligament stem cells (PDLSCs) are largely unknown. Thus, the purpose of this study was to determine the cytotoxic effect of nicotine by means of nicotinic acetylcholine receptor (nAChR) activation in PDLSCs. We first detected α7 and β4 nAChRs in PDLSCs. The gene expressions of α7 and β4 nAChR were increased by nicotine administration. Nicotine significantly decreased cell viability at a concentration higher than 10⁻⁵ M. DNA fragmentation was also detected at high doses of nicotine treatment. Moreover, the detection of sub G1 phase and TUNEL assay demonstrated that nicotine significantly induced apoptotic cell death at 10⁻² M concentration. Western blot analysis confirmed that p53 proteins were phosphorylated by nicotine. Under various doses of nicotine, a decrease in the anti-apoptotic protein Bcl-2, but an increase in p53 and cleaved caspase-3 protein levels, was detected in a dose-dependent manner. However, the apoptotic effect of nicotine was inhibited by the pretreatment of α-bungarotoxin, a selective α7 nAChR antagonist or mecamylamine, a non-selective nAChR antagonist. Finally, increases in the subG1 phase and DNA fragmentation by nicotine was attenuated by each nAChR antagonist. Collectively, the presence of α7 and β4 nAChRs in PDLSCs supports a key role of nAChRs in the modulation of nicotine-induced apoptosis.     

Functional expression of α7 nicotinic acetylcholine receptors in human periodontal ligament fibroblasts and rat periodontal tissues

Tobacco smoking is the main risk factor associated with chronic periodontitis, but the mechanisms that underlie this relationship are largely unknown. Recent reports proposed that nicotine plays an important role in tobacco-related morbidity by acting through the nicotinic acetylcholine receptors (nAChRs) expressed by non-neuronal cells. The aim of this study was to investigate whether α7 nAChR was expressed in periodontal tissues and whether it functions by regulating IL-1β in the process of periodontitis. In vitro, human periodontal ligament (PDL) cells were cultured with 10⁻¹² M of nicotine and/or 10⁻⁹ M of alpha-bungarotoxin (α-Btx), a α7 nAChR antagonist. The expression of α7 nAChR and IL-1β in PDL cells and the effects of nicotine/α-Btx administration on their expression were explored. In vivo, an experimental periodontitis rat model was established, and the effects of nicotine/α-Btx administration on expression of α7 nAChR and development of periodontitis were evaluated. We found that α7 nAChR was present in human PDL cells and rat periodontal tissues. The expressions of α7 nAChR and IL-1β were significantly increased by nicotine administration, whereas α-Btx treatment partially suppressed these effects. This study was the first to demonstrate the functional expression of α7 nAChR in human PDL cells and rat periodontal tissues. Our results may be pertinent to a better understanding of the relationships among smoking, nicotine, and periodontitis.     

Nicotine-induced Ca<Superscript>2+</Superscript>-myristoyl Switch of Neuronal Ca<Superscript>2+</Superscript> Sensor VILIP-1 in Hippocampal Neurons: A Possible Crosstalk Mechanism for Nicotinic Receptors

Visinin-like protein (VILIP-1) belongs to the neuronal Ca²⁺ sensor family of EF-hand Ca²⁺-binding proteins that regulate a variety of Ca²⁺-dependent signal transduction processes in neurons. It is an interaction partner of α4β2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca²⁺ concentration by Ca²⁺-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca²⁺-myristoyl switch. It has been postulated that this will lead to co-localization of the proteins at cell membranes, where VILIP-1 can influence functional activity of α4-containing nAChRs. In order to test this hypothesis we have investigated whether a nicotine-induced and reversible Ca²⁺-myristoyl switch of VILIP-1 exists in primary hippocampal neurons and whether pharmacological agents, such as antagonist specific for distinct nAChRs, can interfere with the Ca²⁺-dependent membrane localization of VILIP-1. Here we report, that only α7- but not α4-containing nAChRs are able to elicit a Ca²⁺-dependent and reversible membrane-translocation of VILIP-1 in interneurons as revealed by employing the specific receptor antagonists dihydro-beta-erythroidine and methylallylaconitine. The nAChRs are associated with processes of synaptic plasticity in hippocampal neurons and they have been implicated in the pathology of CNS disorders, including Alzheimer’s disease and schizophrenia. VILIP-1 might provide a novel functional crosstalk between α4- and α7-containing nAChRs.     

Involvement of Cyclic AMP in ABA- and Ca2--Mediated Signal Transduction of Stomatal Regulation in Vicia faba

The focus of this study is to investigate possible involvementof cyclic AMP in regulation of Vicia stomatal movements. Thepresence of 0.1 mM 8-Br-cAMP, a membrane-permeable analogueof cAMP, alone in the incubation medium did not affect stomatalopening in the light in leaf epidermal peel experiments. However,addition of 0.1 mM 8-Br-cAMP completely reversed exogenous ABA-and C^a2+-induced inhibition of stomatal opening. Consistentwith these results, patch-clamping experiments showed that intracellularaddition of 0.5 mM or 1 mM cAMP significantly reversed the inhibitionof whole-cell inward K⁺ currents by internally supplied 13 µMCa²⁺ or 10 µM ABA in stomatal guard cell protoplasts,respectively. Furthermore, intracellular addition of either10 µM prostaglandin E₁ (PGE1, an adenylate cyclase activator)or 1 mM 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesteraseinhibitor) mimicked the effect of exogenous cAMP on the removalof ABA- or Ca²⁺ inhibition of inward K⁺-current. These resultssuggest that a cAMP signaling pathway is involved in signaltransduction in stomatal regulation by interacting with ABAand Ca²⁺ signaling cascades. A hypothetical mechanism by whichcAMP may regulate K⁺ in stomatal guard cells is also discussed. (Received May 6, 1999; Accepted August 27, 1999)     

Environmental regulation of stomatal response in the <Emphasis Type="Italic">Arabidopsis</Emphasis> Cvi-0 ecotype

The Arabidopsis Cape Verde Islands (Cvi-0) ecotype is known to differ from other ecotypes with respect to environmental stress responses. We analyzed the stomatal behavior of Cvi-0 plants, in response to environmental signals. We investigated the responses of stomatal conductance and aperture to high [CO₂] in the Cvi-0 and Col-0 ecotypes. Cvi-0 showed constitutively higher stomatal conductance and more stomatal opening than Col-0. Cvi-0 stomata opened in response to light, but the response was slow. Under low humidity, stomatal opening was increased in Cvi-0 compared to Col-0. We then assessed whether low humidity affects endogenous ABA levels in Cvi-0. In response to low humidity, Cvi-0 had much higher ABA levels than Col-0. However, epidermal peels experiments showed that Cvi-0 stomata were insensitive to ABA. Measurements of organic and inorganic ions in Cvi-0 guard cell protoplasts indicated an over-accumulation of osmoregulatory anions (malate and Cl⁻). This irregular anion homeostasis in the guard cells may explain the constitutive stomatal opening phenotypes of the Cvi-0 ecotype, which lacks high [CO₂]-induced and low humidity-induced stomatal closure.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.     

Physiochemical and immunological properties of acetylcholine receptors from human muscle

Summary The acetylcholine receptor protein from human muscle was extracted with the non-ionic detergent Triton X-100 and purified by affinity chromatography on -Naja toxin sepharose 4B. Further purification on Dicap-MP sepharose 4B, a choline analog compound, led to ACHR preparations with specific activities of 2–7 nmol/mg protein. The isolated receptor, labeled with ¹²⁵I--bungarotoxin was characterized by different methods and compared to ACHRs from Torpedo californica electroplax and rat-denervated skeletal muscle. Gel filtration on Ultrogel AcA 34 resulted in a stokes radius of 70 Å for the receptor monomer and 99 Å for the dimeric form. Sucrose density gradient centrifugation showed sedimentation coefficients of 9.1 S and 13.5 S. From these data the molecular weight of the ACHR monomer was estimated as 254 000 D and 540 000 D for the receptor dimer. The isoelectric point of the ¹²⁵I--bgt-ACHR complex was determined by thin-layer isoelectric focussing to be pH 5.Purified ACHRs were used for immunization of rats and mice which developed an EAMG as verified by clinical observation and electrophysical measurements. Sera from the immunized animals as well as from myasthenia gravis patients were subsequently used to compare the cross-reactivity of ACHR preparations from different sources. While antibodies of rats immunized with Torpedo ACHRs cross-reacted with ACHR preparations from rat and human skeletal muscle, antibodies from mice immunized with rat ACHR only reacted with preparations from rats and mice. Antibodies from mice immunized with ACHR of human origin exhibited a broad cross-reactivity, as did antibodies from MG patients.Abbreviations AB antibody - ACHR nicotinic acetylcholine receptor - BSA bovine serum albumin - Dicap-MP methyl-[N-(6-aminocaproyl-6aminocaproyl)-3-amino]pyridinbromide - EAMG experimental autoimmune myasthenia gravis - EDTA ethylenediaminetetraaceticacid - MG myasthenia gravis - PMSF phenylmethylsulfonylfluoride Recipient of a postdoctoral grant from Deutsche Forschungsgemeinschaft; present address: Neurologische Klinik, Medizinische Einrichtungen der Universität Düsseldorf.     

Nicotinic acetylcholine receptors: from structure to brain function

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels and can be divided into two groups: muscle receptors, which are found at the skeletal neuromuscular junction where they mediate neuromuscular transmission, and neuronal receptors, which are found throughout the peripheral and central nervous system where they are involved in fast synaptic transmission. nAChRs are pentameric structures that are made up of combinations of individual subunits. Twelve neuronal nAChR subunits have been described, α2–α10 and β2–β4; these are differentially expressed throughout the nervous system and combine to form nAChRs with a wide range of physiological and pharmacological profiles. The nAChR has been proposed as a model of an allosteric protein in which effects arising from the binding of a ligand to a site on the protein can lead to changes in another part of the molecule. A great deal is known about the structure of the pentameric receptor. The extracellular domain contains binding sites for numerous ligands, which alter receptor behavior through allosteric mechanisms. Functional studies have revealed that nAChRs contribute to the control of resting membrane potential, modulation of synaptic transmission and mediation of fast excitatory transmission. To date, ten genes have been identified in the human genome coding for the nAChRs. nAChRs have been demonstrated to be involved in cognitive processes such as learning and memory and control of movement in normal subjects. Recent data from knockout animals has extended the understanding of nAChR function. Dysfunction of nAChR has been linked to a number of human diseases such as schizophrenia, Alzheimer's and Parkinson's diseases. nAChRs also play a significant role in nicotine addiction, which is a major public health concern. A genetically transmissible epilepsy, ADNFLE, has been associated with specific mutations in the gene coding for the α4 or β2 subunits, which leads to altered receptor properties. Electronic Publication     

The acetylcholine receptor of the adrenal medulla.

Abstract— The acetylcholine receptor of the bovine adrenal medulla was studied by specific binding of [¹²⁵1]α-bungarotoxin to membrane fractions and by perfusion of the isolated gland. The subcellular distribution of the acetylcholine receptor paralleled the distribution of the plasma membrane markers, acetylcholinesterase and calciumstimulated ATPase. The dissociation constant for the binding of α-bungarotoxin to a purified plasma membrane fraction was calculated from Scatchard plots to be 1.6 nM, with a concentration of 190 fmol of binding sites/mg of membrane protein. Correcting for recovery, this corresponds to 0.9 pmol acetylcholine receptor/g adrenal medulla. In decreasing order of effectiveness, d-tubocurarine, nicotine, acetylcholine, carbamylcholine, acetate plus choline, decamethonium, atropine and hexamethonium inhibited binding of α-bungarotoxin. Perfusion experiments showed the acetylcholine receptor to be entirely nicotinic. Stimulation by nicotine was inhibited by atropine and decamethonium, as well as by hexamethonium. Calculated dissociation constants for these antagonist-receptor interactions were in the range of 1 to 3 × 10^?5 m. α-Bungarotoxin failed to inhibit nicotine-stimulated catecholamine release in the perfused adrenal, most likely because of its limited diffusion into the gland.