Nicotinic acetylcholine receptor is involved in acetylcholine regulating stomatal movement

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Inactivation (Desensitization) of the acetylcholine receptor inElectrophorus electricus membrane vesicles by carbamylcholine: Comparison between ion flux and α-bungarotoxin binding

Summary The inactivation (desensitization) of the acetylcholine receptor by carbanylcholine, a stable analogue of acetylcholine, has been investigated in eel Ringer's solution, pH 7.0, 0°C, by measurements of (i) ion flux and (ii) the kinetics of the reaction of [¹²⁵I]--bungarotoxin with the receptor. The effect of preincubation with carbamylcholine is significantly different in the two types of measurement. In both the receptor-controlled flux of inorganic ions and the toxin-binding kinetics a biphasic process has been observed (Hess, G.P., Lipkowitz, S., Struve, G.E., 1978,Proc. Nat. Acad. Sci. USA 75:1703; Hess, G.P. et al., 1975,Biochem. Biophys. Res. Commun. 64: 1018; Bulger, J.E. et al., 1977,Biochemistry 16: 684), only the initial fast phase of which is inhibited and the subsequent slow phase persists. However, preincubation with carbamylcholineper se has no effect on the toxin reaction. The results obtained are consistent with the proposal of Katz and Thesleff (Katz, B., Thesleff, S., 1957,J. Physiol. (London) 138: 65) that the active form of the receptor is converted to an inactive form in the presence of acetylcholine receptor ligands, and with our previous experiments (Hess et al., 1978) which indicated that one receptor form is responsible for the initial fast phase of both the receptor-controlled ion flux and the toxin binding reaction, and that its conversion to the other form results in the slow phases in these two measurements.     

[14C]Chloroacetylcholine as an Advantageous Affinity Label of the Acetylcholine Receptor

Abstract

The alkylating agent [¹⁴C]chloroacetylcholine perchlorate ([¹⁴C] ClACh) was synthesized and used for affinity labelling of the nicotinic acetylcholine receptor from Torpedo marmorata. Solubilized and affinity-purified receptor proteins were reduced and alkylated according to the bromoacetylcholine-method. Covalent binding of [¹⁴C] ClACh to the cholinergic receptor proved to be specific and saturable, and occurred exclusively to the α- subunit. Halogen substitution of acetylcholine by chlorine and insertion of a ¹⁴C-isotope instead of the widely used ³H resulted in favourable properties of the affinity label.     

Antibodies Directed against Functional Sites on the Acetylcholine Receptor from Torpedo Marmorata

Abstract

Antibodies directed against functional sites on the acetylcholine receptor from Torpedo marmorata have been obtained by the following two procedures: (i) Our library of monoclonal antibodies raised against the whole receptor protein was screened for antibodies competing with cholinergic agonists, antagonists and local anesthetics for receptor binding, (ii) antibodies were raised against short peptides matching the sequence of predetermined sites on the receptor protein. In this way, a topographic map of the functional sites on the receptor surface can be constructed.     

Identification of Purine Binding Sites on Torpedo Acetylcholine Receptor

Abstract

Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[³H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the β-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of local anesthetics.     

Introductory Lecture: Allosteric Modulation of Torpedo Nicotinic Acetylcholine Receptor Ion Channel Activity by Noncompetitive Agonists

Abstract

Similar to other neuroreceptors of the vertebrate central nervous system, the nicotinic acetylcholine receptor (nAChR) is subject to modulatory control by allosterically acting ligands. Of particular interest in this regard are allosteric ligands that enhance the sensitivity of the receptor to its natural agonist acetylcholine (ACh), as such ligands could be useful as drugs in diseases associated with impaired nicotinic neurotransmission. Here we discuss the action of a novel class of nAChR ligands which act as allosterically potentiating ligands (APL) on the nicotinic responses induced by ACh and competitive agonists. In addition, APLs also act as noncompetitive agonists of very low efficacy, and as direct blockers of ACh-activated channels. These actions are observed with nAChRs from brain, muscle and electric tissue, and they depend on the structure of the APL and the concentration range applied. We focus here on Torpedo nAChR because (i) the unusual pharmacology of these ligands was first discovered with this system, and (ii) large quantities of this receptor are readily available for biochemical studies.     

烟碱型乙酰胆碱受体在面神经支配的口轮匝肌和躯体神经支配的腓肠肌中的表达差异研究

目的:研究烟碱型乙酰胆碱受体在在面神经支配的口轮匝肌和躯体神经支配的腓肠肌运动终板处的表达差异及可能的原因。方法:分离SD大鼠的口轮匝肌和腓肠肌,通过免疫共沉淀技术计算口轮匝肌和腓肠肌肌肉特异性激酶(Muscle Specific Kinase,MuSK)的表达以及MuSK磷酸化水平。对MuSK的上游信号通路中能使其发生磷酸化的集聚蛋白Agrin、低密度脂蛋白受体相关蛋白4(low-density lipoprotein receptor-related protein 4,Lrp4)以及表皮生长因子家族受体ErbB2、ErbB3和ErbB4(epidermal growth factor receptor)免疫荧光染色,计算这两条不同通路中的蛋白在运动终板处的表达水平。结果:口轮匝肌中MuSK磷酸化水平显著高于腓肠肌(P<0.05)。口轮匝肌与腓肠肌的运动终板处的Agrin和Lrp4表达没有显著差异(P>0.05)。口轮匝肌ErbB2、ErbB3、ErbB4的表达显著高于其在腓肠肌运动终板处的表达(P<0.01)。结论:口轮匝肌和腓肠肌运动终板ErbB、ErbB3、ErbB4的差异表达造成MuSK磷酸化水平不同,可能是两种肌肉运动终板处烟碱型乙酰胆碱亚基表达量不同的原因。     

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.     

Biochemistry and Function of Nematode Steroids

Abstract

Elucidation of the molecular mechanisms that govern ligand-receptor recognition is essential to the rational design of specific pharmacological reagents. Whereas often the receptor and its binding site are the target of investigation, study of the ligand in its free and bound state can also reveal important information regarding this recognition process. Nuclear magnetic resonance (NMR) spectroscopy can be extremely useful for such studies. In this review, we discuss the attributes of NMR in the study of ligand receptor interactions. The cholinergic receptor and its binding to the neurotransmitter, acetylcholine, and cholinergic antagonists serve as a model system, illustrating the power of ligand analysis by NMR. The results discussed prove that the region of residues a 180–205 of the nicotinic acetylcholine receptor are an essential component of the cholinergic binding site and that ligand binding involves a positively charged hydrophobic motif.     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

Initial Characterization of the Nicotinic Acetylcholine Receptors in Rat Hippocampal Neurons

Abstract

The properties of the neuronal nicotinic acetylcholine receptor in primary cultures of hippocampal cells from fetal rats (17–18 days gestation) were studied using the whole-cell patch-clamp technique in Na⁺-external, Cs⁺-internal and nominally Mg²⁺-free solutions. The nicotinic agonists acetylcholine, (+)anatoxin-a, and (-) and (+)nicotine all evoked inward whole-cell currents in hippocampal neurons that were voltage clamped near their resting potentials. Sensitivity to (+)anatoxin-a was first detected at around day 6, and thereafter the magnitude of the response increased as a function of number of days in culture up to about 40 days. The whole-cell current waveforms consisted of more than one peak whose relative amplitude depended on the agonist concentration. These currents were reversibly blocked by micromolar concentrations of d-tubocurarine, mecamylamine, and dihydro-β-erythroidine. At nanomolar concentrations, neuronal bungarotoxin, α-bungarotoxin and α-cobratoxin caused an irreversible blockade of the currents but they were unaffected by tetrodotoxin, atropine, DL-2-amino-5-phosphonovaleric acid, Mg²⁺, and 6,7-dinitroquinoxaline-2,3-dione. In addition, the currents were also blocked in a reversible manner by methyllycaconitine at picomolar concentration. The current-voltage plots elicited by both (+)anatoxin-a and acetylcholine revealed larger inward currents and smaller or no outward currents. The present results demonstrate the existence of an inwardly rectifying, snake neurotoxin-sensitive functional nicotinic acetylcholine receptor ion channel in rat hippocampal neurons.     

Drastic decrease in dopamine receptor levels in the striatum of acetylcholinesterase knock-out mouse

BackgroundThe acetylcholinesterase knock-out mouse lives to adulthood despite 60-fold elevated acetylcholine concentrations in the brain that are lethal to wild-type animals. Part of its mechanism of survival is a 50% decrease in muscarinic and nicotinic receptors and a 50% decrease in adrenoceptor levels.HypothesisThe hypothesis was tested that the dopaminergic neuronal system had also adapted.MethodsRadioligand binding assays measured dopamine receptor level and binding affinity in the striatum. Immunohistochemistry of brain sections with specific antibodies visualized dopamine transporter. Effects on the intracellular compartment were measured as cAMP content, PI-phospholipase C activity.ResultsDopamine receptor levels were decreased 28-fold for the D₁-like, and more than 37-fold for the D₂-like receptors, though binding affinity was normal. Despite these huge changes in receptor levels, dopamine transporter levels were not affected. The intracellular compartment had normal levels of cAMP and PI-phospholipase C activity.ConclusionSurvival of the acetylcholinesterase knock-out mouse could be linked to adaptation of many neuronal systems during development including the cholinergic, adrenergic and dopaminergic. These adaptations balance the overstimulation of cholinergic receptors caused by high acetylcholine concentrations and thus maintain homeostasis inside the cell, allowing the animal to live.     

Monoclonal Anti-Acetycholine Receptor Antibodies as Probes for Human Acetylcholine-Receptor in Myasthenia Gravis

Abstract

Monoclonal antibodies have been shown to bind to five regions on human acetylcholine receptor, each probably consisting of a discrete epitope on the extracellular surface. Two of these regions are equivalent to the ‘main immunogenic region’, and the other three appear to be close to the a-Bungarotoxin binding sites. These antibodies have been used to probe differences in myasthenia gravis anti-acetylcholine receptor antibodies, to locate acetylcholine receptor in thymic tissue, and to look for naturally-occurring anti-idiotype antibodies.

Anti-acetylcholine receptor antibody specificities differ between groups of patients defined by their age of onset, thymic pathology and HLA associations. Anti-AChR synthesised by the thymus in young onset patients has similar specificity to that found in the individual's serum, and may be stimulated by the presence of AChR on thymic myoid cells. However, myoid cells (defined by staining with anti-troponin and anti-myosin antibodies) do not appear to differ between control and myasthenia gravis patients and show no obvious involvement in an immunological reaction.

There was no convincing evidence for the presence of anti-idiotype antibodies in myasthenia gravis patients.     

Minireview: Genetic Manipulations of Cholinergic Communication Reveal Trans-Acting Control Mechanisms Over Acetylcholine Receptors

Abstract

Several approaches have been developed for genetic modulations of receptor expression. These initiated with gene cloning and heterologous expression in microinjected Xenopus oocytes, and proceeded through transgenic expression and genomic disruption of receptor genes in mice. In addition, antisense treatments have reduced receptor levels in a transient, reversible manner. Integration of foreign DNA with host genomic sequences yields both cis- and trans-acting responses. These may depend on the DNA integration site, host cells condition and, most importantly, the affected signal transduction circuit. For example, acetylcholinesterase (AChE) overexpression in microinjected Xenopus tadpoles has been shown to upregulate α-bungarotoxin binding levels, indicating trans-acting control conferring overproduction of muscle nicotinic acetylcholine receptors. In transgenic mice expressing human AChE, the hypothermic response to oxotremorine was suppressed, reflecting modified levels of brain muscarinic receptors. To dissociate the feedback processes occurring in transfected cells from responses related to DNA integration, we examined the endogenous expression of the α₇ neuronal nicotinic acetylcholine receptor in PC 12 cells transfected with DNA vectors carrying alternative splicing variants of human AChE mRNA. Our findings demonstrate suppression of α₇ receptor levels associated with the accumulation of foreign DNA in the transfected cells. Acetylcholine receptor levels thus depend on multiple elements, each of which should be considered when genetic interventions are employed.     

M1胆碱受体选择性别构激动剂的虚拟筛选和体外活性研究

目的:M1毒蕈碱型乙酰胆碱受体(M1受体)在改善学习和记忆等高级认知功能障碍中起重要作用,本文利用计算机辅助药物设计和高表达各M受体亚型的CHO细胞(Chinese hamster ovary cell,中国仓鼠卵巢细胞),以期筛选获得新型M1受体选择性别构激动剂。方法:通过计算机辅助药物设计方法,对已知具有M1受体选择性作用别构激动剂与M1受体的晶体结构进行对接,确定活性对接口袋,据此进行化合物库虚拟筛选;利用高表达各M受体亚型的CHO细胞,对化合物进行体外活性检测。结果:虚拟筛选得到184个化合物,其中,体外实验显示化合物AJ-292和AG-205-6对M1受体有明显的激动效果,而对M3、M5受体则无影响。结论:综合利用虚拟筛选、结构分析以及特异性活性分析,筛选出具有M1受体高选择性激动作用的化合物AJ-292和AG-205-6,为设计开发新型的M1选择性别构激动剂奠定了基础。     

Molecular Cloning of the Antagonist-Binding Subunit of the Glycine Receptor

Abstract

The postsynaptic receptor for the inhibitory neurotransmitter glycine is a heterooligomeric membrane protein which, after affinity purification on an antagonist column, contains three polypeptides of 48K, 58K and 93K. Sequencing of cDNA clones of the antagonist-binding 48K subunit revealed a structural organization similar to and significant amino acid homology with nicotinic acetylcholine receptor proteins. Our data suggest the existence of a set of related genes encoding transmembrane channel-forming neurotransmitter receptor polypeptides of excitable membranes.     

Comparative study of chemical pathology sample collection tubes at the largest hospital in South Africa

BackgroundBarricor^TM Lithium heparin plasma tubes are new blood tubes that have been introduced to overcome the effects of gel in serum separator tubes (SST) and the shortcomings of standard Lithium heparin plasma. We aimed to evaluate Barricor^TM tubes as an alternative to serum separator tubes and compare the stability between the tubes.MethodsForty-four paired samples were collected using both Barricor^TM and SST. We compared five analytes at baseline (<6 h) and after every 24 h using the PassingBablok and Bland-Altman plots. Aspartate aminotransferase (AST), potassium (K), phosphate (PO4) , lactate dehydrogenase (LDH), and creatinine were analysed in both tubes. We calculated the percentage difference for each analyte between the baseline and time intervals to assess analyte stability. The percentage difference was compared to the desirable specification for bias and reference change value (RCV).ResultsAll analytes were comparable at baseline. Statistical differences (p<0.001) became evident after 24 h. PO4, K, and creatinine had a mean difference that exceeded the desirable specification for bias (-9.59%, - 9.35%, and -4.59%, respectively). Potassium was stable up to 24 h in both tubes. LDH showed better stability in SST (144 h vs 96 h). PO4 concentrations were more stable in both tubes with the SST (96 h vs 72 h). Creatinine and AST had the longest stability in both tubes compared to other analytes (144 h).ConclusionsData demonstrated variability and similarities in analyte concentrations and stability, respectively, in both tubes.     

Separation of Muscarinic Acetylcholine Receptor Entities from Rat Cerebral Cortex by Ion Exchange Chromatography

Abstract

Digitonin solubilized muscarinic acetylcholine receptor (mAChR) of rat cerebral cortex membranes were chromatographed on the FPLC anion exchanger Mono Q. [³H]QNB (quinuclidinyl benzilate) and [³H]PZ (pirenzepine) binding activity was retarded from a NaCl free elution buffer and thereby separated from a part of the accompanying proteins. Elution of the column with a continuously increasing NaCl concentration desorbed the radioligand binding activities forming several peaks, two of which were nearly completely separated. Our data show that the mAChR in rat cerebral cortex consists of several entities with different electrical charges.     

Nicotinic Acetylcholine Receptors have Ligand-Specific Attachment Point Patterns

Abstract

Employing a panel of synthetic peptides as representative structural elements of the nicotinic acetylcholine receptor from Torpedo electric organ, we recently identified three sequence regions of the receptor (α55–74, α134–153 and α181–200) serving as subsites for the binding of high molecular weight antagonists of acetylcholine (Conti-Tronconi et al. 1990). The relative binding affinities to these subsites of α-bungarotoxin and three competitive antibodies varied in a ligand-specific fashion. Employing a set of homologous synthetic peptides differing from α181–200 by the exchange of single amino acid residues along the sequence, we now find that ligand binding crucially depends on the presence of particular amino acids within the subsite while others influence binding only marginally if at all. The existence of ligand-specific attachment points may account for the wide range in binding and kinetic parameters, pharmacological specificity and distinct mean open times of the receptor-integral cation channel observed for cholinergic ligands.     

Independent sites of low and high affinity for agonists on Torpedocalifornica acetylcholine receptor

It is demonstrated that two classes of binding site for acetylcholine are present on $\text{Torpedo}\text{californica}$ acetylcholine receptor. One class is the well documented site on each of the two subunits of 40,000 daltons, which can be covalently modified by bromocetylcholine. Both in the absence and in the presence of bromoacetylcholine another binding site is shown to exist by virtue of acetylcholine dependent fluorescence changes in the receptor covalently modified by 4-[N-(iodoacetoxy)ethyl-N-methyl]-amino-7-Nitrobenz-2-oxa-1,3 diazole (IANBD). This site has a low affinity for acetylcholine (K_d ～ 80 μM) that corresponds closely with the known concentration dependence of acetylcholine mediated activation of this receptor and we conclude that it may represent a site of association that participates in channel opening in this system.