Tobacco Nitrosamine N-nitrosonornicotine as Inhibitor of Neuronal Nicotinic Acetylcholine Receptors

Nitrosamines are well known for their carcinogenic potential. Recently, it was found that some of them may also interact with human nicotinic acetylcholine receptor (nAChR) subtypes. This work studied the effects of N-nitrosonornicotine (NNN) on recombinant rat α3β4 nAChR in HEK cells as well as on nAChR endogenously expressed in PC12 pheochromocytoma cells and in BC3H1 muscle-type cells. Whole-cell recording in combination with the cell-flow technique for agonist and inhibitor application in the millisecond time region revealed that NNN inhibits the activity of neuronal nAChR expressed in HEK or PC12, whereas weak inhibitory effects on muscle-type nAChR were observed at NNN concentrations up to 3 mM. Pharmacological actions of NNN and the inhibition mechanism were studied in detail using recombinant α3β4 nAChR expressed in HEK cells as a model. NNN-induced inhibition of nicotine-evoked α3β4 nAChR activity was dose-dependent with an inhibitory constant (IC₅₀) of 0.92?±?0.05 mM. Analysis based on mathematical models indicated a noncompetitive inhibition mechanism of the rat α3β4 nAChR by NNN. NNN's mechanism of action involves acceleration of conversion of the receptor from active to desensitized forms. In summary, this work shows that NNN inhibits rat α3β4 nAChR in a noncompetitive way and interacts weakly with muscular nAChR.     

Palmitoylation of Nicotinic Acetylcholine Receptors

It is well established that nicotinic acetylcholine receptors (nAChRs) undergo a number of different posttranslational modifications, such as disulfide bond formation, glycosylation, and phosphorylation. Recently, our laboratory has developed more sensitive assays of protein palmitoylation that have allowed us and others to detect the palmitoylation of relatively low abundant proteins such as ligand-gated ion channels. Here, we present evidence that palmitoylation is prevalent on many subunits of different nAChR subtypes, both muscle-type nAChRs and the neuronal “α₄β₂” and “α₇” subtypes most abundant in brain. The loss of ligand binding sites that occurs when palmitoylation is blocked with the inhibitor bromopalmitate suggests that palmitoylation of α₄β₂ and α₇ subtypes occurs during subunit assembly and regulates the formation of ligand binding sites. However, additional experiments are needed to test whether nAChR subunit palmitoylation is involved in other aspects of nAChR trafficking or whether palmitoylation regulates nAChR function. Further investigation would be aided by identifying the sites of palmitoylation on the subunits, and here we propose a mass spectrometry strategy for identification of these sites.     

乙酰胆碱受体抗体与大鼠中枢神经元烟碱型乙酰胆碱受体之间免疫结合反应

本研究旨在探讨长期以来无定论的重症肌无力（MG）患者血和脑脊液（CSF）中的乙酰胆碱受体抗体（AChRab）能否与中枢神经元烟碱型乙酰胆碱受体（神经-nAChR）结合，并引起中枢神经系统（CNS）功能障碍。用免疫亲和层析法从AChRab阳性的全身型MG患者血中提取纯化AChRab，然后用免疫组化法探讨AChRab与大鼠中枢神经-nAChR之间的免疫结合反应。结果首次表明，AChRab与神经-nAChR之间的阳性免疫结合反应广泛分布于大鼠大脑皮层、脑干颅神经运动核团、脊髓前角运动神经元等部位，提示MG患者AChRab不仅可与神经肌接头（NMJ）处肌-nAChR结合引起肌无力等症状，还可与CNS神经-nAChR结合，并可能引起CNS功能障碍。     

Ethanol Depression of Cerebellar Purkinje Neuron Firing Involves Nicotinic Acetylcholine Receptors

Local application of ethanol (EtOH) has been reported to inhibit Purkinje neuron firing. EtOH-induced depressions can be antagonized by bicuculline, suggesting involvement of GABA_Areceptors. Since there is evidence from other studies indicating that nicotine may interact with EtOH responses, in this study we investigated whether nicotinic acetylcholine receptors (nAChR's) might be also involved in EtOH-induced depressions of these neurons in urethane-anesthetized Sprague–Dawley rats. Using local application (micropressure ejection) of drugs onto cerebellar Purkinje neurons while recording extracellular firing rates, we found that depressant responses to EtOH could be potentiated by subdepressant doses of nicotine. Furthermore, EtOH-induced depressions of firing could be antagonized by mecamylamine, a nicotinic acetylcholine receptor (nAChR) antagonist. Results from the present study indicate that EtOH-induced depressions may involve nAChRs in the cerebellum.     

Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat α₃β₄, chick α_<3/β₂, α₄β₂, α₄β₄, α₇and α₈, expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca²⁺ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of α₈ nAChR had a slope conductance of 23 pS, whereas two conductance classes (19–23 and 32–45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric α₇ and α₈ nAChRs (6–7 ms) than for heteromeric nAChRs (1–3 ms), with the exception of α₃α₄nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (α₃α₄and α₃α₂) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChRs channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.     

Expression of Neuronal Nicotinic Acetylcholine Receptor Subunit Genes in the Rat Autonomic Nervous System

In the autonomic nervous system efferent signals are relayed in sympathetic and parasympathetic ganglia. Fast synaptic transmission between pre- and postsynaptic neurons is achieved by neuronal nicotinic acetylcholine receptors (nAChRs). There is still little known about the subunit composition of these receptors. Establishing the subunit composition of native neuronal nAChRs is important for the understanding of their functional properties both in vivo and after expression in heterologous expression systems. We have combined in situ hybridization and autoradiography to detect the presence of mRNAs encoding subunits of neuronal nAChRs in sympathetic and parasympathetic ganglia. Inspection of the autoradiographs showed that the hybridization signal of five riboprobes (α3, 014–1, α7, β2 and β4) was significantly higher than the unspecific signal obtained with sense riboprobes. The distribution of α7 was tissue-dependent: a7 riboprobe binding was detected in the neurons of the superior cervical ganglion, adrenal medulla and ciliary ganglion. In contrast, the α7 hybridization signal was found only in a small fraction (1 -3%) of the neurons of the sphenopalatine and otic ganglia. Our results are consistent with the idea that α3 mRNA expression levels are somewhat higher than those of α7, α4 -1, β2 and β4.     

Association Study of Polymorphisms in Neuronal Nicotinic Acetylcholine Receptor Subunit Genes With Schizophrenia in the Han Chinese Population

ObjectiveTo investigate the relation between nicotinic acetylcholine receptor subunit (nAChR) genes and schizophrenia, and the relation between tag single nucleotide polymorphism (rs1317286, rs1044396, rs6494212, rs16969968, and rs684513) and schizophrenia in Han Chinese people. MethodsThe protein-protein interaction (PPI) network among nAChR protein and 350 proteins encoded by schizophrenia-related susceptibility genes was constructed through the String database to explore whether nAChR genes were associated with schizophrenia in these known databases. Then, five single nucleotide polymorphisms (SNPs) of CHRNA3 (rs1317286), CHRNA4 (rs1044396), CHRNA7 (rs6494212), and CHRNA5 (rs16969968, rs684513) were analyzed in a sample of 1,035 schizophrenic patients and 816 healthy controls. The interaction between the markers was analyzed using multifactor dimensionality reduction (MDR) software. Power analysis was performed using the Quanto program. ResultsThere are no significant differences in genotype or allele distribution were identified between the patients and controls (p>0.05). The haplotypes constructed by four markers rs1317286, rs6494212, rs16969968, and rs684513 were not associated with schizophrenia either. However, a significant association between models made of rs1317286, rs1044396, rs6494212, and rs684513 and schizophrenia was revealed in interaction analysis (p<0.05). ConclusionThe nAChR protein may have effects on the development of schizophrenia through the interaction with proteins encoded by schizophrenia-related susceptibility genes, but no relation was found between selected polymorphisms and schizophrenia in the collected Han Chinese people. However, interaction analysis suggested four-SNP model has an important effect on schizophrenia.     

Choline is a Selective Agonist of α7 Nicotinic Acetylcholine Receptors in the Rat Brain Neurons

In the present study, we demonstrate that choline, a precursor of acetylcholine (ACh) and a product of acetylcholine hydrolysis by acetylcholinesterase (AChE), acts as an efficient and relatively selective agonist of α7–containing nicotinic acetylcholine receptors (nAChR) in neurons cultured from the rat hippocampus, olfactory bulb and thalamus as well as in PC12 cells. Choline was able to activate postsynaptic and presynaptic α7 nAChRs, with the latter action resulting in the release of other neurotransmitters. Although choline was approximately one order of magnitude less potent than ACh (EC₅₀ of 1.6 mM for choline and 0.13 mM for ACh), it acted as a full agonist at α7 nAChRs. In contrast, choline did not activate α4β2 agonist-bearing nAChRs on hippocampal neurons, and acted as a partial agonist at α3β4-containing nAChRs on PC12 cells. The ethyl alcohol moiety of choline is required for the selective action on α7 nAChR. Exposure of cultured hippocampal neurons for 10 min to choline (10–100 μM) resulted in desensitization of the native α7 nAChRs. Moreover, chronic exposure (10 days) of the cultured hippocampal neurons to a desensitizing concentration of choline (∼30 μM) decreased their responsiveness to ACh. The selective action of choline on native α7 nAChRs suggests that this naturally occurring compound may act in vivo as an endogenous ligand for these receptors. Putative physiological actions of choline include retrograde messenger activity during the development of the mammalian central nervous system and during periods of elevated synaptic activity that leads to long-term potentiation.     

Boundary Lipids In The Nicotinic Acetylcholine Receptor Microenvironment

The structural and functional properties of the nicotinic acetylcholine receptor (AChR), the archetype molecule in the superfamily of Cys-looped ligand-gated ion channels, are strongly dependent on the lipids in the vicinal microenvironment. The influence on receptor properties is mainly exerted by the AChR-vicinal (“shell” or “annular”) lipids, which occur in the liquid-ordered phase as opposed to the more disordered and “fluid” bulk membrane lipids. Fluorescence studies from our laboratory have identified discrete sites for fatty acids, phospholipids, and cholesterol on the AChR protein, and electron-spin resonance spectroscopy has enabled the establishment of the stoichiometry and selectivity of the shell lipid for the AChR and the disclosure of lipid sites in the AChR transmembrane region. Experimental evidence supports the notion that the interface between the protein moiety and the adjacent lipid shell is the locus of a variety of pharmacologically relevant processes, including the action of steroids and other lipids. I surmise that the outermost ring of M4 helices constitutes the boundary interface, most suitable to convey the signals from the lipid microenvironment to the rest of the transmembrane region, and to the channel inner ring in particular.     

Differential Effects of Acetylcholine on Neuronal Activity and Interactions in the Auditory Cortex of the Guinea-pig

During normal brain operations, cortical neurons are subjected to continuous cholinergic modulations. In vitro studies have indicated that, in addition to affecting general cellular excitability, acetylcholine also modulates synaptic transmission. Whether these cholinergic mechanisms lead to a modulation of functional connectivity in vivo is not yet known. Herein, the effects were studied of an iontophoretic application of acetylcholine and of the muscarinic agonist, carbachol, on the ongoing activity and co-activity of neurons simultaneously recorded in the auditory cortex of the anaesthetized guinea-pig. Iontophoresis of cholinergic agonists mainly affected the spontaneous firing rates of auditory neurons, affected autocorrelations less (in most cases their central peak areas were reduced), and rarely affected cross-correlations. These findings are consistent with cholinergic agonists primarily affecting the excitability of cortical neurons rather than the strength of cortical connections. However, when changes of cross-correlations occurred, they were usually not correlated with concomitant changes in average firing rates nor with changes in autocorrelations, which suggests a secondary cholinergic effect on specific cortico-cortical or thalamo-cortical connections.     

Physiological Properties of Neuronal Nicotinic Receptors Reconstituted from the Vertebrate β2 Subunit and Drosophilaα Subunits

Three cDNAs (ALS, Dα2 and ARD) isolated from the nervous system of Drosophila and encoding putative nicotinic acetylcholine receptor subunits were expressed in Xenopus oocytes in order to study their functional properties. Functional receptors could not be reconstituted from any of these subunits taken singly or in twos and threes. In contrast, large evoked currents (in the μA range) were consistently observed upon agonist application on oocytes co-injected with ALS or Dα2 in combination with the chick β2 structural subunit. The ALS/β2 and Dα2/β2 receptors are highly sensitive to acetylcholine and nicotine, and their physiological properties resemble those of native or reconstituted receptors from vertebrates. Although the physiological properties of ALS/β2 and Dα2/β2 receptors are quite similar, clear differences appear in their pharmacological profiles. The ALS/β2 receptor is highly sensitive to α-bungarotoxin while the Dα2/β2 receptor is totally insensitive to this agent. These results demonstrate that the Drosophila ALS and Dα2 cDNAs encode neuronal nicotinic subunits responding to physiological concentrations of the agonists acetylcholine and nicotine.