Home About us Contact
|
Home About us Contact | ||
|
|
||
nAChR Subunits (nachr + subunit)
Selected AbstractsActivity of nAChRs containing ,9 subunits modulates synapse stabilization via bidirectional signaling programsDEVELOPMENTAL NEUROBIOLOGY, Issue 14 2009Vidya Murthy Abstract Although the synaptogenic program for cholinergic synapses of the neuromuscular junction is well known, little is known of the identity or dynamic expression patterns of proteins involved in non-neuromuscular nicotinic synapse development. We have previously demonstrated abnormal presynaptic terminal morphology following loss of nicotinic acetylcholine receptor (nAChR) ,9 subunit expression in adult cochleae. However, the molecular mechanisms underlying these changes have remained obscure. To better understand synapse formation and the role of cholinergic activity in the synaptogenesis of the inner ear, we exploit the nAChR ,9 subunit null mouse. In this mouse, functional acetylcholine (ACh) neurotransmission to the hair cells is completely silenced. Results demonstrate a premature, effusive innervation to the synaptic pole of the outer hair cells in ,9 null mice coinciding with delayed expression of cell adhesion proteins during the period of effusive contact. Collapse of the ectopic innervation coincides with an age-related hyperexpression pattern in the null mice. In addition, we document changes in expression of presynaptic vesicle recycling/trafficking machinery in the ,9 null mice that suggests a bidirectional information flow between the target of the neural innervation (the hair cells) and the presynaptic terminal that is modified by hair cell nAChR activity. Loss of nAChR activity may alter transcriptional activity, as CREB binding protein expression is decreased coincident with the increased expression of N-Cadherin in the adult ,9 null mice. Finally, by using mice expressing the nondesensitizing ,9 L9,T point mutant nAChR subunit, we show that increased nAChR activity drives synaptic hyperinnervation. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] The Caenorhabditis elegans lev-8 gene encodes a novel type of nicotinic acetylcholine receptor , subunitJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Paula R. Towers Abstract We have cloned Caenorhabditis elegans lev-8 and demonstrated that it encodes a novel nicotinic acetylcholine receptor (nAChR) subunit (previously designated ACR-13), which has functional roles in body wall and uterine muscles as part of a levamisole-sensitive receptor. LEV-8 is an ,,subunit and is the first to be described from the ACR-8-like group, a new class of nAChR with atypical acetylcholine-binding site (loop C) and channel-lining motifs. A single base pair change in the first intron of lev-8 in lev-8(x15) mutants leads to alternative splicing and the introduction of a premature stop codon. lev-8(x15) worms are partially resistant to levamisole-induced egg laying and paralysis, phenotypes rescued by expression of the wild-type gene. lev-8(x15) worms also show reduced rates of pharyngeal pumping. Electrophysiological recordings from body wall muscle show that currents recorded in response to levamisole have reduced amplitude in lev-8(x15) compared with wild-type animals. Consistent with these phenotypic observations, green fluorescent protein fused to LEV-8 is expressed in body wall and uterine muscle, motor neurons and epithelial-derived socket cells. Thus, LEV-8 is a levamisole receptor subunit and exhibits the most diverse expression pattern of any invertebrate nAChR subunit studied to date. [source] A Polymorphism in the ,4 Nicotinic Receptor Gene (Chrna4) Modulates Enhancement of Nicotinic Receptor Function by EthanolALCOHOLISM, Issue 5 2003Christopher M. Butt Background: Several studies indicate that ethanol enhances the activity of ,4,2 nicotinic acetylcholine receptors (nAChR). Our laboratory has identified a polymorphism in the ,4 gene that results in the substitution of an alanine (A) for threonine (T) at amino acid position 529 in the second intracellular loop of the ,4 protein. Mouse strains expressing the A variant have, in general, greater nAChR-mediated 86Rb+ efflux in response to nicotine than strains with the T variant. However, the possibility of the polymorphism modulating the effects of ethanol on the 86Rb+ efflux response has not been investigated. Methods: We have used the 86Rb+ efflux method to study the acute effects of ethanol on the function of the ,4,2 nAChR in the thalamus in six different mouse strains. Experiments were also performed on tissue samples taken from F2 intercross animals. The F2 animals were derived from A/J mice crossed with a substrain of C57BL/6J mice that carried a null mutation for the gene encoding the ,2 nAChR subunit. Results: In strains carrying the A polymorphism (A/J, AKR/J, C3H/Ibg), coapplication of ethanol (10,100 mM) with nicotine (0.03,300 ,M) increased maximal ion flux when compared with nicotine alone with no effect on agonist potency. In contrast, ethanol had little effect on the nicotine concentration-response curve in tissue prepared from strains carrying the T polymorphism (Balb/Ibg, C57BL/6J, C58/J). Experiments with the F2 hybrids demonstrated that one copy of the A polymorphism was sufficient to produce a significant enhancement of nAChR function by ethanol (50 mM) in animals that were also ,2 +/+. Ethanol had no effect on nicotine concentration-response curves in T/T ,2 +/+ animals. Conclusions: The results suggest that the A/T polymorphism influences the initial sensitivity of the ,4,2 nAChR to ethanol. [source] Aromatic residues at position 55 of rat ,7 nicotinic acetylcholine receptors are critical for maintaining rapid desensitizationTHE JOURNAL OF PHYSIOLOGY, Issue 4 2008Elaine A. Gay The rat ,7 nicotinic acetylcholine receptor (nAChR) can undergo rapid onset of desensitization; however, the mechanisms of desensitization are largely unknown. The contribution of a tryptophan (W) residue at position 55 of the rat ,7 nAChR subunit, which lies within the ,2 strand, was studied by mutating it to other hydrophobic and/or aromatic amino acids, followed by voltage-clamp experiments in Xenopus oocytes. When mutated to alanine, the ,7-W55A nAChR desensitized more slowly, and recovered from desensitization more rapidly, than wildtype ,7 nAChRs. The contribution of desensitization was validated by kinetic modelling. Mutating W55 to other aromatic residues (phenylalanine or tyrosine) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (alanine, cysteine or valine) significantly decreased the rate of onset and increased the rate of recovery from desensitization. To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of W55 by mutating W55 to cysteine (,7-W55C) and testing the ability of various sulfhydryl reagents to react with this cysteine. Several positively charged sulfhydryl reagents blocked ACh-induced responses for ,7-W55C nAChRs, whereas a neutral sulfhydryl reagent potentiated responses; residue C55 was not accessible for modification in the desensitized state. These data suggest that W55 plays an important role in both the onset and recovery from desensitization in the rat ,7 nAChR, and that aromatic residues at position 55 are critical for maintaining rapid desensitization. Furthermore, these data suggest that W55 may be a potential target for modulatory agents operating via hydrophobic interactions. [source] A New Chrna4 Mutation with Low Penetrance in Nocturnal Frontal Lobe EpilepsyEPILEPSIA, Issue 7 2003Tobias Leniger Summary: Purpose: To identify and characterize the mutation(s) causing nocturnal frontal lobe epilepsy in a German extended family. Methods: Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes were screened by direct sequencing. Once a CHRNA4 mutation was identified, its biophysical and pharmacologic properties were characterized by expression experiments in Xenopus oocytes. Results: We report a new CHRNA4 mutation, causing a ,4-T265I amino acid exchange at the extracellular end of the second transmembrane domain (TM). Functional studies of ,4-T265I revealed an increased ACh sensitivity of the mutated receptors. ,4-T265I is associated with an unusual low penetrance of the epilepsy phenotype. Sequencing of the TM1-TM3 parts of the 1 known nAChR subunits did not support a two-locus model involving a second nAChR sequence variation. Conclusions: nAChR mutations found in familial epilepsy are not always associated with an autosomal dominant mode of inheritance. ,4-T265I is the first nAChR allele showing a markedly reduced penetrance consistent with a major gene effect. The low penetrance of the mutation is probably caused by unknown genetic or environmental factors or both. [source] Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2003L. M. Marubio Abstract The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the ,4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the ,4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125I ,-conotoxin MII. These receptors are comprised of the ,4, ,2 and ,6 nAChR subunits and non-,4, ,2, and ,6 nAChR subunits. Non-,4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and ,4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and non-habituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that ,4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs. [source] Localization of nAChR subunit mRNAs in the brain of Macaca mulattaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000Zhi-Yan Han Abstract We present here a systematic mapping of nAChR subunit mRNAs in Macaca mulatta brain. A fragment, from the transmembrane segments MIII to MIV of Macaca neuronal nAChR subunits was cloned, and shown to exhibit high identity (around 95%) to the corresponding human subunits. Then, specific oligodeoxynucleotides were synthesized for in situ hybridization experiments. Both ,4 and ,2 mRNA signals were widely distributed in the brain, being stronger in the thalamus and in the dopaminergic cells of the mesencephalon. Most brain nuclei displayed both ,4 and ,2 signals with the exception of some basal ganglia regions and the reticular thalamic nucleus which were devoid of ,4 signal. ,6 and ,3 mRNA signals were selectively concentrated in the substantia nigra and the medial habenula. The strongest signals for ,3 or ,4 mRNAs were found in the epithalamus (medial habenula and pineal gland), whereas there were no specific ,3 or ,4 signals in mesencephalic dopaminergic nuclei. ,5 and ,7 mRNA signals were found in several brain areas, including cerebral cortex, thalamus and substantia nigra, although at a lower level than ,4 and ,2. The distribution of ,3, ,4, ,5, ,6, ,7, ,2, ,3 and ,4 subunit mRNAs in the monkey is substantially similar to that observed in rodent brain. Surprisingly, ,2 mRNA signal was largely distributed in the Macaca brain, at levels comparable with those of ,4 and ,2. This observation represents the main difference between rodent and Macaca subunit mRNA distribution and suggests that, besides ,4,2*, ,2,2* nAChRs constitute a main nAChR isoform in primate brain. [source] Cholinergic modulation of angiogenesis: Role of the 7 nicotinic acetylcholine receptorJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Jenny C.F. Wu Abstract Pathological angiogenesis contributes to tobacco-related diseases such as malignancy, atherosclerosis and age-related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except ,2, ,4, ,, and , in four different types of ECs. Using siRNA methodology, we found that the ,7 nAChR plays a dominant role in nicotine-induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine-activated EC functions (proliferation, survival, migration, and tube formation). The ,9 and ,7 nAChRs have opposing effects on nicotine-induced cell proliferation and survival. Our studies reveal a critical role for the ,7 nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis. J. Cell. Biochem. 108: 433,446, 2009. © 2009 Wiley-Liss, Inc. [source] Role of endogenous acetylcholine in the control of the dopaminergic system via nicotinic receptorsJOURNAL OF NEUROCHEMISTRY, Issue 3 2010Uwe Maskos J. Neurochem. (2010) 114, 641,646. Abstract Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane protein receptors activated by the addictive drug, nicotine. However, sometimes underestimated, under physiological conditions the endogenous neurotransmitter acetylcholine is the agonist. In this mini-review, I will discuss the evidence in favour of an important role for this cholinergic activation of the dopaminergic (DAergic) system. I will focus on the literature implicating the action of acetylcholine on the somato-dendritic compartment of these neurons. This modulation is responsible for a variety of phenotypes in knock-out animals of nAChR subunits. These include locomotion, exploratory behaviour, dopamine (DA) release, and DA neuron firing patterns. The novel techniques brought to bear on these analyses, lentiviral re-expression, and repression, of nAChR subunits, and transgenic expression of hypersensitive receptors will be discussed. [source] Amino acids outside of the loops that define the agonist binding site are important for ligand binding to insect nicotinic acetylcholine receptorsJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Zewen Liu Abstract Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the , subunits and another three loops (D, E, and F) of the non-, subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-, subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens, a target-site mutation (Y151S) was found within two , subunits (Nl,1 and Nl,3). In Drosophila S2 cells and Xenopus oocytes, Nl,1 can form functional receptors with rat ,2 subunit. However, the same thing was not observed in Nl,3. In the present paper, by exchanging the corresponding regions between Nl,1 and Nl,3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding. [source] Nicotinic acetylcholine receptor-subunit mRNAs in the mouse superior cervical ganglion are regulated by development but not by deletion of distinct subunit genesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2008G. Putz Abstract Mice with deletions of nicotinic ACh receptor (nAChR) subunit genes are valuable models for studying nAChR functions. We could previously show in the mouse superior cervical ganglion (SCG) that the absence of distinct subunits affects the functional properties of receptors. Here, we have addressed the question of whether deletions of the subunits ,5, ,7, or ,2 are compensated at the mRNA level, monitored by reverse transcription and quantitative real-time polymerase chain reaction. Relative to our reference gene, ,3, which is expressed in all SCG nAChRs, mRNA levels of ,4 showed little change from birth until adult ages in intact ganglia of wild-type mice. In contrast, ,4 declined sharply after birth and was barely detectable in adult animals. ,5, ,7, and ,2 subunit message levels also declined, though more slowly and less completely than ,4. The subunits ,6 and ,3 were detected by conventional polymerase chain reaction at very low levels, if at all, whereas ,2 was never seen in any of our samples. The developmental profile of nAChR mRNA levels in the three knockout strains did not differ markedly from that of wild-type mice. Likewise, message levels of nAChR subunits were similar in cultures prepared from either wild-type or knockout animals. Our observations indicate a developmental regulation of nAChR subunit mRNAs in the SCG of mice after birth that was not affected by the three knockouts under investigation. © 2007 Wiley-Liss, Inc. [source] An increase in intracellular free calcium ions by nicotinic acetylcholine receptors in a single cultured rat cortical astrocyteJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2005Hirotaka Oikawa Abstract Neuronal nicotinic acetylcholine receptors (nAChRs) are composed of an assembly between at least seven alpha (,2,,7, ,9) and three beta (,2,,4) subunits in mammals. The addition of 50 mM KCl or 1 mM nicotine immediately increased the number of cells with high fluorescence intensity in rat cortical astrocytes on fluo-3 fluorescence measurement. Nicotine was effective at increasing the fluorescence intensity in astrocytes cultured for 2 days after replating, but not in those used 1 or 5 days after replating, without markedly affecting the cellular viability irrespective of the exposure period. Nicotine markedly increased the fluorescence intensity in a concentration-dependent manner at a concentration range of 10,100 ,M in cultured astrocytes when analyzed on a responsive single cell. In these responsive single cells, the increase by nicotine was significantly prevented by the heteromeric ,4/,2 subtype antagonist dihydro-,-erythroidine and the homomeric ,7 subtype antagonist methyllycaconitine, as well as by nifedipine and EGTA but not thapsigargin. Methyllycaconitine failed to inhibit further the increase by nicotine in the presence of nifedipine, however, whereas the expression of mRNA was seen for all mammalian neuronal nAChR subunits in cultured rat cortical astrocytes as well as neurons. These results suggest that nicotine may increase intracellular free Ca2+ through the influx of extracellular Ca2+ across L-type voltage-gated Ca2+ channels rather than Ca2+ release from intracellular stores, in a manner related to the ,4/,2 and/or ,7 nAChR channels functionally expressed in cultured rat cortical astrocytes. © 2005 Wiley-Liss, Inc. [source] Acetylcholine and Alcohol Sensitivity of Neuronal Nicotinic Acetylcholine Receptors: Mutations in Transmembrane DomainsALCOHOLISM, Issue 12 2002Cecilia M. Borghese Background The effect of n-alcohols on glycine and ,-aminobutyric acid type A receptors depends on two specific amino acids (AAs) located in the transmembrane domains (TM) 2 and 3. Our aim was to assess whether the corresponding AAs in the neuronal nicotinic acetylcholine receptor (nAChR) also formed a binding pocket for alcohols. Methods We made single AA substitutions in the homologous sites in rat neuronal nAChR ,2 and ,4 (,L261 and ,L283) and expressed them in Xenopus laevis oocytes in combination with ,4 wild type. The effect of different n-alcohols was studied in ,4(L261A),4 and ,4(L283A),4 nAChRs. The effect of ethanol, propanol, and octanol on acetylcholine (ACh) responses was studied in ,2(L261X),4 and ,2(L283X),4 nAChRs. Results Most of the mutations in the ,2 subunit, in either the 261 or the 283 position, induced changes in ACh sensitivity and increased alcohol action, but none was able to reduce ethanol potentiation. In ,4(L283A),4, enhancement of potentiation by short-chain alcohols was observed, as well as a change from inhibition to potentiation for long-chain alcohols. The exposure of the AAs was assessed through the action of a charged thiol-specific reagent on ,2(L261C),4 and ,2(L283C),4, and these experiments suggest that the AA in TM2 is located in a water-accessible position, whereas the AA in TM3 is inaccessible. However, a noncharged thiol-specific reagent did not affect either ACh responses or ethanol effect on ,2(L261C),4. Conclusions The AAs located at positions 261 and 283 of the ,2 and ,4 nAChR subunits do not seem to form a binding pocket for alcohols. Additional studies are required to determine whether alcohols act on a site near these AAs or on sites unrelated to the TM2-TM3 site found in glycine and ,-aminobutyric acid type A receptors. [source] Reduced expression of nicotinic , subunits 3, 7, 9 and 10 in lesional and nonlesional atopic dermatitis skin but enhanced expression of , subunits 3 and 5 in mast cellsBRITISH JOURNAL OF DERMATOLOGY, Issue 4 2008F. Kindt Summary Background, The skin cholinergic signalling system is modulated in atopic dermatitis (AD). Objectives, To investigate of the role of nicotinic acetylcholine receptors (nAChRs) in the pathogenesis of AD. Methods, We investigated the expression and localization of nAChR , subunits in AD by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry of biopsies from lesional and nonlesional areas of AD skin and of skin biopsies from healthy control persons. Results, Our data demonstrate the presence of mRNA and protein of the nAChR , subunits 3, 5, 7, 9 and 10 in keratinocytes and mast cells in healthy and AD skin. Expression of the , subunits 3, 7, 9 and 10 was generally reduced in the skin of patients with AD whereas mast cells in AD but not in healthy skin showed ,3 and ,5 subunit immunoreactivity. Differences in the subunit mRNA levels between lesional and nonlesional skin were obtained for the , subunits 3, 9 and 10 with higher levels of ,3 but lower levels of ,10 subunit mRNA in lesional areas. No differences in the expression of the , subunits was found between the groups of extrinsic, intrinsic or mixed AD types, between genders and between smokers and nonsmokers. Conclusions, This supports the idea that the cholinergic system is dysregulated independently from inflammation in AD and that inflammation further modulates individual nAChR subunits. [source] Region-specific effects of N,N,-dodecane-1,12-diyl-bis-3-picolinium dibromide on nicotine-induced increase in extracellular dopamine in vivoBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2008S Rahman Background and purpose: Systemic administration of N,N,-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), an antagonist of nicotinic acetylcholine receptors (nAChRs) attenuated the nicotine-induced increase in dopamine levels in nucleus accumbens (NAcc). Experimental approach: Using in vivo microdialysis, we investigated the effects of local perfusion of the novel nAChR antagonist bPiDDB into the NAcc or ventral tegmental area (VTA) on increased extracellular dopamine in NAcc, induced by systemic nicotine. We also examined the concentration-dependent effects of bPiDDB on the acetylcholine (ACh)-evoked response of specific recombinant neuronal nAChR subtypes expressed in Xenopus oocytes, using electrophysiological methods. Key results: Nicotine (0.4 mg kg,1, s.c.) increased extracellular dopamine in NAcc, which was attenuated by intra-VTA perfusion of mecamylamine (100 ,M). Intra-VTA perfusion of bPiDDB (1 and 10 ,M) reduced nicotine-induced increases in extracellular dopamine in NAcc. In contrast, intra-NAcc perfusion of bPiDDB (1 or 10 ,M) failed to alter the nicotine-induced increase in dopamine in NAcc. Intra-VTA perfusion of bPiDDB alone did not alter basal dopamine levels, compared to control, nor the increased dopamine in NAcc following amphetamine (0.5 mg kg,1, s.c.). Using Xenopus oocytes, bPiDDB (0.01,100 ,M) inhibited the response to ACh on specific combinations of rat neuronal nAChR subunits, with highest potency at ,3,4,3 and lowest potency at ,6/3,2,3. Conclusions and implications: bPiDDB-Sensitive nAChRs involved in regulating nicotine-induced dopamine release are located in the VTA, rather than in the NAcc. As bPiDDB has properties different from the prototypical nAChR antagonist mecamylamine, further development may lead to novel nAChR antagonists for the treatment of tobacco dependence. British Journal of Pharmacology (2008) 153, 792,804; doi:10.1038/sj.bjp.0707612; published online 3 December 2007 [source] | |||||||||||||