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Sample records for acetylcholine receptor expression

  1. Functional expression of human α7 nicotinic acetylcholine receptor in human embryonic kidney 293 cells.

    PubMed

    Gong, Yuan; Jiang, Ji-Hong; Li, Shi-Tong

    2016-09-01

    The functional expression of recombinant α7 nicotinic acetylcholine receptors in human embryonic kidney (HEK) 293 cells has presented a challenge. Resistance to inhibitors of cholinesterase 3 (RIC‑3) has been confirmed to act as a molecular chaperone of nicotinic acetylcholine receptors. The primary objectives of the present study were to investigate whether the co‑expression of human (h)RIC‑3 with human α7 nicotinic acetylcholine receptor in HEK 293 cells facilitates functional expression of the α7 nicotinic acetylcholine receptor. Subsequent to transfection, western blotting and polymerase chain reaction were used to test the expression of α7 nicotinic acetylcholine receptor and RIC-3. The α7 nicotinic acetylcholine receptor was expressed alone or co‑expressed with hRIC‑3 in the HEK 293 cells. Drug‑containing solution was then applied to the cells via a gravity‑driven perfusion system. Calcium influx in the cells was analyzed using calcium imaging. Nicotine did not induce calcium influx in the HEK 293 cells expressing human α7 nicotinic acetylcholine receptor only. However, in the cells co‑expressing human RIC‑3 and α7 nicotinic acetylcholine receptor, nicotine induced calcium influx via the α7 nicotinic acetylcholine receptor in a concentration‑dependent manner (concentration required to elicit 50% of the maximal effect=29.21 µM). Taken together, the results of the present study suggested that the co‑expression of RIC‑3 in HEK 293 cells facilitated the functional expression of the α7 nicotinic acetylcholine receptor. PMID:27430244

  2. Increased expression of the nicotinic acetylcholine receptor in stimulated muscle.

    PubMed

    O'Reilly, Clare; Pette, Dirk; Ohlendieck, Kay

    2003-01-10

    Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts. PMID:12504123

  3. Expression of somatostatin receptor genes and acetylcholine receptor development in rat skeletal muscle during postnatal development.

    PubMed

    Peng, M; Conforti, L; Millhorn, D E

    1998-05-01

    Our laboratory reported previously that somatostatin (SST) is transiently expressed in rat motoneurons during the first 14 days after birth. We investigated the possibility that the SST receptor (SSTR) is expressed in skeletal muscle. We found that two of the five subtypes of SSTR (SSTR3 and SSTR4) are expressed in skeletal muscle with a time course that correlates with the transient expression of SST in motoneurons. In addition, SSTR2A is expressed from birth to adulthood in skeletal muscle. Both SSTR2A and SSTR4 are also expressed in L6 cells, a skeletal muscle cell line. Somatostatin acting through its receptors has been shown to stimulate tyrosine phosphatase activity in a number of different tissues. We found that several proteins (50, 65, 90, 140, 180 and 200 kDa) exhibited a reduced degree of tyrosine phosphorylation following SST treatment. Inhibition of tyrosine phosphatase activity with sodium orthovanadate increased expression of the nicotinic acetyl-choline receptor (nAChR) epsilon subunit mRNA by three fold. Somatostatin reversed the elevated epsilon mRNA following orthovanadate treatment. These findings show that SSTR is expressed in skeletal muscle and that SST acting via the SSTR regulates tyrosine phosphorylation and expression of the epsilon subunit of the AChR in the rat skeletal muscle. PMID:9852305

  4. Functional Expression of Two Neuronal Nicotinic Acetylcholine Receptors from cDNA Clones Identifies a Gene Family

    NASA Astrophysics Data System (ADS)

    Boulter, Jim; Connolly, John; Deneris, Evan; Goldman, Dan; Heinemann, Steven; Patrick, Jim

    1987-11-01

    A family of genes coding for proteins homologous to the α subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by α -bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

  5. Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia

    SciTech Connect

    Weiner, D.M. Howard Hughes Medical Inst., Bethesda, MD ); Levey, A.I. Johns Hopkins Univ., Baltimore, MD ); Brann, M.R. )

    1990-09-01

    Within the basal ganglia, acetylcholine and dopamine play a central role in the extrapyramidal control of motor function. The physiologic effects of these neurotransmitters are mediated by a diversity of receptor subtypes, several of which have now been cloned. Muscarinic acetylcholine receptors are encoded by five genes (m1-m5), and of the two known dopamine receptor subtypes (D1 and D2) the D2 receptor gene has been characterized. To gain insight into the physiological roles of each of these receptor subtypes, the authors prepared oligodeoxynucleotide probes to localize receptor subtype mRNAs within the rat striatum and substantia nigra by in situ hybridization histochemistry. Within the striatum, three muscarinic (m1, m2, m4) receptor mRNAs and the D2 receptor mRNA were detected. The m1 mRNA was expressed in most neurons; the m2 mRNA, in neurons which were both very large and rare; and the m4 and D2 mRNAs, in 40-50% of the neurons, one-third of which express both mRNAs. Within the substantia nigra, pars compacta, only the m5 and D2 mRNAs were detected, and most neurons expressed both mRNAs. These data provide anatomical evidence for the identity of the receptor subtypes which mediate the diverse effects of muscarinic and dopaminergic drugs on basal ganglia function.

  6. Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system

    SciTech Connect

    Wadsworth, S.C.; Rosenthal, L.S.; Kammermeyer, K.L.; Potter, M.B.; Nelson, D.J.

    1988-02-01

    The authors isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobasae poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.

  7. Muscarinic Acetylcholine Receptors in Macaque V1 Are Most Frequently Expressed by Parvalbumin-Immunoreactive Neurons

    PubMed Central

    Disney, Anita A.; Aoki, Chiye

    2010-01-01

    Acetylcholine (ACh) is believed to underlie mechanisms of arousal and attention in mammals. ACh also has a demonstrated functional effect in visual cortex that is both diverse and profound. We have reported previously that cholinergic modulation in V1 of the macaque monkey is strongly targeted toward GABAergic interneurons. Here we examine the localization of m1 and m2 muscarinic receptor subtypes across subpopulations of GABAergic interneurons—identified by their expression of the calcium-binding proteins parvalbumin, calbindin, and calretinin—using dual-immunofluorescence confocal microscopy in V1 of the macaque monkey. In doing so, we find that the vast majority (87%) of parvalbumin-immunoreactive neurons express m1-type muscarinic ACh receptors. m1 receptors are also expressed by 60% of calbindin-immunoreactive neurons and 40% of calretinin-immunoreactive neurons. m2 AChRs, on the other hand, are expressed by only 31% of parvalbumin neurons, 23% of calbindin neurons, and 25% of calretinin neurons. Parvalbumin-immunoreactive cells comprise ≈75% of the inhibitory neuronal population in V1 and included in this large subpopulation are neurons known to veto and regulate the synchrony of principal cell spiking. Through the expression of m1 ACh receptors on nearly all of these PV cells, the cholinergic system avails itself of powerful control of information flow through and processing within the network of principal cells in the cortical circuit. PMID:18265004

  8. Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

    PubMed Central

    Marshall, J; Buckingham, S D; Shingai, R; Lunt, G G; Goosey, M W; Darlison, M G; Sattelle, D B; Barnard, E A

    1990-01-01

    We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA. PMID:1702381

  9. Nicotinic acetylcholine receptor expression in human airway correlates with lung function.

    PubMed

    Lam, David Chi-Leung; Luo, Susan Yang; Fu, Kin-Hang; Lui, Macy Mei-Sze; Chan, Koon-Ho; Wistuba, Ignacio Ivans; Gao, Boning; Tsao, Sai-Wah; Ip, Mary Sau-Man; Minna, John Dorrance

    2016-02-01

    Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChRs) on bronchial epithelial cells, can regulate cellular signaling and inflammatory processes. Delineation of nAChR subtypes and their responses to nicotine stimulation in bronchial epithelium may provide information for therapeutic targeting in smoking-related inflammation in the airway. Expression of nAChR subunit genes in 60 bronchial epithelial biopsies and immunohistochemical staining for the subcellular locations of nAChR subunit expression were evaluated. Seven human bronchial epithelial cell lines (HBECs) were exposed to nicotine in vitro for their response in nAChR subunit gene expression to nicotine exposure and removal. The relative normalized amount of expression of nAChR α4, α5, and α7 and immunohistochemical staining intensity of nAChR α4, α5, and β3 expression showed significant correlation with lung function parameters. Nicotine stimulation in HBECs resulted in transient increase in the levels of nAChR α5 and α6 but more sustained increase in nAChR α7 expression. nAChR expression in bronchial epithelium was found to correlate with lung function. Nicotine exposure in HBECs resulted in both short and longer term responses in nAChR subunit gene expression. These results gave insight into the potential of targeting nAChRs for therapy in smoking-related inflammation in the airway. PMID:26608528

  10. Effects of varenicline on alpha4-containing nicotinic acetylcholine receptor expression and cognitive performance in mice.

    PubMed

    Lange-Asschenfeldt, Christian; Schäble, Sandra; Suvorava, Tatsiana; Fahimi, Ehsan Gholamreza; Bisha, Marion; Stermann, Torben; Henning, Uwe; Kojda, Georg

    2016-08-01

    Nicotinic acetylcholine receptor (nAChR) subtypes containing the α4 subunit, particularly α4β2 nAChRs, play an important role in cognitive functioning. The impact of the smoking cessation aid varenicline, a selective partial α4β2 nAChR agonist, on (1) changes of central protein and mRNA expression of this receptor and (2) on memory deficits in a mouse model of cognitive impairment was investigated. Protein and mRNA expression of both the α4 and β2 receptor subunits in mouse brain endothelial and hippocampal cells as well as hippocampus and neocortex tissues were determined by western blot and realtime PCR, respectively. The β2 antibody showed low specificity, though. Tissues were examined following a 2-week oral treatment with various doses of varenicline (0.01, 0.1, 1, 3 mg/kg/day) or vehicle. In addition, episodic memory of mice was assessed following this treatment with an object recognition task using (1) normal mice and (2) animals with anticholinergic-induced memory impairment (i.p. injection of 0.5 mg/kg scopolamine). Varenicline dose-dependently increased protein expression of both the α4 and β2 subunit in cell cultures and brain tissues, respectively, but had no effect on mRNA expression of both subunits. Scopolamine injection induced a significant reduction of object memory in vehicle-treated mice. By contrast, cognitive performance was not altered by scopolamine in varenicline-treated mice. In conclusion, a 2-week oral treatment with varenicline prevented memory impairment in the scopolamine mouse model. In parallel, protein, but not mRNA expression was upregulated, suggesting a posttranscriptional mechanism. Our findings suggest a beneficial effect of varenicline on cognitive dysfunction. PMID:27012889

  11. Airway-related vagal preganglionic neurons express multiple nicotinic acetylcholine receptor subunits

    PubMed Central

    Dehkordi, Ozra; Kc, Prabha; Balan, Kannan V.; Haxhiu, Musa A.

    2007-01-01

    Nicotine acting centrally increases bronchomotor tone and airway secretion, suggesting that airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) express nicotinic acetylcholine receptors (nAChRs). In the present study, we examined the three main functionally characterized subtypes of nAChRs in the CNS, the α7 homomeric and α4β2 heteromeric receptors. First, we characterized the expression of these subunits at the message (mRNA) and protein levels in brain tissues taken from the rNA region, the site where AVPNs are located. In addition, double labeling fluorescent immunohistochemistry and confocal laser microscopy were used to define the presence of α7, α4, and β2 nAChRs on AVPNs that were retrogradely labeled with cholera toxin h subunit (CTb), injected into the upper lung lobe (n = 4) or extrathoracic trachea (n = 4). Our results revealed expression of all three studied subunits at mRNA and protein levels within the rNA region. Furthermore, virtually all identified AVPNs innervating intrapulmonary airways express α7 and α4 nAChR subunits. Similarly, a majority of labeled AVPNs projecting to extrathoracic trachea contain α7 and β2 subunits, but less than half of them show detectable α4 nAChR traits. These results suggest that AVPNs express three major nAChR subunits (α7, α4, and β2) that could assemble into functional homologous or heterologous pentameric receptors, mediating fast and sustained nicotinic effects on cholinergic outflow to the airways. PMID:16616705

  12. The subpopulation of microglia expressing functional muscarinic acetylcholine receptors expands in stroke and Alzheimer's disease.

    PubMed

    Pannell, Maria; Meier, Maria Almut; Szulzewsky, Frank; Matyash, Vitali; Endres, Matthias; Kronenberg, Golo; Prinz, Vincent; Waiczies, Sonia; Wolf, Susanne A; Kettenmann, Helmut

    2016-03-01

    Microglia undergo a process of activation in pathology which is controlled by many factors including neurotransmitters. We found that a subpopulation (11 %) of freshly isolated adult microglia respond to the muscarinic acetylcholine receptor agonist carbachol with a Ca(2+) increase and a subpopulation of similar size (16 %) was observed by FACS analysis using an antibody against the M3 receptor subtype. The carbachol-sensitive population increased in microglia/brain macrophages isolated from tissue of mouse models for stroke (60 %) and Alzheimer's disease (25 %), but not for glioma and multiple sclerosis. Microglia cultured from adult and neonatal brain contained a carbachol-sensitive subpopulation (8 and 9 %), which was increased by treatment with interferon-γ to around 60 %. This increase was sensitive to blockers of protein synthesis and correlated with an upregulation of the M3 receptor subtype and with an increased expression of MHC-I and MHC-II. Carbachol was a chemoattractant for microglia and decreased their phagocytic activity. PMID:25523105

  13. Spatiotemporal calcium signaling in a Drosophila melanogaster cell line stably expressing a Drosophila muscarinic acetylcholine receptor.

    PubMed

    Cordova, D; Delpech, V Raymond; Sattelle, D B; Rauh, J J

    2003-11-01

    A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca(2+)](i)) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca(2+)](i) transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx. PMID:12827518

  14. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    SciTech Connect

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  15. Olfactory discrimination varies in mice with different levels of α7-nicotinic acetylcholine receptor expression

    PubMed Central

    Hellier, Jennifer L.; Arevalo, Nicole L.; Blatner, Megan J.; Dang, An K.; Clevenger, Amy C.; Adams, Catherine E.; Restrepo, Diego

    2010-01-01

    Previous studies have shown that schizophrenics have decreased expression of α7-nicotinic acetylcholine (α7) receptors in the hippocampus and other brain regions, paranoid delusions, disorganized speech, deficits in auditory gating (i.e., inability to inhibit neuronal responses to repetitive auditory stimuli), and difficulties in odor discrimination and detection. Here we use mice with decreased α7 expression that also show a deficit in auditory gating to determine if these mice have similar deficits in olfaction. In the adult mouse olfactory bulb (OB), α7 expression localizes in the glomerular layer; however, the functional role of α7 is unknown. We show that inbred mouse strains (i.e., C3H and C57) with varying α7 expression (e.g., α7 wild-type [α7+/+], α7 heterozygous knock-out [α7+/−] and α7 homozygous knockout mice [α7−/−]) significantly differ in odor discrimination and detection of chemically related odorant pairs. Using [125I] α-bungarotoxin (α-BGT) autoradiography, α7 expression was measured in the OB. As previously demonstrated, α-BGT binding was localized to the glomerular layer. Significantly more expression of α7 was observed in C57 α7+/+ mice compared to C3H α7+/+ mice. Furthermore, C57 α7+/+ mice were able to detect a significantly lower concentration of an odor in a mixture compared to C3H α7+/+ mice. Both C57 and C3H α7+/+ mice discriminated between chemically related odorants sooner than α7+/− or α7−/− mice. These data suggest that α7-nicotinic-receptors contribute strongly to olfactory discrimination and detection in mice and may be one of the mechanisms producing olfactory dysfunction in schizophrenics. PMID:20713028

  16. Expression of acetylcholine receptor genes in human thymic epithelial cells: implications for myasthenia gravis.

    PubMed

    Wakkach, A; Guyon, T; Bruand, C; Tzartos, S; Cohen-Kaminsky, S; Berrih-Aknin, S

    1996-10-15

    The intrathymic presence of the muscle acetylcholine receptor (AChR) is controversial, and the nature of the cell(s) expressing it is unclear. We thus analyzed the molecular expression of muscle AChR in human thymi. mRNA studies indicated that the two isoforms (P3A+ and P3A-) of the alpha-subunit were present in thymic extracts and in cultured thymic epithelial cells (TEC), while expression in thymocytes was low and not consistently detectable. The amount of mRNA coding for the alpha-subunit, evaluated by means of quantitative PCR, was about 20 times less in TEC than in muscle, and was similar in TEC from normal subjects and from patients with myasthenia gravis (MG). The beta- and epsilon-subunits present in adult AChR were also expressed in TEC (but not in thymocytes), while the embryonic subunit (gamma) was absent. In TEC cultures, the AChR alpha- and epsilon-subunit mRNA levels were down-regulated by forskolin, as also observed in the TE671 rhabdomyosarcoma cell line, suggesting similar regulation of AChR subunits in thymus and muscle. Protein expression was evidenced on TEC (but not on thymocytes), by Western blotting as well as by immunofluorescence, thus demonstrating AChR expression on human thymic epithelial cells. There was no difference in the expression of AChR between TEC from MG patients and controls, meaning that the expression of AChR subunits alone is not sufficient to explain the onset of MG. PMID:8871679

  17. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms

    PubMed Central

    Li, Ben-Wen; Rush, Amy C.; Weil, Gary J.

    2015-01-01

    Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of “classical” anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of Vas deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects

  18. Expression of nicotinic acetylcholine receptor subunits from parasitic nematodes in Caenorhabditis elegans.

    PubMed

    Sloan, Megan A; Reaves, Barbara J; Maclean, Mary J; Storey, Bob E; Wolstenholme, Adrian J

    2015-11-01

    The levamisole-sensitive nicotinic acetylcholine receptor present at nematode neuromuscular junctions is composed of multiple different subunits, with the exact composition varying between species. We tested the ability of two well-conserved nicotinic receptor subunits, UNC-38 and UNC-29, from Haemonchus contortus and Ascaris suum to rescue the levamisole-resistance and locomotion defects of Caenorhabditis elegans strains with null deletion mutations in the unc-38 and unc-29 genes. The parasite cDNAs were cloned downstream of the relevant C. elegans promoters and introduced into the mutant strains via biolistic transformation. The UNC-38 subunit of H. contortus was able to completely rescue both the locomotion defects and levamisole resistance of the null deletion mutant VC2937 (ok2896), but no C. elegans expressing the A. suum UNC-38 could be detected. The H. contortus UNC-29.1 subunit partially rescued the levamisole resistance of a C. elegans null mutation in unc-29 VC1944 (ok2450), but did cause increased motility in a thrashing assay. In contrast, only a single line of worms containing the A. suum UNC-29 subunit showed a partial rescue of levamisole resistance, with no effect on thrashing. PMID:26747395

  19. Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression.

    PubMed Central

    Yu, L; LaPolla, R J; Davidson, N

    1986-01-01

    Clones coding for the mouse nicotinic acetylcholine receptor (AChR) gamma subunit precursor have been selected from a cDNA library derived from a mouse myogenic cell line and sequenced. The deduced protein sequence consists of a signal peptide of 22 amino acid residues and a mature gamma subunit of 497 amino acid residues. There is a high degree of sequence conservation between this mouse sequence and published human and calf AChR gamma subunits and, after allowing for functional amino acid substitutions, also to the more distantly related chicken and Torpedo AChR gamma subunits. The degree of sequence conservation is especially high in the four putative hydrophobic membrane spanning regions, supporting the assignment of these domains. RNA blot hybridization showed that the mRNA level of the gamma subunit increases by 30 fold or more upon differentiation of the two mouse myogenic cell lines, BC3H-1 and C2C12, suggesting that the primary controls for changes in gene expression during differentiation are at the level of transcription. One cDNA clone was found to correspond to a partially processed nuclear transcript containing two as yet unspliced intervening sequences. Images PMID:3010242

  20. Immunisation with Torpedo acetylcholine receptor.

    PubMed

    Elfman, L

    1984-01-01

    Acetylcholine mediates the transfer of information between neurons in the electric organ of, for example, Torpedo as well as in vertebrate skeletal muscle. The nicotinic acetylcholine receptor complex translates the binding of acetylcholine into ion permeability changes. This leads to an action potential in the muscle fibre. The nicotinic acetylcholine receptor protein has been purified from Torpedo by use of affinity chromatography. The receptor is an intrinsic membrane glycoprotein composed of five polypeptide chains. When various animals are immunised with the receptor they demonstrate clinical signs of severe muscle weakness coincident with high antibody titres in their sera. The symptoms resemble those found in the autoimmune neuromuscular disease myasthenia gravis in humans. This animal model has constituted a unique model for studying autoimmune diseases. This paper reviews some of the work using Torpedo acetylcholine receptor in order to increase the understanding of the motor nervous system function and myasthenia gravis. It is now known that the nicotinic acetylcholine receptor protein is the antigen involved in myasthenia gravis. The mechanism of immune damage involves a direct block of the receptor function. This depends on the presence of antibodies which crosslink the postsynaptic receptors leading to their degradation. The questions to be answered in the future are; (a) what initiates or triggers the autoimmune response, (b) how do the antibodies cause the symptoms--is there a steric hindrance of the interaction of acetylcholine and the receptor, (c) why is there not a strict relationship between antibody titre and severity of symptoms, and (d) why are some muscles affected and other spared? With help of the experimental model, answers to these questions may result in improved strategies for the treatment of the autoimmune disease myasthenia gravis. PMID:6097937

  1. Propofol and AZD3043 Inhibit Adult Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

    PubMed Central

    Jonsson Fagerlund, Malin; Krupp, Johannes; Dabrowski, Michael A.

    2016-01-01

    Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs) has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1β1δε, α3β2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1β1δε, α3β2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses. PMID:26861354

  2. Nicotinic acetylcholine receptors regulate type 1 inositol 1,4,5-trisphosphate receptor expression via calmodulin kinase IV activation.

    PubMed

    Mizuno, Koji; Kurokawa, Kazuhiro; Ohkuma, Seitaro

    2015-04-01

    Type 1 inositol 1,4,5-trisphosphate receptors (IP3 R-1) are among the important calcium channels regulating intracellular Ca(2+) concentration in the central nervous system. In a previous study, we showed that drugs of abuse, such as cocaine, methamphetamine, and ethanol, induced IP3 R-1 upregulation via the calcium signal transduction pathway in psychological dependence. Although nicotine, a major component in tobacco smoke, participates in psychological and/or physical dependence, it has not yet been clarified how nicotine alters IP3 R-1 expression. The present study, therefore, seeks to clarify the mechanism bgy which nicotine modifies IP3 R-1 expression by using mouse cerebral cortical neurons in primary culture. Nicotine induced dose- and time-dependent upregulation of IP3 R-1 protein following its mRNA increase, and the latter was significantly suppressed by a nonselective nicotinic acetylcholine receptors (nAChR) antagonist, mecamylamine. Both cFos and phosphorylated-cJun (p-cJun) were immediately increased in the nucleus, together with an increase of calmodulin kinase (CaMK) IV but not CaMKII expression after nicotine exposure. A nonselective inhibitor of CaMKs, KN-93, and a calcium chelating regent, BAPTA-AM, completely suppressed the expression of cFos and p-cJun in the nucleus as well as the nicotine-induced IP3 R-1 upregulation. These results indicate that nAChR activation by nicotine upregulates IP3 R-1 via increase of activator protein-1, which is a cFos and cJun dimmer, in the nucleus, with activation of Ca(2+) signaling transduction processes. PMID:25430056

  3. Expression of the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants.

    PubMed Central

    Gershoni, J M

    1987-01-01

    Restriction fragments of DNA derived from a cDNA clone of the alpha subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2. Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound alpha-bungarotoxin. Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria. This provides a new approach for gene selection via functional expression--i.e., ligand overlays of colony blots. Images PMID:3295881

  4. Ligand binding properties of muscarinic acetylcholine receptor subtypes (m1-m5) expressed in baculovirus-infected insect cells.

    PubMed

    Dong, G Z; Kameyama, K; Rinken, A; Haga, T

    1995-07-01

    Five subtypes of muscarinic acetylcholine receptors (m1-m5) have been expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system. Up to 6 nmol of muscarinic acetylcholine receptors were produced by 1 liter culture; 0.3 to 0.6 (human m1), 3 to 6 (human m2), 2 to 4 (rat m3), 1 to 2 (rat m4) and 0.5 to 1 (human m5) nmol. Pirenzepine, AF-DX116 and hexahidrosiladifenidol showed the highest affinity for the m1, m2 and m3 subtype, respectively, indicating that these receptors expressed in Sf9 cells retain the same substrate specificity as those in mammalian tissues or cultured cells. Among 32 kinds of muscarinic ligands examined in the present studies, prifinium was found to have the highest affinity for the m4 subtype, and pilocarpine, oxotremorine, McN-A343 and promethazine the highest affinity for the m5 subtype, although the differences in the affinities among the five subtypes were less than 10-fold. Alcuronium increased the binding of [3H]N-methylscopalamine to the m2 subtype, but not the m1, m4 and m5 subtypes and only slightly to the m3 subtype. Similar but smaller effects of fangchinoline and tetrandrine were found for [3H]N-methylscopalamine binding to only the m3 subtype. These effects may also be useful for the discrimination of individual subtypes. PMID:7616422

  5. Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors.

    PubMed Central

    Nef, P; Oneyser, C; Alliod, C; Couturier, S; Ballivet, M

    1988-01-01

    Four genes encode the related protein subunits that assemble to form the nicotinic acetylcholine receptor (nAChR) at the motor endplate of vertebrates. We have isolated from the chicken genome four additional members of the same gene family whose protein products, termed alpha 2, alpha 3, alpha 4 and n alpha (non-alpha) probably define three distinct neuronal nAChR subtypes. The neuronal nAChR genes have identical structures consisting of six protein-coding exons and specify proteins that are best aligned with the chicken endplate alpha subunit, whose gene we have also characterized. mRNA transcripts encoding alpha 4 and n alpha are abundant in embryonic and in adult avian brain, whereas alpha 2 and alpha 3 transcripts are much scarcer. The same set of neuronal genes probably exists in all vertebrates since their counterparts have also been identified in the rat genome. Images PMID:3267226

  6. [Rhabdomyosarcoma lysis by T cells expressing a human autoantibody based chimeric receptor targeting the fetal acetylcholine receptors].

    PubMed

    Gattenlöhner, S

    2006-01-01

    Rhabdomyosarcomas (RMSs) are the most frequent malignant soft tissue tumors of childhood. Since even aggressive multimodality treatments including autologous stem cell rescue have failed to improve the < 20 % overall survival rate of children with metastatic RMS, novel treatment approaches are urgently needed. Looking for potential targets for immunotherapies, we identified the gamma subunit of the fetal acetylcholine receptor (fAChR) as a specific and overexpressed membrane antigen in RMS. Additionally we established a duplex RT-PCR with simultaneous amplification of alpha and gamma subunit message of the fAChR and the quantification of both transcripts resulting in alpha/gammaAChR ratio > 1 was 100% sensitive in alveolar and embryonal rhabdomyosarcoma. Since the fAChR was the first extracellular tumor marker that can distinguish rhabdomyosarcomas from nonrhabdomyomatous tumors and from normal muscle and therefore implies, that the fAChR may be a target for immunotherapeutic strategies, we synthesized a scFv antibody fragment directed against the fAChR and enigineered both a Pseudomonas exotoxin A based immunotoxin as well as a chimeric T cell receptor composed of the antigen-binding domain of the scFv fragment joined to the signaling domain of the T cell receptor zeta chain. The interaction of fAChzeta-transduced T cells with several RMS cell lines but not with fAChR-negative controls induced strong T cell activation, characterized by secretion of high amounts of interferon-gamma. Moreover after co-incubations with RMS cell lines fAChRzeta-transduced T cells as well fAChR specific immunotoxin induced specific receptor-concentration dependent tumor cell lysis. Therefore, fAChRzeta-transduced T cells and the fAChR specific immunotoxin respectively are promising new tools for the immunotherapy of rhabdomyosarcomas and may provide an effective complementary approach to eradicate residual or metastatic RMS cells in patients, since 1. RMS-direceted chemotherapies

  7. "Warming yang and invigorating qi" acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis.

    PubMed

    Huang, Hai-Peng; Pan, Hong; Wang, Hong-Feng

    2016-03-01

    Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction. "Warming yang and invigorating qi" acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis. However, few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction. Here, we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following "warming yang and invigorating qi" acupuncture therapy. Needles were inserted at acupressure points Shousanli (LI10), Zusanli (ST36), Pishu (BL20), and Shenshu (BL23) once daily for 7 consecutive days. The treatment was repeated after 1 day of rest. We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment. This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide. These findings suggest that "warming yang and invigorating qi" acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis. PMID:27127487

  8. “Warming yang and invigorating qi” acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis

    PubMed Central

    Huang, Hai-peng; Pan, Hong; Wang, Hong-feng

    2016-01-01

    Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction. “Warming yang and invigorating qi” acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis. However, few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction. Here, we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following “warming yang and invigorating qi” acupuncture therapy. Needles were inserted at acupressure points Shousanli (LI10), Zusanli (ST36), Pishu (BL20), and Shenshu (BL23) once daily for 7 consecutive days. The treatment was repeated after 1 day of rest. We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment. This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide. These findings suggest that “warming yang and invigorating qi” acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis. PMID:27127487

  9. Molecular properties of muscarinic acetylcholine receptors

    PubMed Central

    HAGA, Tatsuya

    2013-01-01

    Muscarinic acetylcholine receptors, which comprise five subtypes (M1-M5 receptors), are expressed in both the CNS and PNS (particularly the target organs of parasympathetic neurons). M1-M5 receptors are integral membrane proteins with seven transmembrane segments, bind with acetylcholine (ACh) in the extracellular phase, and thereafter interact with and activate GTP-binding regulatory proteins (G proteins) in the intracellular phase: M1, M3, and M5 receptors interact with Gq-type G proteins, and M2 and M4 receptors with Gi/Go-type G proteins. Activated G proteins initiate a number of intracellular signal transduction systems. Agonist-bound muscarinic receptors are phosphorylated by G protein-coupled receptor kinases, which initiate their desensitization through uncoupling from G proteins, receptor internalization, and receptor breakdown (down regulation). Recently the crystal structures of M2 and M3 receptors were determined and are expected to contribute to the development of drugs targeted to muscarinic receptors. This paper summarizes the molecular properties of muscarinic receptors with reference to the historical background and bias to studies performed in our laboratories. PMID:23759942

  10. Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

    PubMed Central

    Cheng, Hao; Fan, Chen; Zhang, Si-wei; Wu, Zhong-shan; Cui, Zhi-cheng; Melcher, Karsten; Zhang, Cheng-hai; Jiang, Yi; Cong, Yao; Xu, H Eric

    2015-01-01

    Aim: To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target. Methods: α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis. Results: Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel. Conclusion: We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis. PMID:26073323

  11. Heterologous expression and nonsense suppression provide insights into agonist behavior at α6β2 nicotinic acetylcholine receptors.

    PubMed

    Post, Michael R; Limapichat, Walrati; Lester, Henry A; Dougherty, Dennis A

    2015-10-01

    The α6-containing subtypes of the nicotinic acetylcholine receptor (nAChR) are localized to presynaptic terminals of the dopaminergic pathways of the central nervous system. Selective ligands for these nAChRs are potentially useful in both Parkinson's disease and addiction. For these and other goals, it is important to distinguish the binding behavior of agonists at the α6-β2 binding site versus other subtypes. To study this problem, we apply nonsense suppression-based non-canonical amino acid mutagenesis. We report a combination of four mutations in α6β2 that yield high-level heterologous expression in Xenopus oocytes. By varying mRNA injection ratios, two populations were observed with unique characteristics, likely due to differing stoichiometries. Responses to nine known nAChR agonists were analyzed at the receptor, and their corresponding EC50 values and efficacies are reported. The system is compatible with nonsense suppression, allowing structure-function studies between Trp149 - a conserved residue on loop B found to make a cation-π interaction at several nAChR subtypes - and several agonists. These studies reveal that acetylcholine forms a strong cation-π interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the α6β2 nAChR. PMID:25908401

  12. CHARACTERIZATION OF NICOTINE ACETYLCHOLINE RECEPTOR SUBUNITS IN THE COCKROACH Periplaneta americana MUSHROOM BODIES REVEALS A STRONG EXPRESSION OF β1 SUBUNIT: INVOLVEMENT IN NICOTINE-INDUCED CURRENTS.

    PubMed

    Taillebois, Emiliane; Thany, Steeve H

    2016-09-01

    Nicotinic acetylcholine receptors are ligand-gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real-time polymerase chain reaction (PCR) experiments demonstrated that β1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against β1 subunit revealed that inhibition of β1 expression strongly decreases nicotine-induced currents amplitudes. Moreover, co-application with 0.5 μM α-bungarotoxin completely inhibited nicotine currents whereas 10 μM d-tubocurarine had a partial effect demonstrating that β1-containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α-bungarotoxin. PMID:27357353

  13. Altered ultrastructure, density and cathepsin K expression in bone of female muscarinic acetylcholine receptor M3 knockout mice.

    PubMed

    Lips, Katrin Susanne; Kneffel, Mathias; Willscheid, Fee; Mathies, Frank Martin; Kampschulte, Marian; Hartmann, Sonja; Panzer, Imke; Dürselen, Lutz; Heiss, Christian; Kauschke, Vivien

    2015-11-01

    High frequency of osteoporosis is found in postmenopausal women where several molecular components were identified to be involved in bone loss that subsequently leads to an increased fracture risk. Bone loss has already been determined in male mice with gene deficiency of muscarinic acetylcholine receptor M3 (M3R-KO). Here we asked whether bone properties of female 16-week old M3R-KO present similarities to osteoporotic bone loss by means of biomechanical, radiological, electron microscopic, cell- and molecular biological methods. Reduced biomechanical strength of M3R-KO correlated with cortical thickness and decreased bone mineral density (BMD). Femur and vertebrae of M3R-KO demonstrated a declined trabecular bone volume, surface, and a higher trabecular pattern factor and structure model index (SMI) compared to wild type (WT) mice. In M3R-KO, the number of osteoclasts as well as the cathepsin K mRNA expression was increased. Osteoclasts of M3R-KO showed an estimated increase in cytoplasmic vesicles. Further, histomorphometrical analysis revealed up-regulation of alkaline phosphatase. Osteoblasts and osteocytes showed a swollen cytoplasm with an estimated increase in the amount of rough endoplasmatic reticulum and in case of osteocytes a reduced pericellular space. Thus, current results on bone properties of 16-week old female M3R-KO are related to postmenopausal osteoporotic phenotype. Stimulation and up-regulation of muscarinic acetylcholine receptor subtype M3 expression in osteoblasts might be a possible new option for prevention and therapy of osteoporotic fractures. Pharmacological interventions and the risk of side effects have to be determined in upcoming studies. PMID:26002583

  14. Pesticide exposure during pregnancy, like nicotine, affects the brainstem α7 nicotinic acetylcholine receptor expression, increasing the risk of sudden unexplained perinatal death.

    PubMed

    Lavezzi, Anna Maria; Cappiello, Achille; Pusiol, Teresa; Corna, Melissa Felicita; Termopoli, Veronica; Matturri, Luigi

    2015-01-15

    This study indicates the impact of nicotine and pesticides (organochlorine and organophosphate insecticides used in agriculture) on neuronal α7-nicotinic acetylcholine receptor expression in brainstem regions receiving cholinergic projections in human perinatal life. An in-depth anatomopathological examination of the autonomic nervous system and immunohistochemistry to analyze the α7-nicotinic acetylcholine receptor expression in the brainstem from 44 fetuses and newborns were performed. In addition, the presence of selected agricultural pesticides in cerebral cortex samples of the victims was determined by specific analytical procedures. Hypodevelopment of brainstem structures checking the vital functions, frequently associated with α7-nicotinic acetylcholine receptor immunopositivity and smoke absorption in pregnancy, was observed in high percentages of victims of sudden unexpected perinatal death. In nearly 30% of cases however the mothers never smoked, but lived in rural areas. The search for pesticides highlighted in many of these cases traces of both organochlorine and organophosphate pesticides. We detain that exposition to pesticides in pregnancy produces homologous actions to those of nicotine on neuronal α7-nicotinic acetylcholine receptor, allowing to developmental alterations of brainstem vital centers in victims of sudden unexplained death. PMID:25433450

  15. Age-related hearing loss: GABA, nicotinic acetylcholine and NMDA receptor expression changes in spiral ganglion neurons of the mouse.

    PubMed

    Tang, X; Zhu, X; Ding, B; Walton, J P; Frisina, R D; Su, J

    2014-02-14

    Age-related hearing loss - presbycusis - is the number one communication disorder and most prevalent neurodegenerative condition of our aged population. Although speech understanding in background noise is quite difficult for those with presbycusis, there are currently no biomedical treatments to prevent, delay or reverse this condition. A better understanding of the cochlear mechanisms underlying presbycusis will help lead to future treatments. Objectives of the present study were to investigate GABAA receptor subunit α1, nicotinic acetylcholine (nACh) receptor subunit β2, and N-methyl-d-aspartate (NMDA) receptor subunit NR1 mRNA and protein expression changes in spiral ganglion neurons (SGN) of the CBA/CaJ mouse cochlea, that occur in age-related hearing loss, utilizing quantitative immunohistochemistry and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) techniques. We found that auditory brainstem response (ABR) thresholds shifted over 40dB from 3 to 48kHz in old mice compared to young adults. DPOAE thresholds also shifted over 40dB from 6 to 49kHz in old mice, and their amplitudes were significantly decreased or absent in the same frequency range. SGN density decreased with age in basal, middle and apical turns, and SGN density of the basal turn declined the most. A positive correlation was observed between SGN density and ABR wave 1amplitude. mRNA and protein expression of GABAAR α1 and AChR β2 decreased with age in SGNs in the old mouse cochlea. mRNA and protein expression of NMDAR NR1 increased with age in SGNs of the old mice. These findings demonstrate that there are functionally-relevant age-related changes of GABAAR, nAChR, NMDAR expression in CBA mouse SGNs reflecting their degeneration, which may be related to functional changes in cochlear synaptic transmission with age, suggesting biological mechanisms for peripheral age-related hearing loss. PMID:24316061

  16. α6* nicotinic acetylcholine receptor expression and function in a visual salience circuit

    PubMed Central

    Mackey, Elisha D.W.; Engle, Staci E.; Kim, Mi Ran; O’Neill, Heidi C.; Wageman, Charles R.; Patzlaff, Natalie E.; Wang, Ying; Grady, Sharon R.; McIntosh, J. Michael; Marks, Michael J.; Lester, Henry A.; Drenan, Ryan M.

    2012-01-01

    Nicotinic ACh receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus coeruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with GFP fused in-frame in the M3–M4 intracellular loop. Inα6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitiveα6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway. PMID:22836257

  17. Acetylcholine affects osteocytic MLO-Y4 cells via acetylcholine receptors.

    PubMed

    Ma, Yuanyuan; Li, Xianxian; Fu, Jing; Li, Yue; Gao, Li; Yang, Ling; Zhang, Ping; Shen, Jiefei; Wang, Hang

    2014-03-25

    The identification of the neuronal control of bone remodeling has become one of the many significant recent advances in bone biology. Cholinergic activity has recently been shown to favor bone mass accrual by complex cellular regulatory networks. Here, we identified the gene expression of the muscarinic and nicotinic acetylcholine receptors (m- and nAChRs) in mice tibia tissue and in osteocytic MLO-Y4 cells. Acetylcholine, which is a classical neurotransmitter and an osteo-neuromediator, not only influences the mRNA expression of the AChR subunits but also significantly induces the proliferation and viability of osteocytes. Moreover, acetylcholine treatment caused the reciprocal regulation of RANKL and OPG mRNA expression, which resulted in a significant increase in the mRNA ratio of RANKL:OPG in osteocytes via acetylcholine receptors. The expression of neuropeptide Y and reelin, which are two neurogenic markers, was also modulated by acetylcholine via m- and nAChRs in MLO-Y4 cells. These results indicated that osteocytic acetylcholine receptors might be a new valuable mediator for cell functions and even for bone remodeling. PMID:24508663

  18. Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

    PubMed

    Mousavi, M; Hellström-Lindahl, E; Guan, Z Z; Bednar, I; Nordberg, A

    2001-12-21

    Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent. PMID:11811902

  19. Up-regulated expression of the alpha7 nicotinic acetylcholine receptor subunit on inflammatory infiltrates during Dictyocaulus viviparus infection.

    PubMed

    Lazari, O; Kipar, A; Johnson, D R; Selkirk, M E; Matthews, J B

    2006-09-01

    Cholinergic signalling is known to affect immune cell function, but few studies have addressed its relevance during nematode infection. We therefore analysed the anatomical distribution and expression pattern of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in lungs obtained from Dictyocaulus viviparus-infected and uninfected control cattle. The analysis was performed on trachea and lung parenchyma from uninfected animals and animals necropsied at 15, 22 and 43 days post-infection (DPI). Localization of the alpha7 nAChR was evaluated by immunohistology and mRNA expression analysed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR). In uninfected animals, tracheal, bronchial and bronchiolar epithelium and smooth muscle cells constitutively expressed the alpha7 nAChR, as did type I and II alveolar epithelial cells and alveolar macrophages and a few infiltrating leucocytes. By 15 DPI, immunohistology revealed a massive influx of alpha7 nAChR+ inflammatory cells into the lung parenchyma and tracheal wall. This was reflected in the RT-PCR results. At later time points, both parenchyma and tracheal wall contained large numbers of alpha7 nAChR+ leucocytes, but detection of transcript was restricted to the trachea. Recruitment of nAChR-containing leucocytes to the lungs of D. viviparus-infected cattle suggests that these cells may represent possible downstream targets for parasite-secreted acetylcholinesterases. PMID:16916366

  20. Novel role for cyclin-dependent kinase 2 in neuregulin-induced acetylcholine receptor epsilon subunit expression in differentiated myotubes.

    PubMed

    Lu, Gang; Seta, Karen A; Millhorn, David E

    2005-06-10

    Cyclin-dependent kinases (CDKs) are a family of evolutionarily conserved serine/threonine kinases. CDK2 acts as a checkpoint for the G(1)/S transition in the cell cycle. Despite a down-regulation of CDK2 activity in postmitotic cells, many cell types, including muscle cells, maintain abundant levels of CDK2 protein. This led us to hypothesize that CDK2 may have a function in postmitotic cells. We show here for the first time that CDK2 can be activated by neuregulin (NRG) in differentiated C2C12 myotubes. In addition, this activity is required for expression of the acetylcholine receptor (AChR) epsilon subunit. The switch from the fetal AChRgamma subunit to the adult-type AChRepsilon is required for synapse maturation and the neuromuscular junction. Inhibition of CDK2 activity with either the specific CDK2 inhibitory peptide Tat-LFG or by RNA interference abolished neuregulin-induced AChRepsilon expression. Neuregulin-induced activation of CDK2 also depended on the ErbB receptor, MAPK, and PI3K, all of which have previously been shown to be required for AChRepsilon expression. Neuregulin regulated CDK2 activity through coordinating phosphorylation of CDK2 on Thr-160, accumulation of CDK2 in the nucleus, and down-regulation of the CDK2 inhibitory protein p27 in the nucleus. In addition, we also observed a novel mechanism of regulation of CDK2 activity by a low molecular weight variant of cyclin E in response to NRG. These findings establish CDK2 as an intermediate molecule that integrates NRG-activated signals from both the MAPK and PI3K pathways to AChRepsilon expression and reveal an undiscovered physiological role for CDK2 in postmitotic cells. PMID:15824106

  1. Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells

    PubMed Central

    Elgueta, Claudio; Vielma, Alex H.; Palacios, Adrian G.; Schmachtenberg, Oliver

    2015-01-01

    Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine >> RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca2+ accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca2+ stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing. PMID:25709566

  2. Nicotinic acetylcholine receptors and cancer

    PubMed Central

    DANG, NINGNING; MENG, XIANGUANG; SONG, HAIYAN

    2016-01-01

    Nicotine, the primary addictive constituent of cigarettes, is believed to contribute to cancer promotion and progression through the activation of nicotinic acetylcholine receptors (nAChRs), which are membrane ligand-gated cation channels. nAChRs activation can be triggered by the neurotransmitter Ach, or certain other biological compounds, such as nicotine. In recent years, genome-wide association studies have indicated that allelic variation in the α5-α3-β4 nAChR cluster on chromosome 15q24-15q25.1 is associated with lung cancer risk. The role of nAChRs in other types of cancer has also been reported. The present review highlights the role of nAChRs in types of human cancer. PMID:27123240

  3. Nicotinic Acetylcholine Receptors in Sensory Cortex

    ERIC Educational Resources Information Center

    Metherate, Raju

    2004-01-01

    Acetylcholine release in sensory neocortex contributes to higher-order sensory function, in part by activating nicotinic acetylcholine receptors (nAChRs). Molecular studies have revealed a bewildering array of nAChR subtypes and cellular actions; however, there is some consensus emerging about the major nAChR subtypes and their functions in…

  4. Mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells

    SciTech Connect

    Musil, L.S.; Frail, D.E.; Merlie, J.P. )

    1989-05-01

    Torpedo electric organ and vertebrate neuromuscular junctions contain the receptor-associated protein of the synapse (RAPsyn) (previously referred to as the 43K protein), a nonactin, 43,000-Mr peripheral membrane protein associated with the cytoplasmic face of postsynaptic membranes at areas of high nicotinic acetylcholine receptor (AChR) density. Although not directly demonstrated, several lines of evidence suggest that RAPsyn is involved in the synthesis and/or maintenance of such AChR clusters. Microscopic and biochemical studies had previously indicated that RAPsyn expression is restricted to differentiated, AChR-synthesizing cells. Our recent finding that RAPsyn is also produced in undifferentiated myocytes led to to examine whether RAPsyn is synthesized in cell types that never express AChR (i.e., cells of other than skeletal muscle origin). Various primary and established rodent cell lines were metabolically labeled with (35S)methionine, and extracts were immunoprecipitated with a monospecific anti-RAPsyn serum. Analysis of these immunoprecipitates by SDS-PAGE revealed detectable RAPsyn synthesis in some (notably fibroblast and Leydig tumor cell lines and primary cardiac cells) but not all (hepatocyte- and lymphocyte-derived) cell types. These results were further substantiated by peptide mapping studies of RAPsyn immunoprecipitated from different cells and quantitation of RAPsyn-encoding mRNA levels in mouse tissues. RAPsyn synthesized in both muscle and nonmuscle cells was shown to be tightly associated with membranes. These findings demonstrate that RAPsyn is not specific to skeletal muscle-derived cells and imply that it may function in a capacity either in addition to or instead of AChR clustering.

  5. Agonist actions of clothianidin on synaptic and extrasynaptic nicotinic acetylcholine receptors expressed on cockroach sixth abdominal ganglion.

    PubMed

    Thany, Steeve H

    2009-11-01

    Clothianidin is new neonicotinoid insecticide acting selectively on insect nicotinic acetylcholine receptors (nAChRs). Its effects on nAChRs expressed on cercal afferent/giant interneuron synapses and DUM neurons have been studied using mannitol-gap and whole-cell patch-clamp techniques, respectively. Bath-application of clothianidin-induced dose-dependent depolarizations of cockroach cercal afferent/giant interneuron synapses which were not reversed after wash-out suggesting a strong desensitization of postsynaptic interneurons at the 6th abdominal ganglion (A6). Clothinidin activity on the nerve preparation was characterized by an increased firing rate of action potentials which then ceased when the depolarization reached a peak. Clothianidin responses were insensitive to all muscarinic antagonists tested but were blocked by co-application of specific nicotinic antagonists methyllicaconitine, alpha-bungarotoxin and d-tubocurarine. In a second round of experiment, clothianidin actions were tested on DUM neurons isolated from the A6. There was a strong desensitization of nAChRs which was not affected by muscarinic antagonists, pirenzepine and atropine, but was reduced with nicotinic antagonist alpha-bungarotoxin. In addition, clothianidin-induced currents were completely blocked by methyllicaconitine suggesting that (1) clothianidin acted as a specific agonist of nAChR subtypes and (2) a small proportion of receptors blocked by MLA was insensitive to alpha-bungarotoxin. Moreover, because clothianidin currents were blocked by d-tubocurarine and mecamylamine, we provided that clothianidin was an agonist of both nAChRs: imidacloprid-sensitive nAChR1 and -insensitive nAChR2 subtypes. PMID:19583978

  6. Prenatal cigarette smoke exposure effects on apoptotic and nicotinic acetylcholine receptor expression in the infant mouse brainstem.

    PubMed

    Vivekanandarajah, Arunnjah; Chan, Yik Lung; Chen, Hui; Machaalani, Rita

    2016-03-01

    Infants exposed to cigarette smoked during pregnancy into infancy have increased respiratory and cardiac abnormalities. Nicotine, the major neurotoxic component of cigarette smoke, induces its actions by binding to nicotinic acetylcholine receptors (nAChR), with one downstream effect being increased apoptosis. Using a pre- into post- natal cigarette smoke exposure mouse model (SE), we studied the immunohistochemical expression of nAChR subunits α2, α3, α4, α5, α7, α9, β1 and β2 and two markers of apoptosis, active caspase-3 and TUNEL, in seven nuclei of the medulla and facial nucleus of the pons in male mice. Pups of dams exposed to two cigarettes (nicotine ≤1.2mg, CO ≤15mg) twice daily for six weeks prior to mating, during gestation and lactation (n=5; SE), were compared to pups exposed to air under the same condition (n=5; SHAM) at P20. Results showed that the hypoglossal nucleus had increased α3, α4, α7, α9, Casp-3 and TUNEL, dorsal motor nucleus of the vagus had increased α3, α5, α7, β1 and Casp-3, nucleus of the solitary tract had increased α3 but decreased α4, α5, β1 and apoptosis, cuneate nucleus had increased α3, β2 and Casp- 3, but decreased α5, nucleus of the spinal trigeminal tract had increased α3, α7, β1, lateral reticular nucleus had decreased β1, inferior olivary nucleus had increased β1 but decreased apoptosis, and the facial had increased α2, α3 and α7. This is the first study to demonstrate that nAChR subunits are affected following pre- into post-natal SE and that they simultaneously coincided with changes in apoptotic expression. PMID:26746805

  7. Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

    PubMed Central

    Disney, Anita A; Alasady, Hussein A; Reynolds, John H

    2014-01-01

    Background In the mammalian neocortex, cells that express parvalbumin (PV neurons) comprise a dominant class of inhibitory neuron that substantially overlaps with the fast/narrow-spiking physiological phenotype. Attention has pronounced effects on narrow-spiking neurons in the extrastriate cortex of macaques, and more consistently so than on their broad-spiking neighbors. Cortical neuromodulation by acetylcholine (ACh) is a candidate mechanism for aspects of attention and in the primary visual cortex (V1) of the macaque, receptors for ACh (AChRs) are strongly expressed by inhibitory neurons. In particular, most PV neurons in macaque V1 express m1 muscarinic AChRs and exogenously applied ACh can cause the release of γ-aminobutyric acid. In contrast, few PV neurons in rat V1 express m1 AChRs. While this could be a species difference, it has also been argued that macaque V1 is anatomically unique when compared with other cortical areas in macaques. Aims The aim of this study was to better understand the extent to which V1 offers a suitable model circuit for cholinergic anatomy in the macaque occipital lobe, and to explore cholinergic modulation as a biological basis for the changes in circuit behavior seen with attention. Materials and methods We compared expression of m1 AChRs by PV neurons between area V1 and the middle temporal visual area (MT) in macaque monkeys using dual-immunofluorescence confocal microscopy. Results and conclusion We find that, as in V1, most PV neurons in MT express m1 AChRs but, unlike in V1, it appears that so do most excitatory neurons. This provides support for V1 as a model of cholinergic modulation of inhibition in macaque visual cortex, but not of cholinergic modulation of visual cortical circuits in general. We also propose that ACh acting via m1 AChRs is a candidate underlying mechanism for the strong effects of attention on narrow-spiking neurons observed in behaving animals. PMID:24944872

  8. Immobilization with Atrophy Induces Increased Expression of Neuronal Nicotinic α7 Acetylcholine Receptors in Muscle Contributing to Neurotransmission

    PubMed Central

    Lee, Sangseok; Yang, Hong-seuk; Sasakawa, Tomoki; Khan, Mohammed A. S.; Khatri, Ashok; Kaneki, Masao; Jeevendra Martyn, J. A.

    2014-01-01

    Background Mature acetylcholine receptor (AChR) isoform normally mediates muscle contraction. The hypothesis that α7AChRs upregulate during immobilization and contribute to neurotransmission was tested pharmacologically using specific blockers to mature (waglerin-1), immature (αA-OIVA), and α7AChRs methyllycaconitine, and non-specific muscle AChR antagonist, α-bungarotoxin. Methods Mice were immobilized; contralateral limb was control. Fourteen days later, anesthetized mice were mechanically ventilated. Nerve-stimulated tibialis muscle contractions on both sides were recorded, and blockers enumerated above sequentially administered via jugular vein. Data are mean ± S.E. Results Immobilization (N=7) induced tibialis muscle atrophy (40.6 ± 2.8 vs 52.1 ± 2.0 mg, p<0.01) and decrease of twitch tension (34.8 ± 1.1 vs. 42.9 ± 1.5 g, p< 0.01). Waglerin-1 (0.3 ± 0.05 μg/g) significantly (p=0.001, N=9) depressed twitch tension on contralateral (≥ 97%) versus immobilized side (~45%). Additional waglerin-1 (total dose 1.06 ± 0.12 μg/g or ~15.0 X ED50 in normals) could not depress twitch ≥ 80% on immobilized side. Immature AChR blocker, αA-OIVA (17.0 ± 0.25 μg/g) did not change tension bilaterally. Administration of α-bungarotoxin (N=4) or methyllycaconitine (N=3) caused ≥ 96% suppression of the remaining twitch tension on immobilized side. Methyllycaconitine, administered first (N=3), caused equipotent inhibition by waglerin-1 on both sides. Protein expression of α7AChRs was significantly (N=3, p<0.01) increased on the immobilized side. Conclusions Ineffectiveness of waglerin-1 suggests the twitch tension during immobilization is maintained by receptors other than mature AChRs. Since αA-OIVA caused no changes, immature AChRs contribute minimally to neurotransmission. During immobilization ~20% of twitch tension is maintained by upregulation of α-bungarotoxin- and methyllycaconitine-sensitive α7AChRs. PMID:24126263

  9. Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor:spinosyn interaction.

    PubMed

    Perry, Trent; Somers, Jason; Yang, Ying Ting; Batterham, Philip

    2015-09-01

    Insecticide research has often relied on model species for elucidating the resistance mechanisms present in the targeted pests. The accuracy and applicability of extrapolations of these laboratory findings to field conditions varies but, for target site resistance, conserved mechanisms are generally the rule rather than the exception (Perry et al., 2011). The spinosyn class of insecticides appear to fit this paradigm and are a pest control option with many uses in both crop and animal protection. Resistance to spinosyns has been identified in both laboratory-selected and field-collected pest insects. Studies using the model insect, Drosophila melanogaster, have identified the nicotinic acetylcholine receptor subunit, Dα6 as an important target of the insecticide spinosad (Perry et al., 2007; Watson et al., 2010). Field-isolated resistant strains of several agricultural pest insects provide evidence that resistance cases are often associated with mutations in orthologues to Dα6 (Baxter et al., 2010; Puinean et al., 2013). The expression of these receptors is difficult in heterologous systems. In order to examine the biology of the Dα6 receptor subunit further, we used Drosophila as a model and developed an in vivo rescue system. This allowed us to express four different isoforms of Dα6 and show that each is able to rescue the response to spinosad. Regulatory sequences upstream of the Dα6 gene able to rescue the resistance phenotype were identified. Expression of other D. melanogaster subunits revealed that the rescue phenotype appears to be Dα6 specific. We also demonstrate that expression of pest insect orthologues of Dα6 from a variety of species are capable of rescuing the spinosad response phenotype, verifying the relevance of this receptor to resistance monitoring in the field. In the absence of a robust heterologous expression system, this study presents an in vivo model that will be useful in analysing many other aspects of these receptors and

  10. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    PubMed Central

    Jakubík, Jan; El-Fakahany, Esam E.

    2010-01-01

    An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine). Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer’s disease and other disorders involving impaired cognitive function.

  11. A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression.

    PubMed

    Watson, Gerald B; Chouinard, Scott W; Cook, Kevin R; Geng, Chaoxian; Gifford, Jim M; Gustafson, Gary D; Hasler, James M; Larrinua, Ignacio M; Letherer, Ted J; Mitchell, Jon C; Pak, William L; Salgado, Vincent L; Sparks, Thomas C; Stilwell, Geoff E

    2010-05-01

    Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dalpha6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dalpha6, providing convincing evidence that Dalpha6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dalpha6 alone, co-expression of Dalpha6 with an additional nAChR subunit, Dalpha5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR. PMID:19944756

  12. Kinetics of unliganded acetylcholine receptor channel gating.

    PubMed Central

    Jackson, M B

    1986-01-01

    Open- and closed-state lifetimes of unliganded acetylcholine receptor channel activity were analyzed by the method of likelihood maximazation. For both open times and closed times, the best-fitting density is most often a sum of two exponentials. These multiple open states cannot depend on the number of receptor binding sites occupied since they are observed in the absence of ligand. The rate of spontaneous opening and the faster decay constant of closing increased as the membrane was hyperpolarized. The voltage dependence of the rate of spontaneous opening is stronger than that for curare-liganded channels. Evidence that the acetylcholine receptor channel can open spontaneously in the absence of ligand has been presented previously (Sanchez et al, 1983; Brehm et al, 1984; Jackson, 1984). To add to this evidence, alpha-bungarotoxin was added to the patch electrode, causing the frequency of openings to decay with time. The rate constant determined from this decay is similar to rate constants reported for the binding of iodinated alpha-bungarotoxin to the acetylcholine receptor. The frequency of unliganded channel opening has been estimated as 2 X 10(-3) s-1 per receptor. A comparison of carbamylcholine-liganded and spontaneous gating transition rates suggests that ligand binding increases the rate of opening by a factor of 1.4 X 10(7). Carbamylcholine binding increases the mean open time by a factor of 5. Thus, a cholinergic agonist activates the acetylcholine receptor by destabilizing the closed state. The liganded and unliganded channel gating rates were used to analyze the energetics of ligand activation of the acetylcholine receptor channel, and to relate the open channel dissociation constant to the closed channel dissociation constant. PMID:2421793

  13. External imaging of cerebral muscarinic acetylcholine receptors

    SciTech Connect

    Eckelman, W.C.; Reba, R.C.; Rzeszotarski, W.J.; Gibson, R.E.; Hill, T.; Holman, B.L.; Budinger, T.; Conklin, J.J.; Eng, R.; Grissom, M.P.

    1984-01-20

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  14. External Imaging of Cerebral Muscarinic Acetylcholine Receptors

    NASA Astrophysics Data System (ADS)

    Eckelman, William C.; Reba, Richard C.; Rzeszotarski, Waclaw J.; Gibson, Raymond E.; Hill, Thomas; Holman, B. Leonard; Budinger, Thomas; Conklin, James J.; Eng, Robert; Grissom, Michael P.

    1984-01-01

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  15. Effects of antihistamines on the function of human α7-nicotinic acetylcholine receptors.

    PubMed

    Sadek, Bassem; Khanian, Seyedeh Soha; Ashoor, Abrar; Prytkova, Tatiana; Ghattas, Mohammad A; Atatreh, Noor; Nurulain, Syed M; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Oz, Murat

    2015-01-01

    Effects of the histamine H₁ receptor (H1R) antagonists (antihistamines), promethazine (PMZ), orphenadrine (ORP), chlorpheniramine (CLP), pyrilamine (PYR), diphenhydramine (DPH), citerizine (CTZ), and triprolidine (TRP) on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes were investigated. Antihistamines inhibited the α7-nicotinic acetylcholine receptor in the order PYR>CLP>TRP>PMZ>ORP≥DPH≥CTZ. Among the antihistamines, PYR showed the highest reversible inhibition of acetylcholine (100 µM)-induced responses with IC₅₀ of 6.2 µM. PYR-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. Specific binding of [¹²⁵I] α-bungarotoxin, a selective antagonist for α7-nicotinic acetylcholine receptor, was not changed in the presence of PYR suggesting a non-competitive inhibition of nicotinic receptors. In line with functional experiments, docking studies indicated that PYR can potentially bind allosterically with the α7 transmembrane domain. Our results indicate that the H₂-H₄ receptor antagonists tested in this study (10 µM) showed negligible inhibition of α7-nicotinic acetylcholine receptors. On the other hand, H₁ receptor antagonists inhibited the function of human α7-nicotinic acetylcholine receptor, with varying potencies. These results emphasize the importance of α7-nicotinic acetylcholine receptor for future pharmacological/toxicological profiling. PMID:25445036

  16. Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

    PubMed

    Zhang, Yixi; Wang, Xin; Yang, Baojun; Hu, Yuanyuan; Huang, Lixin; Bass, Chris; Liu, Zewen

    2015-11-01

    Target-site resistance is commonly caused by qualitative changes in insecticide target-receptors and few studies have implicated quantitative changes in insecticide targets in resistance. Here we show that resistance to imidacloprid in a selected strain of Nilaparvata lugens is associated with a reduction in expression levels of the nicotinic acetylcholine receptor (nAChR) subunit Nlα8. Synergism bioassays of the selected strain suggested resistance was conferred, in part, by a target-site mechanism. Sequencing of N. lugens nAChR subunit genes identified no mutations associated with resistance, however, a decrease in mRNA and protein levels of Nlα8 was observed during selection. RNA interference knockdown of Nlα8 decreased the sensitivity of N. lugens to imidacloprid, demonstrating that a decrease in Nlα8 expression is sufficient to confer resistance in vivo. Radioligand binding assays revealed that the affinity of the high-affinity imidacloprid-binding site of native nAChRs was reduced by selection, and reducing the amount of Nlα8 cRNA injected into Xenopus oocytes significantly decreased imidacloprid potency on recombinant receptors. Taken together, these results provide strong evidence that a decrease in Nlα8 levels confers resistance to imidacloprid in N. lugens, and thus provides a rare example of target-site resistance associated with a quantitative rather than qualitative change. In insects, target-site mutations often cause high resistance to insecticides, such as neonicotinoids acting on nicotinic acetylcholine receptors (nAChRs). Here we found that a quantitative change in target-protein level, decrease in mRNA and protein levels of Nlα8, contributed importantly to imidacloprid resistance in Nilaparvata lugens. This finding provides a new target-site mechanism of insecticide resistance. PMID:26259922

  17. Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors.

    PubMed

    Rozman, Klara Bulc; Araoz, Romulo; Sepcić, Kristina; Molgo, Jordi; Suput, Dusan

    2010-09-01

    Nicotinic acetylcholine receptors are implicated in different nervous system-related disorders, and their modulation could improve existing therapy of these diseases. Parazoanthoxanthin A (ParaA) is a fluorescent pigment of the group of zoanthoxanthins. Since it is a potent acetylcholinesterase inhibitor, it may also bind to nicotinic acetylcholine receptors (nAChRs). For this reason its effect on Torpedo nAChR (alpha1(2)betagammadelta) transplanted to Xenopus laevis oocytes was evaluated, using the voltage-clamp technique. ParaA dose-dependently reduced the acetylcholine-induced currents. This effect was fully reversible only at lower concentrations. ParaA also reduced the Hill coefficient and the time to peak current, indicating a channel blocking mode of action. On the other hand, the combined effect of ParaA and d-tubocurarine (d-TC) on acetylcholine-induced currents exhibited only partial additivity, assuming a competitive mode of action of ParaA on nAChR. These results indicate a dual mode of action of ParaA on the Torpedo AChR. PMID:20230806

  18. α-Conotoxins Identify the α3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells.

    PubMed

    Hone, Arik J; McIntosh, J Michael; Azam, Layla; Lindstrom, Jon; Lucero, Linda; Whiteaker, Paul; Passas, Juan; Blázquez, Jesús; Albillos, Almudena

    2015-11-01

    Ligands that selectively inhibit human α3β2 and α6β2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3β4 and α6β4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3β2 than α3β4 and 165-fold more potent on human α6/α3β2β3 than α6/α3β4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with β2 ligand-binding sites. In contrast, the β4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained β4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3β4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, β2, and β4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits. PMID:26330550

  19. [Sites of synthesis of acetylcholine receptors in denervated muscles].

    PubMed

    Giacobini Robecchi, M G; Garelli, M; Filogamo, G

    1980-09-01

    Muscle fibres binding with 125I alpha-bungarotoxine from Bungarus Multicinctus, after treatment with saponine, shows (in electron microscope autoradiography) intracellular binding sites identifying sites of acetylcholine receptor synthesis. In innervated muscle, the acetylcholine receptor is located only at the neuromuscular junction. In denervated muscle the receptor is distributed along the whole sarcolemma; in this situation the acetylcholine receptor is synthesized "ex novo" in the membrane system over the whole length of the muscle fibre. PMID:7214035

  20. Functional expression of human α9* nicotinic acetylcholine receptors in X. laevis oocytes is dependent on the α9 subunit 5' UTR.

    PubMed

    Filchakova, Olena; McIntosh, J Michael

    2013-01-01

    Nicotinic acetylcholine receptors (nAChRs) containing the α9 subunit are expressed in a wide variety of non-neuronal tissues ranging from immune cells to breast carcinomas. The α9 subunit is able to assemble into a functional homomeric nAChR and also co-assemble with the α10 subunit into functional heteromeric nAChRs. Despite the increasing awareness of the important roles of this subunit in vertebrates, the study of human α9-containing nAChRs has been severely limited by difficulties in its expression in heterologous systems. In Xenopus laevis oocytes, functional expression of human α9α10 nAChRs is very low compared to that of rat α9α10 nAChRs. When oocytes were co-injected with cRNA of α9 and α10 subunits of human versus those of rat, oocytes with the rat α9 human α10 combination had an ∼-fold higher level of acetylcholine-gated currents (I(ACh)) than those with the human α9 rat α10 combination, suggesting difficulties with human α9 expression. When the ratio of injected human α9 cRNA to human α10 cRNA was increased from 1∶1 to 5∶1, I(ACh) increased 36-fold (from 142±23 nA to 5171±748 nA). Functional expression of human α9-containing receptors in oocytes was markedly improved by appending the 5'-untranslated region of alfalfa mosaic virus RNA4 to the 5'-leader sequence of the α9 subunit cRNA. This increased the functional expression of homomeric human α9 receptors by 70-fold (from 7±1 nA to 475±158 nA) and of human α9α10 heteromeric receptors by 80-fold (from 113±62 nA to 9192±1137 nA). These findings indicate the importance of the composition of the 5' untranslated leader sequence for expression of α9-containing nAChRs. PMID:23717646

  1. Inhibition of the acetylcholine receptor by histrionicotoxin.

    PubMed Central

    Anwyl, R.; Narahashi, T.

    1980-01-01

    1 The action of C5-decahydrohistrionicotoxin (C5-HTX) has been investigated on the extrajunctional acetylcholine (ACh) receptors of denervated rat muscle. 2 C5-HTX causes both a rapid and slow reduction in amplitude of iontophoretic ACh potentials evoked at all frequencies from the extrajunctional receptors. 3 C5-HTX also causes a time-dependent inhibition of the iontophoretic potentials evoked at frequencies greater than 0.02 Hz. This inhibition was observed either alone or superimposed upon desensitization, and may be caused by a similar mechanism to desensitization. PMID:7378635

  2. Relaxation measurements on the acetylcholine receptor.

    PubMed Central

    Sheridan, R E; Lester, H A

    1975-01-01

    In Electrophorus electroplaques, the agonist-induced postsynaptic conductance depends on membrane potential. During steady exposure to agonists, after a voltage step the conductance relaxes on a millisecond time scale, exponentially approaching a new equilibrium value. The relaxation rate constant k is an instantaneous function of voltage, insensitive to the past or present conductance. Two components sum to form k. A concentration-sensitive component increases linearly with agonist concentration and decreases during desensitization or exposure to curare. Thus this component reflects the average frequency at which acetylcholine receptors are opening. The voltage-sensitive component, obtained by extrapolating k to zero agonist concentration, increases at more positive potentials. For acetylcholine, the voltage-sensitive component equals the rate constant for the exponential decay of postsynaptic currents; it thus seems to be the closing rate for active receptors. The voltage-sensitive component has the relative amplitudes acetylcholine less than carbamoylcholine less than decamethonium, and for each agonist equals the closing rate determined from "noise" measurements at neuromuscular junctions. The kinetic data explain several aspects of the steady-state conductance induced by agonists, but shed no light on apparent cooperative effects. PMID:1059136

  3. The gating isomerization of neuromuscular acetylcholine receptors

    PubMed Central

    Auerbach, Anthony

    2010-01-01

    Acetylcholine receptor-channels are allosteric proteins that isomerize (‘gate’) between conformations that have a low vs. high affinity for the transmitter and conductance for ions. In order to comprehend the mechanism by which the affinity and conductance changes are linked it is of value to know the magnitude, timing and distribution of energy flowing through the system. Knowing both the di- and unliganded gating equilibrium constants (E2 and E0) is a foundation for understanding the AChR gating mechanism and for engineering both the ligand and the protein to operate in predictable ways. In adult mouse neuromuscular receptors activated by acetylcholine, E2= 28 and E0≈ 6.5 × 10−7. At each (equivalent) transmitter binding site acetylcholine provides ∼5.2 kcal mol−1 to motivate the isomerization. The partial agonist choline provides ∼3.3 kcal mol−1. The relative time of a residue's gating energy change is revealed by the slope of its rate–equilibrium constant relationship. A map of this parameter suggests that energy propagates as a conformational cascade between the transmitter binding sites and the gate region. Although gating energy changes are widespread throughout the protein, some residues are particularly sensitive to perturbations. Several specific proposals for the structural events that comprise the gating conformational cascade are discussed. PMID:19933754

  4. A Comparison of MyoD1 and Fetal Acetylcholine Receptor Expression in Childhood Tumors and Normal Tissues

    PubMed Central

    Gattenloehner, Stefan; Dockhorn-Dworniczak, Barbara; Leuschner, Ivo; Vincent, Angela; Müller-Hermelink, Hans-Konrad; Marx, Alexander

    1999-01-01

    Detection of minimal residual disease or micrometastases in rhabdomyosarcoma (RMS) has been an unresolved problem in 70 to 80% of RMS patients. In patients with alveolar type RMS, which harbors chromosomal translocations and produces tumor-specific fusion products, polymerase chain reaction (PCR)-based diagnosis is clear-cut. In the more frequent embryonal RMS, however, no such PCR-based marker has been described. Recently it has been suggested that the PCR-based detection of MyoD1 may be a valuable adjunct in the diagnosis of minimal disease in embryonal RMS. We report here that MyoD1 mRNA is not specific for RMS, but can be amplified from ex vivo samples of many other childhood tumors and some normal tissues. By contrast, simultaneous amplification of α and γ subunit message of the fetal type acetylcholine receptor (AChR), by a novel duplex PCR, and the quantification of both transcripts resulting in a α/γAChR ratio <1 was 100% sensitive in alveolar (n = 8) and embryonal (n = 10) RMS. Moreover, γAChR was not detected in other childhood (n = 27) or adult tumors (n = 12), or normal tissues, except thymus. The high sensitivity and specificity of the method were confirmed by the successful detection of five cases of cytologically or molecularly verified RMS bone marrow micrometastases among 47 bone marrow samples from childhood tumor patients. By contrast, MyoD1 showed no amplification because of its low level of transcription. We conclude that mRNA of the fetal type AChR is a more specific and (about 100 times) more sensitive marker for the molecular detection of RMS than MyoD1, and thus appears to be a promising candidate for the detection of minimal disease in RMS lacking tumor-specific translocations. PMID:11272905

  5. Expression of nicotinic acetylcholine receptor subunits in HEp-2 cells for immunodetection of autoantibody specificities in sera from Myasthenia gravis patients.

    PubMed

    George, S; Noack, M; Vanek, M; Rentzsch, J; Röber, N; Conrad, K; Roggenbuck, D; Küpper, J-H

    2015-01-01

    Myasthenia gravis (MG) is an autoimmune disease characterized by the formation of pathogenic autoantibodies mostly targeting the nicotinic acetylcholine receptor (AChR). The AChR is composed of two alpha subunits and one subunit of each beta, delta and gamma (fetal AChR), or epsilon (adult AChR), respectively. Serological diagnostics is commonly done by radioimmunoassay (RIA). Here we used an indirect immunofluorescence assay with MG patient sera on transiently transfected HEp-2 cells expressing selected components of the AChR. Our data show that already 12 out of 13 MG patient sera showed autoantibody binding to HEp-2 cells transfected to express the alpha subunit solely. Interestingly, 11 out of 13 patient sera reacted positive with cells transfected to reconstitute the complete fetal AChR, but only 6 out of 13 sera showed positive signals with cells expressing the components of adult AChR. Moreover, there was no strict correlation of the serum concentration required to obtain clear-cut fluorescence signals to the antibody titer as measured by RIA. It will be an interesting topic to further investigate if the optimal serum dilution for indirect immunofluorescence as well as the autoantibody binding preferences to defined AChR subunits and to the adult versus the fetal receptor variant could provide additional predictive value in MG diagnostics. PMID:26410878

  6. Spontaneous openings of the acetylcholine receptor channel.

    PubMed Central

    Jackson, M B

    1984-01-01

    Patch clamp recordings from embryonic mouse muscle cells in culture revealed spontaneous openings of the acetylcholine receptor channel in the absence of exogenously applied cholinergic agent. The conductance of the spontaneous channel currents was, within experimental error, identical with the conductance of suberyldicholine-activated channel currents. The comparison of channel conductance was made with sodium and with cesium, each at two concentrations, with the same result. Treatment of the cells with alpha-bungarotoxin blocked the spontaneous channel currents. To determine whether the spontaneous openings were caused by an endogenous agent with cholinergic activity a reactive disulfide bond near the receptor binding site was reduced with dithiothreitol and alkylated with N-ethylmaleimide. This chemical modification reduced the effectiveness with which suberyldicholine and curare activated channel currents but did not reduce the frequency of spontaneous openings. These experiments indicate that the acetylcholine receptor briefly and infrequently fluctuates into an active state in the absence of agonist. Agonist activation of the receptor presumably accelerates this spontaneously occurring process. PMID:6328531

  7. [Desensitization of the nicotinic acetylcholine receptor].

    PubMed

    Quiñonez, M; Rojas, L

    1994-01-01

    In biological membranes, ionic channels act speeding up ion movements. Each ionic channel is excited by a specific stimulus (i.e. electric, mechanical, chemical, etc.). Chemically activated ionic channels (CAIC), such as the nicotinic acetylcholine receptor (nAChR), suffer desensitization when the receptor site is still occupied by the agonist molecule. The desensitized CAIC is a non functional channel state regarded as a particular case of receptors rundown. CAIC desensitization only involve reduced activity and not their membrane elimination. Desensitization is important to control synaptic transmission and the development of the nervous system. In this review we discuss results related to its production, modulation and some aspects associated to models that consider it. Finally, an approach combining molecular biology and electrophysiology techniques to understand desensitization and its importance in biological systems is presented. PMID:8525756

  8. An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

    PubMed

    Salmon, A M; Bruand, C; Cardona, A; Changeux, J P; Berrih-Aknin, S

    1998-06-01

    Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subunit AChR gene. In addition to expression in muscle cells, thymic expression was observed in both mouse lines (mainly in myoid cells in Tg1 and myoid cells and epithelial cells in Tg2). After challenge with beta-gal, Tg1 mice produced Th2-dependent anti-beta-gal antibodies, while Tg2 mice were almost unresponsive. By contrast, in a proliferation assay both Tg lines were unresponsive to beta-gal. Cells from Tg1 mice produce Th2-dependent cytokine whereas cells from Tg2 mice were nonproducing in response to beta-gal. These data indicate that the level of expression in Tg1 mice could be sufficient to induce tolerance of Th1 cells but not of Th2 cells, while both populations are tolerated in Tg2 mice. These findings are compatible with the hypothesis that AChR expression is not sufficiently abundant in MG thymus to induce a full tolerance. PMID:9616205

  9. Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

    SciTech Connect

    Machaalani, R.; Ghazavi, E.; Hinton, T.; Waters, K.A.; Hennessy, A.

    2014-05-01

    Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.

  10. A new family of insect muscarinic acetylcholine receptors.

    PubMed

    Xia, R-Y; Li, M-Q; Wu, Y-S; Qi, Y-X; Ye, G-Y; Huang, J

    2016-08-01

    Most currently used insecticides are neurotoxic chemicals that target a limited number of sites and insect cholinergic neurotransmission is the major target. A potential target for insecticide development is the muscarinic acetylcholine receptor (mAChR), which is a metabotropic G-protein-coupled receptor. Insects have A- and B-type mAChRs and the five mammalian mAChRs are close to the A-type. We isolated a cDNA (CG12796) from the fruit fly, Drosophila melanogaster. After heterologous expression in Chinese hamster ovary K1 cells, CG12796 could be activated by acetylcholine [EC50 (half maximal effective concentration), 73 nM] and the mAChR agonist oxotremorine M (EC50 , 48.2 nM) to increase intracellular Ca(2+) levels. Thus, the new mAChR is coupled to Gq/11 but not Gs and Gi/o . The classical mAChR antagonists atropine and scopolamine N-butylbromide at 100 μM completely blocked the acetylcholine-induced responses. The orthologues of CG12796 can also be found in the genomes of other insects, but not in the genomes of the honeybee or parasitoid wasps. Knockdown of CG12796 in the central nervous system had no effect on male courtship behaviours. We suggest that CG12796 represents the first recognized member of a novel mAChR class. PMID:27003873

  11. Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

    PubMed

    Mineur, Y S; Brunzell, D H; Grady, S R; Lindstrom, J M; McIntosh, J M; Marks, M J; King, S L; Picciotto, M R

    2009-04-01

    High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation. PMID:19077117

  12. Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation.

    PubMed

    Imamura, Osamu; Arai, Masaaki; Dateki, Minori; Ogata, Toru; Uchida, Ryuji; Tomoda, Hiroshi; Takishima, Kunio

    2015-12-01

    Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer's disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil

  13. Design and expression of human alpha7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties.

    PubMed

    Zouridakis, Marios; Zisimopoulou, Paraskevi; Eliopoulos, Elias; Poulas, Konstantinos; Tzartos, Socrates J

    2009-02-01

    In order to facilitate structural studies of the extracellular domain (ECD) of human alpha7 nicotinic acetylcholine receptor (nAChR), we designed several mutants, since the wild-type-ECD forms large oligomers and microaggregates, and expressed them in the yeast Pichia pastoris. Mutant design was based on a 3D model of human alpha7-nAChR-ECD, constructed using as templates the X-ray crystal structure of the homologous acetylcholine-binding protein (AChBP) and the electron microscopy structure of the Torpedo alpha-nAChR-ECD. At least one mutant, mut10, carrying six single-point mutations (Phe3Tyr, Val69Thr, Cys116Ser, Ile165Thr, Val177Thr, Phe187Tyr) and the replacement of its Cys-loop with the corresponding and more hydrophilic AChBP Cys-loop, was expressed with a 4-fold higher expression yield (1.2 mg/L) than the wild-type alpha7-ECD, existing exclusively as a soluble oligomeric, probably pentameric, form, at concentrations up to at least 10 mg/mL, as judged by gel filtration and dynamic light scattering. This mutant displayed a significantly improved (125)I-alpha-bungarotoxin-binding affinity (K(d)=24 nM) compared to the wild-type-ECD (K(d)=70 nM), the binding being inhibited by unlabelled alpha-bungarotoxin, d-tubocurarine or nicotine (K(i) of 21.5 nM, 127 microM and 17.5 mM, respectively). Circular dichroism studies of mut10 revealed (a) a similar secondary structure composition ( approximately 5% alpha-helix, approximately 45% beta-sheet) to that of the AChBP, Torpedo alpha-nAChR-ECD, and mouse alpha1-nAChR-ECD, (b) a well-defined tertiary structure and (c) binding of small cholinergic ligands at micromolar concentrations. Furthermore, electron microscopy showed well-assembled, probably pentameric, particles of mut10. Finally, since deglycosylation did not alter its solubility or ligand-binding properties, mut10, in either its glycosylated or deglycosylated form, is a promising alpha7-ECD mutant for structural studies, useful for the rational drug design to

  14. Topographical studies of the nicotinic acetylcholine receptor. [Torpedo californica

    SciTech Connect

    Middlemas, D.S.

    1987-01-01

    All four subunits of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with the photoactivated hydrophobic probe, (/sup 3/H)adamantanediazirine, which selectively labels regions of integral membrane proteins in contact with the hydrocarbon core of the lipid bilayer. All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with (/sup 3/H)cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. Since the photogenerated carbene is situated near the lipid-water interface, this probe has potential as a topographic tool for mapping membrane protein structure. The labeling studies with both (/sup 3/H)adamantanediazirine and (/sup 3/H)cholesteryl diazoacetate support the concept that the acetylcholine receptor is a pseudosymmetric complex of homologous subunits, all of which interact with and span the membrane. The synthesis of the fluorine-containing agonists for the Torpedo californica nicotinic acetylcholine receptor, fluoroacetylcholine bromide and p-fluorophenyltrimethylammonium iodide, are described. It is demonstrated that both are agonists using a cation flux assay with acetylcholine receptor enriched membrane vesicles. The affinity cleavage reagent, p-thiocyanophenyltrimethylammonium iodide, specifically cleaves a peptide bond of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica. It is demonstrated that this reagent is an agonist using a cation flux assay. The cleavage is blocked by stoichiometric quantities of ..cap alpha..-bungarotoxin.

  15. Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection

    PubMed Central

    Rommel, Frank R.; Raghavan, Badrinarayanan; Paddenberg, Renate; Kummer, Wolfgang; Tumala, Susanne; Lochnit, Günter; Gieler, Uwe

    2015-01-01

    Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended. PMID:25673288

  16. Conotoxins Targeting Nicotinic Acetylcholine Receptors: An Overview

    PubMed Central

    Lebbe, Eline K. M.; Peigneur, Steve; Wijesekara, Isuru; Tytgat, Jan

    2014-01-01

    Marine snails of the genus Conus are a large family of predatory gastropods with an unparalleled molecular diversity of pharmacologically active compounds in their venom. Cone snail venom comprises of a rich and diverse cocktail of peptide toxins which act on a wide variety of ion channels such as voltage-gated sodium- (NaV), potassium- (KV), and calcium- (CaV) channels as well as nicotinic acetylcholine receptors (nAChRs) which are classified as ligand-gated ion channels. The mode of action of several conotoxins has been the subject of investigation, while for many others this remains unknown. This review aims to give an overview of the knowledge we have today on the molecular pharmacology of conotoxins specifically interacting with nAChRs along with the structure–function relationship data. PMID:24857959

  17. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    PubMed Central

    Gregory, Karen J; Sexton, Patrick M; Christopoulos, Arthur

    2007-01-01

    Muscarinic acetylcholine receptors (mAChRs) are prototypical Family A G protein coupled-receptors. The five mAChR subtypes are widespread throughout the periphery and the central nervous system and, accordingly, are widely involved in a variety of both physiological and pathophysiological processes. There currently remains an unmet need for better therapeutic agents that can selectively target a given mAChR subtype to the relative exclusion of others. The main reason for the lack of such selective mAChR ligands is the high sequence homology within the acetylcholine-binding site (orthosteric site) across all mAChRs. However, the mAChRs possess at least one, and likely two, extracellular allosteric binding sites that can recognize small molecule allosteric modulators to regulate the binding and function of orthosteric ligands. Extensive studies of prototypical mAChR modulators, such as gallamine and alcuronium, have provided strong pharmacological evidence, and associated structure-activity relationships (SAR), for a “common” allosteric site on all five mAChRs. These studies are also supported by mutagenesis experiments implicating the second extracellular loop and the interface between the third extracellular loop and the top of transmembrane domain 7 as contributing to the common allosteric site. Other studies are also delineating the pharmacology of a second allosteric site, recognized by compounds such as staurosporine. In addition, allosteric agonists, such as McN-A-343, AC-42 and N-desmethylclozapine, have also been identified. Current challenges to the field include the ability to effectively detect and validate allosteric mechanisms, and to quantify allosteric effects on binding affinity and signaling efficacy to inform allosteric modulator SAR. PMID:19305798

  18. [Probable mechanism of recognition of cholinergic ligands by acetylcholine receptors].

    PubMed

    Demushkin, V P; Kotelevtsev, Iu V; Pliashkevich, Iu G; Khramtsov, N V

    1982-01-01

    Dryding's models were used for the conformational analysis of compounds affecting muscarin-specific acetylcholine receptor and nicotin-specific acetylcholine receptor. Ammonium group and ether oxygen (3.6 A apart from the ammonium group) specifically oriented to each other were shown to be necessary structural elements to reveal muscarin-type cholinergic activity. Ammonium group along with carbonyl oxygen or its substituent (5 A distance) are the necessary structural units providing nicotin-type cholinergic activity. The presence of two hydrophobic substituents (one in the ammonium area and the other neighbouring the second active grouping) is the additional factor. The developed principles were justified by the use of a series of synthetic samples. The compounds were obtained likely favouring affinitive modification of acetylcholine receptor (dissociation constants of acetylcholine receptor complexes equalling to 10(-4)--10(-7) M-1). PMID:7070378

  19. Modal gating of muscle nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Vij, Ridhima

    Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (alpha188-alpha199, at the agonist binding site) generate heterogeneity in PO ('modes'). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except alphaY190 and alphaY198) and with different side chain substitutions at alphaP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the alphadelta agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (alphaY93, alphaW149, alphaY190, alphaY198 and deltaW57). Phenylalanine substitutions at all aromatic residues except alphaY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at deltaW57 on the complementary side but not at other aromatics. Also, by substituting four gamma subunit residues into the delta subunit on the complementary beta sheet, I found that

  20. Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

    PubMed Central

    Avilés, Evelyn C.; Pinto, Cristina; Hanna, Patricia; Ojeda, Jorge; Pérez, Viviana; De Ferrari, Giancarlo V.; Zamorano, Pedro; Albistur, Miguel; Sandoval, Daniel; Henríquez, Juan P.

    2014-01-01

    Cumulative evidence indicates that Wnt pathways play crucial and diverse roles to assemble the neuromuscular junction (NMJ), a peripheral synapse characterized by the clustering of acetylcholine receptors (AChR) on postsynaptic densities. The molecular determinants of Wnt effects at the NMJ are still to be fully elucidated. We report here that the Wnt receptor Frizzled-9 (Fzd9) is expressed in developing skeletal muscles during NMJ synaptogenesis. In cultured myotubes, gain- and loss-of-function experiments revealed that Fzd9-mediated signaling impairs the AChR-clustering activity of agrin, an organizer of postsynaptic differentiation. Overexpression of Fzd9 induced the cytosolic accumulation of β-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and β-catenin localize in the postsynaptic domain of embryonic NMJs in vivo. Our findings represent the first evidence pointing to a crucial role of a Fzd-mediated, β-catenin-dependent signaling on the assembly of the vertebrate NMJ. PMID:24860427

  1. Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

    SciTech Connect

    Ma, Xiaoli; Jia, Yanfei; Zu, Shanshan; Li, Ruisheng; Jia, Ying; Zhao, Yun; Xiao, Dongjie; Dang, Ningning; Wang, Yunshan

    2014-07-15

    By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels of α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P < 0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer. - Highlights

  2. Caenorhabditis elegans nicotinic acetylcholine receptors are required for nociception

    PubMed Central

    Cohen, Emiliano; Chatzigeorgiou, Marios; Husson, Steven J.; Steuer-Costa, Wagner; Gottschalk, Alexander; Schafer, William R.; Treinin, Millet

    2014-01-01

    Polymodal nociceptors sense and integrate information on injurious mechanical, thermal, and chemical stimuli. Chemical signals either activate nociceptors or modulate their responses to other stimuli. One chemical known to activate or modulate responses of nociceptors is acetylcholine (ACh). Across evolution nociceptors express subunits of the nicotinic acetylcholine receptor (nAChR) family, a family of ACh-gated ion channels. The roles of ACh and nAChRs in nociceptor function are, however, poorly understood. Caenorhabditis elegans polymodal nociceptors, PVD, express nAChR subunits on their sensory arbor. Here we show that mutations reducing ACh synthesis and mutations in nAChR subunits lead to defects in PVD function and morphology. A likely cause for these defects is a reduction in cytosolic calcium measured in ACh and nAChR mutants. Indeed, overexpression of a calcium pump in PVD mimics defects in PVD function and morphology found in nAChR mutants. Our results demonstrate, for the first time, a central role for nAChRs and ACh in nociceptor function and suggest that calcium permeating via nAChRs facilitates activity of several signaling pathways within this neuron. PMID:24518198

  3. Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with alpha-bungarotoxin-binding activity.

    PubMed

    Paulo, Joao A; Hawrot, Edward

    2009-10-01

    The molluscan acetylcholine-binding protein (AChBP) is a soluble homopentameric homolog of the extracellular domain of various ligand-gated ion channels. Previous studies have reported that AChBP, when fused to the ion pore domain of the serotonin receptor (5HT(3A)R), can form a functional ligand-gated chimeric channel only if the AChBP loop regions between beta-strands beta1 and beta2 (beta1-beta2), beta6 and beta7 (beta6-beta7), and beta8 and beta9 (beta8-beta9) are replaced with those of the 5HT(3A)R. To investigate further the potential interactions among these three important loop regions in a membrane- and detergent-free system, we designed AChBP constructs in which loops beta1-beta2, beta6-beta7, and beta8-beta9 of the AChBP were individually and combinatorially substituted in all permutations with the analogous loops of the 5HT(3A)R. These chimeras were expressed as secreted proteins using the Pichia pastoris yeast expression system. [(125)I]-alpha-Bungarotoxin-binding was detected in the culture media obtained from homologous recombinant clones expressing the wild-type AChBP, the beta1-beta2 loop-only chimera, and the chimera containing all three 5HT(3A)R loop substitutions. The remaining chimeras failed to show [(125)I]-alpha-bungarotoxin binding, and further analysis of cellular extracts allowed us to determine that these binding-negative chimeric constructs accumulated intracellularly and were not secreted into the culture medium. Our results demonstrate that coordinated interactions among loops beta1-beta2, beta6-beta7, and beta8-beta9 are essential for the formation of a functional ligand-binding site, as evidenced by [(125)I]-alpha-bungarotoxin-binding, and for efficient protein secretion. In addition, the constructs described here demonstrate the feasibility of utilizing soluble scaffolds to explore functionally important interactions within the extracellular domain of membrane-bound proteins. PMID:19427904

  4. Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

    PubMed Central

    Courtot, Elise; Charvet, Claude L.; Beech, Robin N.; Harmache, Abdallah; Wolstenholme, Adrian J.; Holden-Dye, Lindy; O’Connor, Vincent; Peineau, Nicolas; Woods, Debra J.; Neveu, Cedric

    2015-01-01

    Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR. PMID:26625142

  5. Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.

    PubMed

    Courtot, Elise; Charvet, Claude L; Beech, Robin N; Harmache, Abdallah; Wolstenholme, Adrian J; Holden-Dye, Lindy; O'Connor, Vincent; Peineau, Nicolas; Woods, Debra J; Neveu, Cedric

    2015-12-01

    Acetylcholine receptors are pentameric ligand-gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR. PMID:26625142

  6. Menthol Binding and Inhibition of α7-Nicotinic Acetylcholine Receptors

    PubMed Central

    Ashoor, Abrar; Nordman, Jacob C.; Veltri, Daniel; Yang, Keun-Hang Susan; Al Kury, Lina; Shuba, Yaroslav; Mahgoub, Mohamed; Howarth, Frank C.; Sadek, Bassem; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-01-01

    Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner. PMID:23935840

  7. Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

    Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. Images Fig. 3 PMID:8633003

  8. Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression

    PubMed Central

    2013-01-01

    Background Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). Results Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 μg L-1 of EMB, a drug concentration tolerated by PT lice, but

  9. Genetic Reconstitution of Functional Acetylcholine Receptor Channels in Mouse Fibroblasts

    NASA Astrophysics Data System (ADS)

    Claudio, Toni; Green, W. N.; Hartman, Deborah S.; Hayden, Deborah; Paulson, Henry L.; Sigworth, F. J.; Sine, Steven M.; Swedlund, Anne

    1987-12-01

    Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.

  10. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Tikkanen, Ritva

    2015-01-01

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles. PMID:25985102

  11. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis. PMID:26711579

  12. Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine

    PubMed Central

    Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M.; DeSimone, John A.; Lyall, Vijay

    2015-01-01

    Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol. PMID:26039516

  13. Halothane shortens acetylcholine receptor channel kinetics without affecting conductance.

    PubMed Central

    Lechleiter, J; Gruener, R

    1984-01-01

    The extracellular patch-clamp technique was used to examine how halothane, a general anesthetic, affects the properties of single nicotinic acetylcholine receptor channels of embryonic Xenopus skeletal muscle cells grown in culture. Under control conditions, single-channel events showed a bimodal distribution on the basis of current amplitudes. This distribution was maintained during exposure to halothane and its washout. In addition, the mean current value of the low-and high-amplitude channels was unaffected by the presence of the anesthetic at clinically relevant concentrations. In contrast, halothane shortened the burst durations of both channel types in a concentration-dependent manner. This shortening of burst durations may be an expression of the more rapid relaxation of the channel protein to the nonconducting state, possibly due to the disordering effect of the anesthetic on membrane lipids in which the receptor protein is embedded. This functional change, in the behavior of the synaptic receptor, provides further direct information on the mode of action of general anesthetics. Images PMID:6326154

  14. Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors

    PubMed Central

    Duguet, Thomas B.; Charvet, Claude L.; Forrester, Sean G.; Wever, Claudia M.; Dent, Joseph A.; Neveu, Cedric; Beech, Robin N.

    2016-01-01

    Helminth parasites rely on fast-synaptic transmission in their neuromusculature to experience the outside world and respond to it. Acetylcholine plays a pivotal role in this and its receptors are targeted by a wide variety of both natural and synthetic compounds used in human health and for the control of parasitic disease. The model, Caenorhabditis elegans is characterized by a large number of acetylcholine receptor subunit genes, a feature shared across the nematodes. This dynamic family is characterized by both gene duplication and loss between species. The pentameric levamisole-sensitive acetylcholine receptor has been characterized from C. elegans, comprised of five different subunits. More recently, cognate receptors have been reconstituted from multiple parasitic nematodes that are found to vary in subunit composition. In order to understand the implications of receptor composition change and the origins of potentially novel drug targets, we investigated a specific example of subunit duplication based on analysis of genome data for 25 species from the 50 helminth genome initiative. We found multiple independent duplications of the unc-29, acetylcholine receptor subunit, where codon substitution rate analysis identified positive, directional selection acting on amino acid positions associated with subunit assembly. Characterization of four gene copies from a model parasitic nematode, Haemonchus contortus, demonstrated that each copy has acquired unique functional characteristics based on phenotype rescue of transgenic C. elegans and electrophysiology of receptors reconstituted in Xenopus oocytes. We found evidence that a specific incompatibility has evolved for two subunits co-expressed in muscle. We demonstrated that functional divergence of acetylcholine receptors, driven by directional selection, can occur more rapidly than previously thought and may be mediated by alteration of receptor assembly. This phenomenon is common among the clade V parasitic

  15. Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors.

    PubMed

    Duguet, Thomas B; Charvet, Claude L; Forrester, Sean G; Wever, Claudia M; Dent, Joseph A; Neveu, Cedric; Beech, Robin N

    2016-07-01

    Helminth parasites rely on fast-synaptic transmission in their neuromusculature to experience the outside world and respond to it. Acetylcholine plays a pivotal role in this and its receptors are targeted by a wide variety of both natural and synthetic compounds used in human health and for the control of parasitic disease. The model, Caenorhabditis elegans is characterized by a large number of acetylcholine receptor subunit genes, a feature shared across the nematodes. This dynamic family is characterized by both gene duplication and loss between species. The pentameric levamisole-sensitive acetylcholine receptor has been characterized from C. elegans, comprised of five different subunits. More recently, cognate receptors have been reconstituted from multiple parasitic nematodes that are found to vary in subunit composition. In order to understand the implications of receptor composition change and the origins of potentially novel drug targets, we investigated a specific example of subunit duplication based on analysis of genome data for 25 species from the 50 helminth genome initiative. We found multiple independent duplications of the unc-29, acetylcholine receptor subunit, where codon substitution rate analysis identified positive, directional selection acting on amino acid positions associated with subunit assembly. Characterization of four gene copies from a model parasitic nematode, Haemonchus contortus, demonstrated that each copy has acquired unique functional characteristics based on phenotype rescue of transgenic C. elegans and electrophysiology of receptors reconstituted in Xenopus oocytes. We found evidence that a specific incompatibility has evolved for two subunits co-expressed in muscle. We demonstrated that functional divergence of acetylcholine receptors, driven by directional selection, can occur more rapidly than previously thought and may be mediated by alteration of receptor assembly. This phenomenon is common among the clade V parasitic

  16. Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene carveol.

    PubMed

    Lozon, Yosra; Sultan, Ahmed; Lansdell, Stuart J; Prytkova, Tatiana; Sadek, Bassem; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Millar, Neil S; Oz, Murat

    2016-04-01

    Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies. PMID:26849939

  17. Expression of a highly antigenic and native-like folded extracellular domain of the human α1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis.

    PubMed

    Niarchos, Athanasios; Zouridakis, Marios; Douris, Vassilis; Georgostathi, Assimina; Kalamida, Dimitra; Sotiriadis, Alexandros; Poulas, Konstantinos; Iatrou, Kostas; Tzartos, Socrates J

    2013-01-01

    We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies. PMID:24376846

  18. Change in desensitization of cat muscle acetylcholine receptor caused by coexpression of Torpedo acetylcholine receptor subunits in Xenopus oocytes.

    PubMed Central

    Sumikawa, K; Miledi, R

    1989-01-01

    Cat muscle acetylcholine receptors (AcChoR) expressed in Xenopus oocytes desensitized more slowly than Torpedo electric organ AcChoRs, also expressed in oocytes. To examine the bases for the different degrees of desensitization, cat-Torpedo AcChoR hybrids were formed by injecting oocytes with cat denervated muscle mRNA mixed with a large excess of cloned Torpedo AcChoR subunit mRNAs. Hybrid AcChoRs formed by coinjection of cat muscle mRNA with the Torpedo beta or delta subunit mRNAs desensitized as slowly as cat AcChoR. In contrast, the hybrid AcChoRs expressed by coinjection with the Torpedo gamma subunit mRNA desensitized much more rapidly than cat AcChoR. The AcChoRs expressed in oocytes injected with cat muscle mRNA together with the Torpedo beta, gamma, and delta subunit mRNAs desensitized as rapidly as Torpedo AcChoR, indicating that the cat alpha subunit does not play an important role in determining the slow rate of desensitization. It is concluded that the difference in the rates of desensitization of cat and Torpedo AcChoRs is determined mainly by differences in their respective gamma subunits. Images PMID:2536157

  19. Serotoninergic dorsal raphe neurons possess functional postsynaptic nicotinic acetylcholine receptors.

    PubMed

    Galindo-Charles, Luis; Hernandez-Lopez, Salvador; Galarraga, Elvira; Tapia, Dagoberto; Bargas, José; Garduño, Julieta; Frías-Dominguez, Carmen; Drucker-Colin, René; Mihailescu, Stefan

    2008-08-01

    Very few neurons in the telencephalon have been shown to express functional postsynaptic nicotinic acetylcholine receptors (nAChRs), among them, the noradrenergic and dopaminergic neurons. However, there is no evidence for postsynaptic nAChRs on serotonergic neurons. In this study, we asked if functional nAChRs are present in serotonergic (5-HT) and nonserotonergic (non-5-HT) neurons of the dorsal raphe nucleus (DRN). In rat midbrain slices, field stimulation at the tegmental pedunculopontine (PPT) nucleus evoked postsynaptic currents (eEPSCs) with different components in DRN neurons. After blocking the glutamatergic and GABAergic components, the remaining eEPSCs were blocked by mecamylamine and reduced by either the selective alpha7 nAChR antagonist methyllycaconitine (MLA) or the selective alpha4beta2 nAChR antagonist dihydro-beta-eritroidine (DHbetaE). Simultaneous addition of MLA and DHbetaE blocked all eEPSCs. Integrity of the PPT-DRN pathway was assessed by both anterograde biocytin tracing and antidromic stimulation from the DRN. Inward currents evoked by the direct application of acetylcholine (ACh), in the presence of atropine and tetrodotoxin, consisted of two kinetically different currents: one was blocked by MLA and the other by DHbetaE; in both 5-HT and non-5-HT DR neurons. Analysis of spontaneous (sEPSCs) and evoked (eEPSCs) synaptic events led to the conclusion that nAChRs were located at the postsynaptic membrane. The possible implications of these newly described nAChRs in various physiological processes and behavioral events, such as the wake-sleep cycle, are discussed. PMID:18512214

  20. Alpha9 nicotinic acetylcholine receptors and the treatment of pain.

    PubMed

    McIntosh, J Michael; Absalom, Nathan; Chebib, Mary; Elgoyhen, Ana Belén; Vincler, Michelle

    2009-10-01

    Chronic pain is a vexing worldwide problem that causes substantial disability and consumes significant medical resources. Although there are numerous analgesic medications, these work through a small set of molecular mechanisms. Even when these medications are used in combination, substantial amounts of pain often remain. It is therefore highly desirable to develop treatments that work through distinct mechanisms of action. While agonists of nicotinic acetylcholine receptors (nAChRs) have been intensively studied, new data suggest a role for selective antagonists of nAChRs. alpha-Conotoxins are small peptides used offensively by carnivorous marine snails known as Conus. A subset of these peptides known as alpha-conotoxins RgIA and Vc1.1 produces both acute and long lasting analgesia. In addition, these peptides appear to accelerate the recovery of function after nerve injury, possibly through immune mediated mechanisms. Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of alpha9alpha10 nAChRs. A recent study also reported that these alpha9alpha10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on alpha9* nAChRs.(1). PMID:19477168

  1. A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1 Acetylcholine Receptors Affects Plasma Membrane Expression

    PubMed Central

    Ehlert, Frederick J.; Shults, Crystal A.

    2010-01-01

    We investigated the functional role of a conserved motif, F(x)6LL, in the membrane proximal C-tail of the human muscarinic M1 (hM1) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence 423FRDTFRLLL431. Wild-type and mutant hM1 receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [3H]N-methylscopolamine binding assays. The plasma membrane expression of hM1 receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM1 receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L430 and L431 play a role in folding hM1 receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V46 and L47, that, when mutated, reduce the plasma membrane expression of hM1 receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM1 receptors to achieve a transport-competent state. PMID:19841475

  2. Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Tochitsky, Ivan; Banghart, Matthew R.; Mourot, Alexandre; Yao, Jennifer Z.; Gaub, Benjamin; Kramer, Richard H.; Trauner, Dirk

    2012-02-01

    Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3β4 and α4β2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.

  3. Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

    PubMed Central

    Tochitsky, Ivan; Banghart, Matthew R.; Mourot, Alexandre; Yao, Jennifer Z.; Gaub, Benjamin

    2016-01-01

    Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3β4 and α4β2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels. PMID:22270644

  4. Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors

    PubMed Central

    Moaddel, Ruin; Abdrakhmanova, Galia; Kozak, Joanna; Jozwiak, Krzysztof; Toll, Lawrence; Jimenez, Lucita; Rosenberg, Avraham; Tran, Thao; Xiao, Yingxian; Zarate, Carlos A.; Wainer, Irving W.

    2012-01-01

    The effect of the (R,S)-ketamine metabolites (R,S)-norketamine, (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)- hydroxynorketamine on the activity of α7 and α3β4 neuronal nicotinic acetylcholine receptors was investigated using patch-clamp techniques. The data indicated that (R,S)-dehydronorketamine inhibited acetylcholine-evoked currents in α7-nicotinic acetylcholine receptor, IC50 = 55 ± 6 nM, and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-norketamine also inhibited α7-nicotinic acetylcholine receptor function at concentrations ≤1μM, while (R,S)-ketamine was inactive at these concentrations. The inhibitory effect of (R,S)-dehydronorketamine was voltage-independent and the compound did not competitively displace selective α7-nicotinic acetylcholine receptor ligands [125I]-α-bungarotoxin and [3H]-epibatidine indicating that (R,S)-dehydronorketamine is a negative allosteric modulator of the α7-nicotinic acetylcholine receptor. (R,S)-Ketamine and (R,S)-norketamine inhibited (S)-nicotine-induced whole-cell currents in cells expressing α3β4-nicotinic acetylcholine receptor, IC50 3.1 and 9.1μM, respectively, while (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were weak inhibitors, IC50 >100μM. The binding affinities of (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine at the NMDA receptor were also determined using rat brain membranes and the selective NMDA receptor antagonist [3H]-MK-801. The calculated Ki values were 38.95 μM for (S)-dehydronorketamine, 21.19 μM for (2S,6S)-hydroxynorketamine and > 100 μM for (2R,6R)-hydroxynorketamine. The results suggest that the inhibitory activity of ketamine metabolites at the α7-nicotinic acetylcholine receptor may contribute to the clinical effect of the drug. PMID:23183107

  5. Primary structure of nicotinic acetylcholine receptor. Final report, 9 April 1989-6 April 1992

    SciTech Connect

    Patrick, J.W.

    1992-05-06

    Signals are transmitted between cells in the brain using neurotransmitters and neurotransmitter receptors. Poisons that interfere with this process stop normal brain function and often kill nerve cells. One of the neurotransmitters used in the mammalian brain is acetylcholine. We discovered that there is a large number of different nicotinic receptors for the neurotransmitter acetylcholine, each with its different properties. We used recombinant DNA technology to clone and sequence the gene transcripts that encode the subunits of these receptors. From these sequences we deduced the primary structures of the nicotinic receptor subunits. We also used the cDNA clones to determine which brain loci express the respective genes. We have expressed the clones in the Xenopus oocyte and have demonstrated that each functional combination of subunits has a unique pharmacology Unlike their homologs at the neuromuscular junction, the nicotinic acetylcholine receptors in the brain are exceptionally permeable to calcium. This property suggests that these receptors may play an important role in regulating calcium-dependent cytoplasmic processes and that they may be important contributors to use-dependent cell death.

  6. Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors.

    PubMed

    Jakubík, J; Bacáková, L; El-Fakahany, E E; Tucek, S

    1997-07-01

    It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in

  7. Riluzole blocks human muscle acetylcholine receptors

    PubMed Central

    Deflorio, Cristina; Palma, Eleonora; Conti, Luca; Roseti, Cristina; Manteca, Alessia; Giacomelli, Elena; Catalano, Myriam; Limatola, Cristina; Inghilleri, Maurizio; Grassi, Francesca

    2012-01-01

    Riluzole, the only drug available against amyotrophic lateral sclerosis (ALS), has recently been shown to block muscle ACh receptors (AChRs), raising concerns about possible negative side-effects on neuromuscular transmission in treated patients. In this work we studied riluzole's impact on the function of muscle AChRs in vitro and on neuromuscular transmission in ALS patients, using electrophysiological techniques. Human recombinant AChRs composed of α1β1δ subunits plus the γ or ɛ subunit (γ- or ɛ-AChR) were expressed in HEK cells or Xenopus oocytes. In both preparations, riluzole at 0.5 μm, a clinically relevant concentration, reversibly reduced the amplitude and accelerated the decay of ACh-evoked current if applied before coapplication with ACh. The action on γ-AChRs was more potent and faster than on ɛ-AChRs. In HEK outside-out patches, riluzole-induced block of macroscopic ACh-evoked current gradually developed during the initial milliseconds of ACh presence. Single channel recordings in HEK cells and in human myotubes from ALS patients showed that riluzole prolongs channel closed time, but has no effect on channel conductance and open duration. Finally, compound muscle action potentials (CMAPs) evoked by nerve stimulation in ALS patients remained unaltered after a 1 week suspension of riluzole treatment. These data indicate that riluzole, while apparently safe with regard to synaptic transmission, may affect the function of AChRs expressed in denervated muscle fibres of ALS patients, with biological consequences that remain to be investigated. PMID:22431338

  8. Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine-Binding Protein as a Structural Surrogate.

    PubMed

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette S; Balle, Thomas

    2016-06-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in the control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for the development of drugs against a number of mental health disorders and for marketed smoking cessation aids. Unfortunately, drug discovery has been hampered by difficulties in obtaining sufficiently selective compounds. Together with functional complexity of the receptors, this has made it difficult to obtain drugs with sufficiently high-target to off-target affinity ratios. The recent and ongoing progress in structural studies holds promise to help understand structure-function relationships of nAChR drugs at the atomic level. This will undoubtedly lead to the design of more efficient drugs with fewer side effects. As a high-resolution structure of a nAChR is yet to be determined, structural studies are to a large extent based on acetylcholine-binding proteins (AChBPs) that despite low overall sequence identity display a high degree of conservation of overall structure and amino acids at the ligand-binding site. Further, AChBPs reproduce relative binding affinities of ligands at nAChRs. Over the past decade, AChBPs have been used extensively as models for nAChRs and have aided the understanding of drug receptor interactions at nAChRs significantly. PMID:26572235

  9. Nicotinic acetylcholine receptors: upregulation, age-related effects and associations with drug use

    PubMed Central

    Melroy-Greif, W. E.; Stitzel, J. A.; Ehringer, M. A.

    2016-01-01

    Nicotinic acetylcholine receptors are ligand-gated ion channels that exogenously bind nicotine. Nicotine produces rewarding effects by interacting with these receptors in the brain’s reward system. Unlike other receptors, chronic stimulation by an agonist induces an upregulation of receptor number that is not due to increased gene expression in adults; while upregulation also occurs during development and adolescence there have been some opposing findings regarding a change in corresponding gene expression. These receptors have also been well studied with regard to human genetic associations and, based on evidence suggesting shared genetic liabilities between substance use disorders, numerous studies have pointed to a role for this system in comorbid drug use. This review will focus on upregulation of these receptors in adulthood, adolescence and development, as well as the findings from human genetic association studies which point to different roles for these receptors in risk for initiation and continuation of drug use. PMID:26351737

  10. Role of dopamine receptor and muscarinic acetylcholine receptor blockade in the antiapomorphine action of neuroleptics

    SciTech Connect

    Zharkovskii, A.M.; Langel, Yu.L.; Chereshka, K.S.; Zharkovskaya, T.A.

    1987-08-01

    The authors analyze the role of dopamine and muscarinic acetylcholine receptor blocking components in the antistereotypic action of neuroleptics with different chemical structure. To determine dopamine-blocking activity in vitro, binding of /sup 3/H-spiperone with membranes of the rat striatum was measured. To study the blocking action of the substances on muscarinic acetylcholine receptors, binding of /sup 3/H-quinuclidinyl benzylate with brain membranes was chosen.

  11. Wnt proteins regulate acetylcholine receptor clustering in muscle cells

    PubMed Central

    2012-01-01

    Background The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish. Results In the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR) cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4. Conclusions These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4. PMID:22309736

  12. END-PLATE ACETYLCHOLINE RECEPTOR: STRUCTURE, MECHANISM, PHARMACOLOGY, AND DISEASE

    PubMed Central

    Sine, Steven M.

    2012-01-01

    The synapse is a localized neurohumoral contact between a neuron and an effector cell and may be considered the quantum of fast intercellular communication. Analogously, the postsynaptic neurotransmitter receptor may be considered the quantum of fast chemical to electrical transduction. Our understanding of postsynaptic receptors began to develop about a hundred years ago with the demonstration that electrical stimulation of the vagus nerve released acetylcholine and slowed the heart beat. During the past 50 years, advances in understanding postsynaptic receptors increased at a rapid pace, owing largely to studies of the acetylcholine receptor (AChR) at the motor endplate. The endplate AChR belongs to a large superfamily of neurotransmitter receptors, called Cys-loop receptors, and has served as an exemplar receptor for probing fundamental structures and mechanisms that underlie fast synaptic transmission in the central and peripheral nervous systems. Recent studies provide an increasingly detailed picture of the structure of the AChR and the symphony of molecular motions that underpin its remarkably fast and efficient chemoelectrical transduction. PMID:22811427

  13. Abundance, distribution, mobility and oligomeric state of M₂ muscarinic acetylcholine receptors in live cardiac muscle.

    PubMed

    Nenasheva, Tatiana A; Neary, Marianne; Mashanov, Gregory I; Birdsall, Nigel J M; Breckenridge, Ross A; Molloy, Justin E

    2013-04-01

    M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHO(M2) cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~1μm(-2), increasing at birth (to ~3μm(-2)) and decreasing back to ~1μm(-2) after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5-10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening. PMID:23357106

  14. Abundance, distribution, mobility and oligomeric state of M2 muscarinic acetylcholine receptors in live cardiac muscle

    PubMed Central

    Nenasheva, Tatiana A.; Neary, Marianne; Mashanov, Gregory I.; Birdsall, Nigel J.M.; Breckenridge, Ross A.; Molloy, Justin E.

    2013-01-01

    M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHOM2 cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~ 1 μm− 2, increasing at birth (to ~ 3 μm− 2) and decreasing back to ~ 1 μm− 2 after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5–10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening. PMID:23357106

  15. A comparison of MyoD1 and fetal acetylcholine receptor expression in childhood tumors and normal tissues: implications for the molecular diagnosis of minimal disease in rhabdomyosarcomas.

    PubMed

    Gattenloehner, S; Dockhorn-Dworniczak, B; Leuschner, I; Vincent, A; Müller-Hermelink, H K; Marx, A

    1999-11-01

    Detection of minimal residual disease or micrometastases in rhabdomyosarcoma (RMS) has been an unresolved problem in 70 to 80% of RMS patients. In patients with alveolar type RMS, which harbors chromosomal translocations and produces tumor-specific fusion products, polymerase chain reaction (PCR)-based diagnosis is clear-cut. In the more frequent embryonal RMS, however, no such PCR-based marker has been described. Recently it has been suggested that the PCR-based detection of MyoD1 may be a valuable adjunct in the diagnosis of minimal disease in embryonal RMS. We report here that MyoD1 mRNA is not specific for RMS, but can be amplified from ex vivo samples of many other childhood tumors and some normal tissues. By contrast, simultaneous amplification of alpha and gamma subunit message of the fetal type acetylcholine receptor (AChR), by a novel duplex PCR, and the quantification of both transcripts resulting in a alpha/gammaAChR ratio <1 was 100% sensitive in alveolar (n = 8) and embryonal (n = 10) RMS. Moreover, gammaAChR was not detected in other childhood (n = 27) or adult tumors (n = 12), or normal tissues, except thymus. The high sensitivity and specificity of the method were confirmed by the successful detection of five cases of cytologically or molecularly verified RMS bone marrow micrometastases among 47 bone marrow samples from childhood tumor patients. By contrast, MyoD1 showed no amplification because of its low level of transcription. We conclude that mRNA of the fetal type AChR is a more specific and (about 100 times) more sensitive marker for the molecular detection of RMS than MyoD1, and thus appears to be a promising candidate for the detection of minimal disease in RMS lacking tumor-specific translocations. PMID:11272905

  16. Immunochemical studies of the muscarinic acetylcholine receptor.

    PubMed

    André, C; Marullo, S; Guillet, J G; Convents, A; Lauwereys, M; Kaveri, S; Hoebeke, J; Strosberg, A D

    1987-01-01

    Muscarinic receptors have been purified from calf forebrain plasma cell membranes by affinity chromatography on a dexetimide-agarose gel. SDS-PAGE analysis showed a single 70 kDa band. Monoclonal antibodies have been prepared against these affinity purified 70 kDa protein(s). One antibody, M-35, immunoprecipitated up to 80% of digitonin-solubilized muscarinic receptors. M-35 had agonist-like effects on guinea-pig myometrium: it increased the intracellular cyclic GMP content, decreased prostaglandin-induced cyclic AMP accumulation and caused muscle contractions. The two first effects were inhibited by atropine. M-35 was used to visualize muscarinic receptors at the surface of human fibroblastic cells. In the particular cell line used, the receptors have a low affinity for pirenzepine, were negatively coupled to adenylate cyclase and mediated increase in the phosphatidyl-inositol breakdown. PMID:3040987

  17. Acetylcholine-induced cation translocation across cell membranes and inactivation of the acetylcholine receptor: chemical kinetic measurements in the millisecond time region.

    PubMed Central

    Cash, D J; Aoshima, H; Hess, G P

    1981-01-01

    Acetylcholine-induced flux of inorganic ions across membranes and inactivation of the acetylcholine receptor were measured at pH 7.0, 1 degrees C, over a 5000-fold concentration range of acetylcholine. Receptor-containing electroplax membrane vesicles prepared from Electrophorus electricus and a quench-flow technique were used, allowing flux to be measured in the 2-msec to 1-min time region. Five different measurements were made: (i) rate of ion translocation with the active state of the receptor, (ii) rate of the slower ion translocation after equilibration of active and inactive receptor states, (iii) rate of inactivation, (iv) equilibrium between active and inactive forms of the receptor, and (v) reactivation of inactivated receptor. The kinetics of the steps in the receptor-controlled ion flux follow single-exponential rate laws, and simple analytical expressions for their ligand concentration dependence can be used. Thus, the rate and equilibrium constants in a scheme that relates the ligand binding steps to ion translocation could be evaluated. It was found that the dependence of the receptor-controlled ion translocation over the concentration range investigated obeys the integrated rate equation based on the proposed mechanism. The flux rate before inactivation was approximately 10(7) ions sec-1 per receptor, which is comparable with that measured electrophysiologically in muscle cells. The half-time of inactivation is approximately 100 msec when the receptor is saturated with acetylcholine. The specific reaction rate of the ion translocation (J) is 3 X 10(7) M-1 sec-1. The results support a minimum reaction mechanism previously proposed on the basis of experiments in which carbamylcholine was used. PMID:6267581

  18. Neuronal nicotinic acetylcholine receptor modulation by general anesthetics.

    PubMed

    Flood, P; Role, L W

    1998-11-23

    1. General anesthetics have been shown to inhibit synaptic transmission in multiple areas of the central and peripheral nervous systems. 2. The mechanism of inhibition is not well understood. 3. It has become clear that general anesthetics modulate the function of members of the ligand gated ion channel superfamily, including receptors for GABA(A), glycine (Harrison et al., Mol. Pharmacol. 44(3), 1993, 628-632) and 5HT3 (Zhou and Lovinger, J. Pharmacol. Exp. Therap. 278(2), 1996, 732-740). 4. Studies of the activity of general anesthetics on recombinant neuronal nicotinic acetylcholine receptors have added this receptor family to those potently inhibited by general anesthetics (Flood et al., Anesthesiology 86(4), 1997, 859-865; Violet et al., Anesthesiology 86(4), 1997, 866-874). 5. Studies of neuronal nicotinic receptors in native neurons suggest that the inhibition of these receptors by general anesthetics at low clinical concentrations may be biologically significant (Nicoll, Science 199(4327), 1978, 451-452). 6. Recent work on neuronal nicotinic acetylcholine receptors in the central nervous system suggests that their primary role may be to modulate synaptic transmission (Role and Berg, Neuron 16(6), 1996, 1077-1085). 7. Thus, inhibition of nicotinic modulation in the central nervous system may result in inhibition of synaptic transmission and some of the behavioral consequences of general anesthesia. PMID:10049135

  19. Use of acetylcholine mustard to study allosteric interactions at the M2 muscarinic receptor

    PubMed Central

    Suga, Hinako; Figueroa, Katherine W.; Ehlert, Frederick J.

    2008-01-01

    We explored the interaction of a nitrogen mustard derivative of acetylcholine with the human M2 muscarinic receptor expressed in CHO cells using the muscarinic radioligand, [3H]N-methylscopolamine. Acetylcholine mustard caused a concentration-dependent, first order loss of [3H]N-methylscopolamine binding at 37°C, with the half maximal rate constant occurring at 24 µM and a maximal rate constant of 0.16 min−1. We examined the effects of various ligands on the rate of alkylation of M2 receptors by acetylcholine mustard. N-methylscopolamine and McN-A-343 (4-(trimethylamino)-2-butynyl-(3-chlorophenyl)carbamate) competitively slowed the rate of alkylation, whereas the inhibition by gallamine reached a plateau at high concentrations, indicating allosteric inhibition. In contrast, WIN 51708 (17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) had no effect. We also measured the inhibition of [3H]NMS binding by acetylcholine mustard at 0°C, conditions under which there is little or no detectable covalent binding. In these experiments, the dissociation constant of the aziridinium ion of acetylcholine mustard was estimated to be 12.3 µM. In contrast, the parent mustard and alcoholic hydrolysis product of acetylcholine mustard were without effect. Our results show that measurement of the effects of ligands on the rate of inactivation of the orthosteric site by a small site-directed electrophile is a powerful method for discriminating competitive inhibition from allosterism. PMID:18682569

  20. Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection: Evaluation in Murine Skin.

    PubMed

    Rommel, Frank R; Raghavan, Badrinarayanan; Paddenberg, Renate; Kummer, Wolfgang; Tumala, Susanne; Lochnit, Günter; Gieler, Uwe; Peters, Eva M J

    2015-05-01

    Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended. PMID:25673288

  1. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit

    SciTech Connect

    Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. )

    1988-03-01

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

  2. Mechanisms of Barbiturate Inhibition of Acetylcholine Receptor Channels

    PubMed Central

    Dilger, James P.; Boguslavsky, Rebecca; Barann, Martin; Katz, Tamir; Vidal, Ana Maria

    1997-01-01

    We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts is 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 μM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 μM, n = 1.09; for barbital, Ki = 1900 μM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. <0.1 ms at the Ki). Pentobarbital binds ≥10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at >100 μM pentobarbital. PMID:9089445

  3. Mechanisms of barbiturate inhibition of acetylcholine receptor channels.

    PubMed

    Dilger, J P; Boguslavsky, R; Barann, M; Katz, T; Vidal, A M

    1997-03-01

    We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts in 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 microM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 microM, n = 1.09; for barbital, Ki = 1900 microM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. < 0.1 ms at the Ki). Pentobarbital binds > or = 10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at > 100 microM pentobarbital. PMID:9089445

  4. Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor.

    PubMed

    Barchan, D; Asher, O; Tzartos, S J; Fuchs, S; Souroujon, M C

    1998-02-01

    Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR alpha subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR alpha subunit (H alpha1-210), and to portions of it that encompass either the MIR (H alpha1-121) or the ligand binding site of AChR (H alpha122-210), and studied their ability to interfere with the immunopathological anti-AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S-transferase. Fragments H alpha1-121 and H alpha1-210 protected AChR in TE671 cells against accelerated degradation induced by the anti-MIR monoclonal antibody (mAb)198 in a dose-dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti-MIR mAb and by polyclonal rat anti-AChR antibodies. Fragments H alpha1-121 and H alpha1-210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment H alpha122-210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen-specific therapy of myasthenia. PMID:9521072

  5. Galantamine-induced amyloid-{beta} clearance mediated via stimulation of microglial nicotinic acetylcholine receptors.

    PubMed

    Takata, Kazuyuki; Kitamura, Yoshihisa; Saeki, Mana; Terada, Maki; Kagitani, Sachiko; Kitamura, Risa; Fujikawa, Yasuhiro; Maelicke, Alfred; Tomimoto, Hidekazu; Taniguchi, Takashi; Shimohama, Shun

    2010-12-17

    Reduction of brain amyloid-β (Aβ) has been proposed as a therapeutic target for Alzheimer disease (AD), and microglial Aβ phagocytosis is noted as an Aβ clearance system in brains. Galantamine is an acetylcholinesterase inhibitor approved for symptomatic treatment of AD. Galantamine also acts as an allosterically potentiating ligand (APL) for nicotinic acetylcholine receptors (nAChRs). APL-binding site is located close to but distinct from that for acetylcholine on nAChRs, and FK1 antibody specifically binds to the APL-binding site without interfering with the acetylcholine-binding site. We found that in human AD brain, microglia accumulated on Aβ deposits and expressed α7 nAChRs including the APL-binding site recognized with FK1 antibody. Treatment of rat microglia with galantamine significantly enhanced microglial Aβ phagocytosis, and acetylcholine competitive antagonists as well as FK1 antibody inhibited the enhancement. Thus, the galantamine-enhanced microglial Aβ phagocytosis required the combined actions of an acetylcholine competitive agonist and the APL for nAChRs. Indeed, depletion of choline, an acetylcholine-competitive α7 nAChR agonist, from the culture medium impeded the enhancement. Similarly, Ca(2+) depletion or inhibition of the calmodulin-dependent pathways for the actin reorganization abolished the enhancement. These results suggest that galantamine sensitizes microglial α7 nAChRs to choline and induces Ca(2+) influx into microglia. The Ca(2+)-induced intracellular signaling cascades may then stimulate Aβ phagocytosis through the actin reorganization. We further demonstrated that galantamine treatment facilitated Aβ clearance in brains of rodent AD models. In conclusion, we propose a further advantage of galantamine in clinical AD treatment and microglial nAChRs as a new therapeutic target. PMID:20947502

  6. Effects of alpha-7 nicotinic acetylcholine receptor positive allosteric modulator on lipopolysaccharide-induced neuroinflammatory pain in mice.

    PubMed

    Abbas, Muzaffar; Rahman, Shafiqur

    2016-07-15

    Evidence indicates that microglial activation contributes to the pathophysiology and maintenance of neuroinflammatory pain involving central nervous system alpha-7 nicotinic acetylcholine receptors. The objective of the present study was to determine the effects of 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS), an alpha-7 nicotinic acetylcholine receptor positive allosteric modulator (PAM), on tactile allodynia and thermal hyperalgesia following lipopolysaccharide (LPS)-induced microglial activation in hippocampus, a neuroinflammatory pain model in mice. In addition, we examined the effects of TQS on microglial activation marker, an ionized calcium-binding adapter molecule 1 (Iba-1), in the hippocampus may be associated with neuroinflammatory pain. Pretreatment of TQS (4mg/kg) significantly reduced LPS (1mg/kg)-induced tactile allodynia and thermal hyperalgesia. Moreover, pretreatment of methyllycaconitine (3mg/kg) significantly reversed TQS-induced antiallodynic and antihyperalgesic responses indicating the involvement of alpha-7 nicotinic acetylcholine receptor. Pretreatment of TQS significantly decreased LPS-induced increased in hippocampal Iba-1 expression. Overall, these results suggest that TQS reduces LPS-induced neuroinflammatory pain like symptoms via modulating microglial activation likely in the hippocampus and/or other brain region by targeting alpha-7 nicotinic acetylcholine receptor. Therefore, alpha-7 nicotinic acetylcholine receptor PAM such as TQS could be a potential drug candidate for the treatment of neuroinflammatory pain. PMID:27154173

  7. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    SciTech Connect

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K.

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  8. Counting Bungarotoxin Binding Sites of Nicotinic Acetylcholine Receptors in Mammalian Cells with High Signal/Noise Ratios

    PubMed Central

    Simonson, Paul D.; DeBerg, Hannah A.; Ge, Pinghua; Alexander, John K.; Jeyifous, Okunola; Green, William N.; Selvin, Paul R.

    2010-01-01

    Nicotinic acetylcholine receptors are some of the most studied synaptic proteins; however, many questions remain that can only be answered using single molecule approaches. Here we report our results from single α7 and neuromuscular junction type nicotinic acetylcholine receptors in mammalian cell membranes. By labeling the receptors with fluorophore-labeled bungarotoxin, we can image individual receptors and count the number of bungarotoxin-binding sites in receptors expressed in HEK 293 cells. Our results indicate that there are two bungarotoxin-binding sites in neuromuscular junction receptors, as expected, and five in α7 receptors, clarifying previous uncertainty. This demonstrates a valuable technique for counting subunits in membrane-bound proteins at the single molecule level, with nonspecialized optics and with higher signal/noise ratios than previous fluorescent protein-based techniques. PMID:21081055

  9. Cholinergic modulation of dopamine pathways through nicotinic acetylcholine receptors.

    PubMed

    de Kloet, Sybren F; Mansvelder, Huibert D; De Vries, Taco J

    2015-10-15

    Nicotine addiction is highly prevalent in current society and is often comorbid with other diseases. In the central nervous system, nicotine acts as an agonist for nicotinic acetylcholine receptors (nAChRs) and its effects depend on location and receptor composition. Although nicotinic receptors are found in most brain regions, many studies on addiction have focused on the mesolimbic system and its reported behavioral correlates such as reward processing and reinforcement learning. Profound modulatory cholinergic input from the pedunculopontine and laterodorsal tegmentum to dopaminergic midbrain nuclei as well as local cholinergic interneuron projections to dopamine neuron axons in the striatum may play a major role in the effects of nicotine. Moreover, an indirect mesocorticolimbic feedback loop involving the medial prefrontal cortex may be involved in behavioral characteristics of nicotine addiction. Therefore, this review will highlight current understanding of the effects of nicotine on the function of mesolimbic and mesocortical dopamine projections in the mesocorticolimbic circuit. PMID:26208783

  10. Coexpressed D1- and D2-Like Dopamine Receptors Antagonistically Modulate Acetylcholine Release in Caenorhabditis elegans

    PubMed Central

    Allen, Andrew T.; Maher, Kathryn N.; Wani, Khursheed A.; Betts, Katherine E.; Chase, Daniel L.

    2011-01-01

    Dopamine acts through two classes of G protein-coupled receptor (D1-like and D2-like) to modulate neuron activity in the brain. While subtypes of D1- and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interacts to modulate the activity of the neuron in which they are expressed. D1- and D2-like dopamine receptors are also coexpressed in the cholinergic ventral-cord motor neurons of Caenorhabditis elegans. To begin to understand how coexpressed dopamine receptors interact to modulate neuron activity, we performed a genetic screen in C. elegans and isolated mutants defective in dopamine response. These mutants were also defective in behaviors mediated by endogenous dopamine signaling, including basal slowing and swimming-induced paralysis. We used transgene rescue experiments to show that defects in these dopamine-specific behaviors were caused by abnormal signaling in the cholinergic motor neurons. To investigate the interaction between the D1- and D2-like receptors specifically in these cholinergic motor neurons, we measured the sensitivity of dopamine-signaling mutants and transgenic animals to the acetylcholinesterase inhibitor aldicarb. We found that D2 signaling inhibited acetylcholine release from the cholinergic motor neurons while D1 signaling stimulated release from these same cells. Thus, coexpressed D1- and D2-like dopamine receptors act antagonistically in vivo to modulate acetylcholine release from the cholinergic motor neurons of C. elegans. PMID:21515580

  11. Acetylcholine receptors and cholinergic ligands: biochemical and genetic aspects in Torpedo californica and Drosophila melanogaster

    SciTech Connect

    Rosenthal, L.S.

    1987-01-01

    This study evaluates the biochemical and genetic aspects of the acetylcholine receptor proteins and cholinergic ligands in Drosophila melanogaster and Torpedo californica. Included are (1) a comparative study of nicotinic ligand-induced cation release from acetylcholine receptors isolated from Torpedo californica and from Drosophila melanogaster, (2) solution studies of the cholinergic ligands, nikethamide and ethamivan, aimed at measuring internal molecular rotational barriers in solvents of different polarity; and (3) the isolation and characterization of the gene(s) for the acetylcholine receptor in Drosophila melasogaster. Acetylcholine receptor proteins isolated from Drosphila melanogaster heads were found to behave kinetically similar (with regards to cholinergic ligand-induced /sup 155/Eu:/sup 3 +/ displacement from prelabeled proteins) to receptor proteins isolated from Torpedo californica electric tissue, providing additional biochemical evidence for the existence of a Drosophila acetylcholine receptor.

  12. Amygdala kindling-induced seizures selectively impair spatial memory. 2. Effects on hippocampal neuronal and glial muscarinic acetylcholine receptor.

    PubMed

    Beldhuis, H J; Everts, H G; Van der Zee, E A; Luiten, P G; Bohus, B

    1992-10-01

    The muscarinic acetylcholine receptor is linked via hydrolysis of phosphoinositides to the protein kinase C pathway. In a preceding paper (Beldhuis, H. J. A., H. G. J. Everts, E. A. Vander Zee, P. G. M. Luiten, and B. Bohus (1992) Amygdala kindling-induced seizures selectively impair spatial memory. 1. Behavioral characteristics and effects on hippocampal neuronal protein kinase C isoforms. Hippocampus 2:397-410), the role of different isoforms of protein kinase C in neurobiological processes associated with plasticity was studied using both a spatial learning paradigm and amygdala kindling in the rat. This study extended the findings on protein kinase C activity to the level of the muscarinic acetylcholine receptor. Rats were trained in a spatial learning paradigm and kindled simultaneously in the amygdala to develop generalized motor convulsions. Control rats were trained only in the spatial learning paradigm to acquire stable working and reference memory performance. Alteration in the expression of the muscarinic acetylcholine receptor was investigated using a monoclonal antibody to muscarinic acetylcholine receptor proteins. Trained control rats that were exposed repeatedly to the spatial learning paradigm showed an increase in immunoreactivity for the muscarinic acetylcholine receptor located in the same hippocampal regions in which the protein kinase C activity was increased. In fully kindled rats, however, this increase located in principal neurons was absent, whereas expression of muscarinic acetylcholine receptor proteins was increased in hippocampal astrocytes. Moreover, fully kindled rats showed an impairment in reference memory performance as compared to trained control rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1308197

  13. Identification of subunits of acetylcholine receptor that interact with a cholesterol photoaffinity probe

    SciTech Connect

    Middlemas, D.S.; Raftery, M.A.

    1987-03-10

    All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with (/sup 3/H)cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. This investigation also demonstrates that this probe is a useful reagent for studying the interaction of cholesterol with membrane proteins.

  14. Cell-surface translational dynamics of nicotinic acetylcholine receptors

    PubMed Central

    Barrantes, Francisco J.

    2014-01-01

    Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor–receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional “trapping,” and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain α7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the α7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain. PMID

  15. Purification of muscarinic acetylcholine receptors by affinity chromatography.

    PubMed Central

    André, C; De Backer, J P; Guillet, J C; Vanderheyden, P; Vauquelin, G; Strosberg, A D

    1983-01-01

    Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein. Images Fig. 3. PMID:6605245

  16. Differential effects of subtype-specific nicotinic acetylcholine receptor agonists on early and late hippocampal LTP.

    PubMed

    Kroker, Katja S; Rast, Georg; Rosenbrock, Holger

    2011-12-01

    Brain nicotinic acetylcholine receptors are involved in several neuropsychiatric disorders, e.g. Alzheimer's and Parkinson's diseases, Tourette's syndrome, schizophrenia, depression, autism, attention deficit hyperactivity disorder, and anxiety. Currently, approaches selectively targeting the activation of specific nicotinic acetylcholine receptors are in clinical development for treatment of memory impairment of Alzheimer's disease patients. These are α4β2 and α7 nicotinic acetylcholine receptor agonists which are believed to enhance cholinergic and glutamatergic neurotransmission, respectively. In order to gain a better insight into the mechanistic role of these two nicotinic acetylcholine receptors in learning and memory, we investigated the effects of the α4β2 nicotinic acetylcholine receptor agonist TC-1827 and the α7 nicotinic acetylcholine receptor partial agonist SSR180711 on hippocampal long-term potentiation (LTP), a widely accepted cellular experimental model of memory formation. Generally, LTP is distinguished in an early and a late form, the former being protein-synthesis independent and the latter being protein-synthesis dependent. TC-1827 was found to increase early LTP in a bell-shaped dose dependent manner, but did not affect late LTP. In contrast, the α7 nicotinic acetylcholine receptor partial agonist SSR180711 showed enhancing effects on both early and late LTP in a bell-shaped manner. Furthermore, SSR180711 not only increased early LTP, but also transformed it into late LTP, which was not observed with the α4β2 nicotinic acetylcholine receptor agonist. Therefore, based on these findings α7 nicotinic acetylcholine receptor (partial) agonists appear to exhibit stronger efficacy on memory improvement than α4β2 nicotinic acetylcholine receptor agonists. PMID:21968142

  17. 6,6-Spiroimine analogs of (-)-gymnodimine A: synthesis and biological evaluation on nicotinic acetylcholine receptors.

    PubMed

    Duroure, Leslie; Jousseaume, Thierry; Aráoz, Rómulo; Barré, Elvina; Retailleau, Pascal; Chabaud, Laurent; Molgó, Jordi; Guillou, Catherine

    2011-12-01

    Simple models of the spiroimine core of (-)-gymnodimine A have been synthesized in racemic and optically active forms. The quaternary carbon of the racemic spiroimines was created by Michael addition of a β-ketoester to acrolein, whereas the asymmetric allylic alkylation of the same β-ketoester was used to access the spiroimines in an enantioselective fashion. Both racemic and enantio-enriched mixtures were tested for their biological activities on Xenopus oocytes either expressing (human α4β2) or having incorporated (Torpedoα1(2)βγδ) nicotinic acetylcholine receptors (nAChRs). These spiroimine analogs of (-)-gymnodimine A inhibited acetylcholine-evoked nicotinic currents, but were less active than the phycotoxin. Our results reveal that the 6,6-spiroimine moiety is important for the blockade of nAChRs and support the hypothesis that it is one of the pharmacophores of this group of toxins. PMID:22024965

  18. Influence of acetylcholine on binding of 4-[125I]iododexetimide to muscarinic brain receptors.

    PubMed

    Weckesser, M; Fixmann, A; Holschbach, M; Müller-Gärtner, H W

    1998-11-01

    The distribution of nicotinic and muscarinic cholinergic receptors in the human brain in vivo has been successfully characterized using radiolabeled tracers and emission tomography. The effect of acetylcholine release into the synaptic cleft on receptor binding of these tracers has not yet been investigated. The present study examined the influence of acetylcholine on binding of 4-[125I]iododexetimide to muscarinic cholinergic receptors of porcine brain synaptosomes in vitro. 4-Iododexetimide is a subtype-unspecific muscarinic receptor antagonist with high affinity. Acetylcholine competed with 4-[125I]iododexetimide in a dose-dependent manner. A concentration of 500 microM acetylcholine inhibited 50% of total specific 4-[125I]iododexetimide binding to synaptosomes when both substances were given simultaneously. An 800 microM acetylcholine solution reduced total specific 4-[125I]iododexetimide binding by about 35%, when acetylcholine was given 60 min after incubation of synaptosomes with 4-[125I]iododexetimide. Variations in the synaptic acetylcholine concentration might influence muscarinic cholinergic receptor imaging in vivo using 4-[123I]iododexetimide. Conversely, 4-[123I]iododexetimide might be an appropriate molecule to investigate alterations of acetylcholine release into the synaptic cleft in vivo using single photon emission computed tomography. PMID:9863566

  19. Schizophrenia and the alpha7 nicotinic acetylcholine receptor.

    PubMed

    Martin, Laura F; Freedman, Robert

    2007-01-01

    In addition to the devastating symptoms of psychosis, many people with schizophrenia also suffer from cognitive impairment. These cognitive symptoms lead to marked dysfunction and can impact employability, treatment adherence, and social skills. Deficits in P50 auditory gating are associated with attentional impairment and may contribute to cognitive symptoms and perceptual disturbances. This nicotinic cholinergic-mediated inhibitory process represents a potential new target for therapeutic intervention in schizophrenia. This chapter will review evidence implicating the nicotinic cholinergic, and specifically, the alpha7 nicotinic receptor system in the pathology of schizophrenia. Impaired auditory sensory gating has been linked to the alpha7 nicotinic receptor gene on the chromosome 15q14 locus. A majority of persons with schizophrenia are heavy smokers. Although nicotine can acutely reverse diminished auditory sensory gating in people with schizophrenia, this effect is lost on a chronic basis due to receptor desensitization. The alpha7 nicotinic agonist 3-(2,4 dimethoxy)benzylidene-anabaseine (DMXBA) can also enhance auditory sensory gating in animal models. DMXBA is well tolerated in humans and a new study in persons with schizophrenia has found that DMXBA enhances both P50 auditory gating and cognition. alpha7 Nicotinic acetylcholine receptor agonists appear to be viable candidates for the treatment of cognitive disturbances in schizophrenia. PMID:17349863

  20. Aporphine metho salts as neuronal nicotinic acetylcholine receptor blockers.

    PubMed

    Iturriaga-Vásquez, Patricio; Pérez, Edwin G; Slater, E Yvonne; Bermúdez, Isabel; Cassels, Bruce K

    2007-05-15

    (S)-Aporphine metho salts with the 1,2,9,10 oxygenation pattern displaced radioligands from recombinant human alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChR) at low micromolar concentrations. The affinity of the nonphenolic glaucine methiodide (4) (vs [(3)H]cytisine) was the lowest at alpha4beta2 nAChR (K(i)=10 microM), and predicentrine methiodide (2) and xanthoplanine iodide (3), with free hydroxyl groups at C-2 or C-9, respectively, had the highest affinity at these receptors (K(i) approximately 1 microM), while the affinity of the diphenolic boldine methiodide (1) was intermediate between these values. At homomeric alpha7 nAChR, xanthoplanine had the highest affinity (K(i)=10 microM) vs [(125)I]alpha-bungarotoxin while the other three compounds displaced the radioligand with K(i) values between 15 and 21 microM. At 100 microM, all four compounds inhibited the responses of these receptors to EC(50) concentrations of ACh. The effects of xanthoplanine iodide (3) were studied in more detail. Xanthoplanine fully inhibited the EC(50) ACh responses of both alpha7 and alpha4beta2 nACh receptors with estimated IC(50) values of 9+/-3 microM (alpha7) and 5+/-0.8 microM (alpha4beta2). PMID:17391965

  1. Critical metabolic roles of β-cell M3 muscarinic acetylcholine receptors

    PubMed Central

    de Azua, Inigo Ruiz; Gautam, Dinesh; Jain, Shalini; Guettier, Jean-Marc; Wess, Jürgen

    2013-01-01

    Muscarinic acetylcholine (ACh) receptors (mAChRs; M1–M5) regulate the activity of an extraordinarily large number of important physiological processes. We and others previously demonstrated that pancreatic β-cells are endowed with M3 mAChRs which are linked to G proteins of the Gq family. The activation of these receptors by ACh or other muscarinic agonists leads to the augmentation of glucose-induced insulin release via multiple mechanisms. Interestingly, in humans, ACh acting on human β-cell mAChRs is released from adjacent α-cells which express both choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (vAChT), indicative of the presence of a non-neuronal cholinergic system in human pancreatic islets. In order to shed light on the physiological roles of β-cell M3 receptors, we recently generated and analyzed various mutant mouse models. Specifically, we carried out studies with mice which overexpressed M3 receptors or mutant M3 receptors in pancreatic β-cells or which selectively lacked M3 receptors or M3-receptor-associated proteins in pancreatic β-cells. Our findings indicate that β-cell M3 receptors play a key role in maintaining proper insulin release and whole body glucose homeostasis and that strategies aimed at enhancing signaling through β-cell M3 receptors may prove useful to improve β-cell function for the treatment of type 2 diabetes (T2D). PMID:22525375

  2. Recent developments in the synthesis of nicotinic acetylcholine receptor ligands.

    PubMed

    Breining, Scott R

    2004-01-01

    The extraordinary pharmacology of nicotine and epibatidine have indicated the potential for nicotinic acetylcholine receptor (nAChR) ligands to serve as a new therapeutic class for a host of CNS disorders. Many such ligands are natural products, or analogs thereof, which represent a significant challenge to the synthetic chemist. Synthesis of such molecules often serves as a showcase to demonstrate the potential of newly developed methodology. This synthetic challenge coupled with the promise of pharmacological activity in compounds possessing the nicotinic pharmacophore has stimulated a great deal of synthetic activity over the last five years. The present report provides an overview of novel synthetic methodology occurring during this period directed toward the synthesis of compounds with presumed affinity for the neuronal nAChR. Syntheses chosen for review here represent the major efforts toward molecules such as epibatidine analogs, anatoxin-a, nicotine and related alkaloids, conformationally constrained nicotine derivatives, cytisine and methyllycaconitine (MLA). PMID:14965298

  3. Inhibitory Learning is Modulated by Nicotinic Acetylcholine Receptors

    PubMed Central

    Meyer, Heidi C.; Putney, Rachel B.; Bucci, David J.

    2014-01-01

    Prior research has established that stimulating nicotinic acetylcholine receptors can facilitate learning and memory. However, most studies have focused on learning to emit a particular behavior, while little is known about the effects of nicotine on learning to withhold a behavioral response. The present study consisted of a dose response analysis of the effects of nicotine on negative occasion setting, a form of learned inhibition. In this paradigm, rats received one type of training trial in which presentation of a tone by itself was followed immediately by food reward. During the other type of trials, the tone was preceded by presentation of a light and no food was delivered after the tone. Rats gradually learned to approach the cup in anticipation of receiving food reward during presentations of the tone alone, but withheld that behavior when the tone was preceded by the light. Nicotine (0.35mg/kg) facilitated negative occasion setting by reducing the number of sessions needed to learn the discrimination between trial types and by reducing the rate of responding on non-reinforced trials. Nicotine also increased the orienting response to the light, suggesting that nicotine may have affected the ability to withhold food cup behavior on non-reinforced trials by increasing attention to the light. In contrast to the effects of nicotine, rats treated with mecamylamine (0.125, 0.5, or 2 mg/kg) needed more training sessions to discriminate between reinforced and non-reinforced trials compared to saline-treated rats. The findings indicate that nicotinic acetylcholine receptors may be active during negative occasion setting and that nicotine can potentiate learned inhibition. PMID:25445487

  4. Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

    SciTech Connect

    Resende, R.R.; Alves, A.S.; Britto, L.R.G; Ulrich, H.

    2008-04-15

    Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G{alpha}{sub q/11}-coupled M{sub 1}, M{sub 3} and M{sub 5} receptors and intracellular calcium stores, whereas G{alpha}{sub i/o}-protein coupled M{sub 2} receptor activity mediated neuronal differentiation.

  5. Purification of the muscarinic acetylcholine receptor from porcine atria.

    PubMed Central

    Peterson, G L; Herron, G S; Yamaki, M; Fullerton, D S; Schimerlik, M I

    1984-01-01

    The muscarinic acetylcholine receptor from porcine atria has been purified 100,000-fold to homogeneity by solubilization in digitonin/cholate and sequential chromatography on wheat germ agglutinin-agarose, diethylaminoethylagarose, hydroxylapatite, and 3-(2'-aminobenzhydryloxy)tropane-agarose. The yield of purified receptor was 4.3% of that found in the membrane fraction, and the purified receptor bound 11.1-12.8 nmol of L-[3H]quinuclidinyl benzilate per mg of protein, corresponding to a binding component Mr of 78,400-90,000. The purified receptor preparation consisted of two polypeptides in approximately equimolar amounts when examined on silver-stained sodium dodecyl sulfate/polyacrylamide gels. The larger polypeptide (Mr 78,000 on 8% polyacrylamide gels) was specifically alkylated with [3H]propylbenzilylcholine mustard, whereas the smaller polypeptide (Mr 14,800) was not labeled. The possibility that the small polypeptide is a contaminant fortuitously appearing in equimolar amounts with the large polypeptide cannot be ruled out at this time. The purified preparation was highly stable, with no measurable change in the number of ligand binding sites or the gel pattern after 1 month's storage on ice. Scatchard analysis showed a single class of binding sites for the antagonist L-[3H]quinuclidinyl benzilate with a dissociation constant of 61 +/- 4 pM. Equilibrium titration experiments demonstrated that the antagonist L-hyoscyamine displaced L-[3H]quinuclidinyl benzilate from a single class of sites (Kd = 475 +/- 30 pM), whereas the agonist carbamoylcholine interacted at two populations of sites (53% +/- 3% high affinity, Kd = 1.1 +/- 0.3 microM; 47% +/- 3% low affinity, Kd = 67 +/- 14 microM). The ligand binding data were very similar to that for the membrane-bound receptor, suggesting that the receptor has not been altered radically during purification. Images PMID:6589642

  6. Immunological relationship between acetylcholine receptor and thymus: a possible significance in myasthenia gravis.

    PubMed Central

    Aharonov, A; Tarrab-Hazdai, R; Abramsky, O; Fuchs, S

    1975-01-01

    A defined immunological cross-reaction was observed between acetylcholine receptor fraction from the electric eel, Electrophorus electricus, and two calf thymus fractions. The cross-reaction was demonstrated on the cellular level by means of the lymphocyte transformation technique, and on the humoral level, by means of the microcomplement fixation assay. In the human disease myasthenia gravis both acetylcholine receptor at the neuromuscular junction and the thymus are affected, probably by an autoimmune mechanism. The immunological cross-reaction between acetylcholine receptor and thymic components may explain the association between endplate and thymus disorders in myasthenia gravis. PMID:1055418

  7. Mechanisms of acetylcholine receptor loss in myasthenia gravis.

    PubMed Central

    Drachman, D B; Adams, R N; Stanley, E F; Pestronk, A

    1980-01-01

    The fundamental abnormality affecting the neuromuscular junctions of myasthenic patients is a reduction of available AChRs, due to an autoimmune attack directed against the receptors. Antibodies to AChR are present in most patients, and there is evidence that they have a predominant pathogenic role in the disease, aided by complement. The mechanism of antibody action involves acceleration of the rate of degradation of AChRs, attributable to cross-linking of the receptors. In addition, antibodies may block AChRs, and may participate in producing destructive changes, perhaps in conjunction with complement. The possibility that cell-mediated mechanisms may play a role in the autoimmune responses of some myasthenic patients remains to be explored. Although the target of the autoimmune attack in myasthenic patients is probably always the acetylcholine receptors, it is not yet clear which of these immune mechanisms are most important. It is likely that the relative role of each mechanism varies from patient to patient. One of the goals of future research will be to identify the relative importance of each of these mechanisms in the individual patient, and to tailor specific immunotherapeutic measures to the abnormalities found. PMID:6249894

  8. Biosynthesis of the Torpedo californica Acetylcholine Receptor α Subunit in Yeast

    NASA Astrophysics Data System (ADS)

    Fujita, Norihisa; Nelson, Nathan; Fox, Thomas D.; Claudio, Toni; Lindstrom, Jon; Riezman, Howard; Hess, George P.

    1986-03-01

    Yeast cells were transformed with a plasmid containing complementary DNA encoding the α subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the α subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The α subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.

  9. Activation of endplate nicotinic acetylcholine receptors by agonists.

    PubMed

    Auerbach, Anthony

    2015-10-15

    The interaction of a small molecule made in one cell with a large receptor made in another is the signature event of cell signaling. Understanding the structure and energy changes associated with agonist activation is important for engineering drugs, receptors and synapses. The nicotinic acetylcholine receptor (AChR) is a ∼300kD ion channel that binds the neurotransmitter acetylcholine (ACh) and other cholinergic agonists to elicit electrical responses in the central and peripheral nervous systems. This mini-review is in two sections. First, general concepts of skeletal muscle AChR operation are discussed in terms of energy landscapes for conformational change. Second, adult vs. fetal AChRs are compared with regard to interaction energies between ACh and agonist-site side chains, measured by single-channel electrophysiology and molecular dynamics simulations. The five aromatic residues that form the core of each agonist binding site can be divided into two working groups, a triad (led by αY190) that behaves similarly at all sites and a coupled pair (led by γW55) that has a large influence on affinity only in fetal AChRs. Each endplate AChR has 5 homologous subunits, two of α(1) and one each of β, δ, and either γ (fetal) or ϵ (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at αδ and either αγ or αϵ subunit interfaces. The receptor undergoes a reversible, global isomerization between structures called C and O. The C shape does not conduct ions and has a relatively low affinity for ACh, whereas O conducts cations and has a higher affinity. When both agonist sites are empty (filled only with water) the probability of taking on the O conformation (PO) is low, <10(-6). When ACh molecules occupy the agonist sites the C→O opening rate constant and C↔O gating equilibrium constant increase dramatically. Following a pulse of ACh at the nerve-muscle synapse, the endplate current rises rapidly

  10. Physiological characterization of human muscle acetylcholine receptors from ALS patients

    PubMed Central

    Palma, Eleonora; Inghilleri, Maurizio; Conti, Luca; Deflorio, Cristina; Frasca, Vittorio; Manteca, Alessia; Pichiorri, Floriana; Roseti, Cristina; Torchia, Gregorio; Limatola, Cristina; Grassi, Francesca; Miledi, Ricardo

    2011-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: “microtransplantation” of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease. PMID:22128328

  11. Histamine H3 receptors regulate acetylcholine release from the guinea pig ileum myenteric plexus

    SciTech Connect

    Poli, E.; Coruzzi, G.; Bertaccini, G. )

    1991-01-01

    The effect of selective histamine H3-receptor agonists and antagonists on the acetylcholine release from peripheral nerves was evaluated in the guinea pig longitudinal muscle-myenteric plexus preparations, preloaded with ({sup 3}H)choline. In the presence of H1 and H2 blockade, histamine and (R)-{alpha}-methylhistamine inhibited the electrically-evoked acetylcholine release, being (R)-{alpha}-methylhistamine more active than histamine, but behaving as a partial agonist. The effect of histamine was completely reversed by selective H3-blocking drugs, thioperamide and impromidine, while only submaximal doses of (R)-{alpha}-methylhistamine were antagonized. Furthermore, thioperamide and impromidine enhanced the electrically-evoked acetylcholine release. On the contrary, the new H3-blocker, HST-7, was found substantially ineffective, both as histamine antagonist and as acetylcholine overflow enhancer. These data suggest that histamine exerts an inhibitory control on the acetylcholine release from intestinal cholinergic nerves through the activation of H3 receptors.

  12. Allosteric modulation of M1 muscarinic acetylcholine receptor internalization and subcellular trafficking.

    PubMed

    Yeatman, Holly R; Lane, J Robert; Choy, Kwok Ho Christopher; Lambert, Nevin A; Sexton, Patrick M; Christopoulos, Arthur; Canals, Meritxell

    2014-05-30

    Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M1 muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M1 mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M1 mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M1 mAChRs, we show that once internalized, M1 mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M1 mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands. PMID:24753247

  13. Megacystis, mydriasis, and ion channel defect in mice lacking the α3 neuronal nicotinic acetylcholine receptor

    PubMed Central

    Xu, Wei; Gelber, Shari; Orr-Urtreger, Avi; Armstrong, Dawna; Lewis, Richard A.; Ou, Ching-Nan; Patrick, James; Role, Lorna; De Biasi, Mariella; Beaudet, Arthur L.

    1999-01-01

    The α3 subunit of the neuronal nicotinic acetylcholine receptor is widely expressed in autonomic ganglia and in some parts of the brain. The α3 subunit can form heteromultimeric ion channels with other α subunits and with β2 and β4 subunits, but its function in vivo is poorly understood. We prepared a null mutation for the α3 gene by deletion of exon 5 and found that homozygous (−/−) mice lacked detectable mRNA on Northern blotting. The −/− mice survive to birth but have impaired growth and increased mortality before and after weaning. The −/− mice have extreme bladder enlargement, dribbling urination, bladder infection, urinary stones, and widely dilated ocular pupils that do not contract in response to light. Detailed histological studies of −/− mice revealed no significant abnormalities in brain or peripheral tissues except urinary bladder, where inflammation was prominent. Ganglion cells and axons were present in bladder and bowel. Bladder strips from −/− mice failed to contract in response to 0.1 mM nicotine, but did contract in response to electrical field stimulation or carbamoylcholine. The number of acetylcholine-activated single-channel currents was severely reduced in the neurons of superior cervical ganglia in −/− mice with five physiologically distinguishable nicotinic acetylcholine receptor subtypes with different conductance and kinetic properties in wild-type mice, all of which were reduced in −/− mice. The findings in the α3-null mice suggest that this subunit is an essential component of the nicotinic receptors mediating normal function of the autonomic nervous system. The phenotype in −/− mice may be similar to the rare human genetic disorder of megacystis–microcolon–intestinal hypoperistalsis syndrome. PMID:10318955

  14. Therapeutic Potential of α7 Nicotinic Acetylcholine Receptors.

    PubMed

    Bertrand, Daniel; Lee, Chih-Hung L; Flood, Dorothy; Marger, Fabrice; Donnelly-Roberts, Diana

    2015-10-01

    Progress in the fields of neuroscience and molecular biology has identified the forebrain cholinergic system as being important in many higher order brain functions. Further analysis of the genes encoding the nicotinic acetylcholine receptors (nAChRs) has highlighted, in particular, the role of α7 nAChRs in these higher order brain functions as evidenced by their peculiar physiologic and pharmacological properties. As this receptor has gained the attention of scientists from academia and industry, our knowledge of its roles in various brain and bodily functions has increased immensely. We have also seen the development of small molecules that have further refined our understanding of the roles of α7 nAChRs, and these molecules have begun to be tested in clinical trials for several indications. Although a large body of data has confirmed a role of α7 nAChRs in cognition, the translation of small molecules affecting α7 nAChRs into therapeutics has to date only progressed to the stage of testing in clinical trials. Notably, however, most recent human genetic and biochemical studies are further underscoring the crucial role of α7 nAChRs and associated genes in multiple organ systems and disease states. The aim of this review is to discuss our current knowledge of α7 nAChRs and their relevance as a target in specific functional systems and disease states. PMID:26419447

  15. Muscarinic acetylcholine receptors: location of the ligand binding site

    SciTech Connect

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-05-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, /sup 3/H-propylbenzilycholine mustard aziridinium ion (/sup 3/H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that /sup 3/H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin.

  16. Crosslinking-Induced Endocytosis of Acetylcholine Receptors by Quantum Dots

    PubMed Central

    Geng, Lin; Peng, H. Benjamin

    2014-01-01

    In a majority of patients with myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies target postsynaptic AChR clusters and thus compromise the membrane integrity of neuromuscular junctions (NMJs) and lead to muscle weakness. Antibody-induced endocytosis of AChRs in the postsynaptic membrane represents the initial step in the pathogenesis of MG; however, the molecular mechanisms underlying AChR endocytosis remain largely unknown. Here, we developed an approach to mimic the pathogenic antibodies for inducing the crosslinking and internalization of AChRs from the postsynaptic membrane. Using biotin-α-bungarotoxin and quantum dot (QD)-streptavidin, cell-surface and internalized AChRs could be readily distinguished by comparing the size, fluorescence intensity, trajectory, and subcellular localization of the QD signals. QD-induced AChR endocytosis was mediated by clathrin-dependent and caveolin-independent mechanisms, and the trafficking of internalized AChRs in the early endosomes required the integrity of microtubule structures. Furthermore, activation of the agrin/MuSK (muscle-specific kinase) signaling pathway strongly suppressed QD-induced internalization of AChRs. Lastly, QD-induced AChR crosslinking potentiated the dispersal of aneural AChR clusters upon synaptic induction. Taken together, our results identify a novel approach to study the mechanisms of AChR trafficking upon receptor crosslinking and endocytosis, and demonstrate that agrin-MuSK signaling pathways protect against crosslinking-induced endocytosis of AChRs. PMID:24587270

  17. Long-term effects of methamphetamine exposure on cognitive function and muscarinic acetylcholine receptor levels in mice

    PubMed Central

    Siegel, Jessica A.; Craytor, Michael J.; Raber, Jacob

    2010-01-01

    Exposure to methamphetamine during brain development impairs cognition in humans and rodents. In mice, these impairments are greater in females than males. Genetic factors, such as apolipoprotein E genotype, may modulate the cognitive effects of methamphetamine. Methamphetamine-induced alterations in the brain acetylcholine system may contribute to the cognitive effects of methamphetamine and may also be modulated by apolipoprotein E isoform. We assessed the long-term effects of methamphetamine exposure during brain development on cognitive function and muscarinic acetylcholine receptors in mice, and whether apolipoprotein E isoform modulates these effects. Mice expressing human apolipoprotein E3 or E4 were exposed to methamphetamine (5 mg/kg) or saline once a day from postnatal day 11-20 and behaviorally tested in adulthood. Muscarinic acetylcholine receptor binding was measured in the hippocampus and cortex. Methamphetamine exposure impaired novel location recognition in female, but not male, mice. Methamphetamine-exposed male and female mice showed impaired novel object recognition and increased number of muscarinic acetylcholine receptors in the hippocampus. The cognitive and cholinergic effects of methamphetamine were similar in apolipoprotein E3 and E4 mice. Thus, the cholinergic system, but not apolipoprotein E isoform, might play an important role in the long-term methamphetamine-induced cognitive deficits in adulthood. PMID:20729719

  18. Enhanced self-administration of alcohol in muscarinic acetylcholine M4 receptor knockout mice.

    PubMed

    de la Cour, Cecilie; Sørensen, Gunnar; Wortwein, Gitta; Weikop, Pia; Dencker, Ditte; Fink-Jensen, Anders; Molander, Anna

    2015-01-01

    Modulation of cholinergic neurotransmission via nicotinic acetylcholine receptors is known to alter alcohol-drinking behavior. It is not known if muscarinic acetylcholine receptor subtypes have similar effects. The muscarinic M4 receptor is highly expressed in the brain reinforcement system and involved in regulation of cholinergic and dopaminergic transmission. Here we investigate, for the first time, the role of the M4 receptor in alcohol consumption using M4 knockout (M4(-/-)) and wild-type (M4(+/+)) mice. Experimentally naïve M4(-/-) and M4(+/+) mice were trained to orally self-administer 5%, 8% and 10% alcohol in 60min sessions, 6 days/week, after having undergone a standard sucrose fading training procedure on a fixed ratio schedule. The mice were further subjected to an extinction period followed by a 1 day reinstatement trial. M4(-/-) mice consumed more alcohol at 5% and 8% compared to their M4(+/+) littermates. The highest alcohol concentration used (10%) did not immediately result in divergent drinking patterns, but after 4 weeks of 10% alcohol self-administration, baseline levels as well as a pattern of M4(-/-) mice consuming more alcohol than their M4(+/+) controls were re-established. Moreover, the M4(-/-) mice displayed a reduced capacity to extinguish their alcohol-seeking behavior. Taken together, alcohol consumption is elevated in M4(-/-) mice, indicating that the M4 receptor is involved in mediating the reinforcing effects of alcohol. The M4 receptor should be further explored as a potential target for pharmacological (positive allosteric modulators or future agonists) treatment of alcohol use disorders. PMID:25445043

  19. A tale of two receptors: Dual roles for ionotropic acetylcholine receptors in regulating motor neuron excitation and inhibition.

    PubMed

    Philbrook, Alison; Barbagallo, Belinda; Francis, Michael M

    2013-07-01

    Nicotinic or ionotropic acetylcholine receptors (iAChRs) mediate excitatory signaling throughout the nervous system, and the heterogeneity of these receptors contributes to their multifaceted roles. Our recent work has characterized a single iAChR subunit, ACR-12, which contributes to two distinct iAChR subtypes within the C. elegans motor circuit. These two receptor subtypes regulate the coordinated activity of excitatory (cholinergic) and inhibitory (GABAergic) motor neurons. We have shown that the iAChR subunit ACR-12 is differentially expressed in both cholinergic and GABAergic motor neurons within the motor circuit. In cholinergic motor neurons, ACR-12 is incorporated into the previously characterized ACR-2 heteromeric receptor, which shows non-synaptic localization patterns and plays a modulatory role in controlling circuit function.(1) In contrast, a second population of ACR-12-containing receptors in GABAergic motor neurons, ACR-12GABA, shows synaptic expression and regulates inhibitory signaling.(2) Here, we discuss the two ACR-12-containing receptor subtypes, their distinct expression patterns, and functional roles in the C. elegans motor circuit. We anticipate our continuing studies of iAChRs in the C. elegans motor circuit will lead to novel insights into iAChR function in the nervous system as well as mechanisms for their regulation. PMID:24778941

  20. Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes.

    PubMed Central

    Lechleiter, J; Hellmiss, R; Duerson, K; Ennulat, D; David, N; Clapham, D; Peralta, E

    1990-01-01

    Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:2124972

  1. R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5

    PubMed Central

    Nakashima, Hiroaki; Ohkawara, Bisei; Ishigaki, Shinsuke; Fukudome, Takayasu; Ito, Kenyu; Tsushima, Mikito; Konishi, Hiroyuki; Okuno, Tatsuya; Yoshimura, Toshiro; Ito, Mikako; Masuda, Akio; Sobue, Gen; Kiyama, Hiroshi; Ishiguro, Naoki; Ohno, Kinji

    2016-01-01

    At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ. PMID:27328992

  2. R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5.

    PubMed

    Nakashima, Hiroaki; Ohkawara, Bisei; Ishigaki, Shinsuke; Fukudome, Takayasu; Ito, Kenyu; Tsushima, Mikito; Konishi, Hiroyuki; Okuno, Tatsuya; Yoshimura, Toshiro; Ito, Mikako; Masuda, Akio; Sobue, Gen; Kiyama, Hiroshi; Ishiguro, Naoki; Ohno, Kinji

    2016-01-01

    At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ. PMID:27328992

  3. Heterogeneity of Drosophila nicotinic acetylcholine receptors: SAD, a novel developmentally regulated alpha-subunit.

    PubMed Central

    Sawruk, E; Schloss, P; Betz, H; Schmitt, B

    1990-01-01

    Two genes, ard and als, are known to encode subunits of the nicotinic acetylcholine receptor (nAChR) in Drosophila. Here we describe the isolation of cDNA clones encoding a novel member (SAD, or alpha 2) of this receptor protein family. The deduced amino acid sequence displays high homology to the ALS protein and shares structural features with ligand binding nAChR alpha-subunits. Sad transcripts accumulate during major periods of neuronal differentiation and, in embryos, are localized in the central nervous system. Expression of SAD cRNA in Xenopus oocytes generates cation channels that are gated by nicotine. These data indicate heterogeneity of nAChRs in Drosophila. Images Fig. 3. Fig. 4. PMID:1697262

  4. Acetylcholine receptor and behavioral deficits in mice lacking apolipoprotein E

    PubMed Central

    Siegel, Jessica A; Benice, Theodore S; Van Meer, Peter; Park, Byung S; Raber, Jacob

    2011-01-01

    Apolipoprotein E (apoE) is involved in the risk to develop sporadic Alzheimer’s disease (AD). Since impaired central acetylcholine (ACh) function is a hallmark of AD, apoE may influence ACh function by modulating muscarinic ACh receptors (mAChRs). To test this hypothesis, mAChR binding was measured in mice lacking apoE and wild type C57BL/6J mice. Mice were also tested on the pre-pulse inhibition, delay eyeblink classical conditioning, and 5-choice serial reaction time tasks, which are all modulated by ACh transmission. Mice were also given scopolamine to challenge central mAChR function. Compared to wild type mice, mice lacking apoE had reduced number of cortical and hippocampal mAChRs. Scopolamine had a small effect on delay eyeblink classical conditioning in wild type mice but a large effect in mice lacking apoE. Mice lacking apoE were also unable to acquire performance on the 5-choice serial reaction time task. These results support a role for apoE in ACh function and suggest that modulation of cortical and hippocampal mAChRs might contribute to genotype differences in scopolamine sensitivity and task acquisition. Impaired apoE functioning may result in cholinergic deficits that contribute to the cognitive impairments seen in AD. PMID:19178986

  5. Early Life Stress, Nicotinic Acetylcholine Receptors and Alcohol Use Disorders.

    PubMed

    Holgate, Joan Y; Bartlett, Selena E

    2015-01-01

    Stress is a major driving force in alcohol use disorders (AUDs). It influences how much one consumes, craving intensity and whether an abstinent individual will return to harmful alcohol consumption. We are most vulnerable to the effects of stress during early development, and exposure to multiple traumatic early life events dramatically increases the risk for AUDs. However, not everyone exposed to early life stress will develop an AUD. The mechanisms determining whether an individual's brain adapts and becomes resilient to the effects of stress or succumbs and is unable to cope with stress remain elusive. Emerging evidence suggests that neuroplastic changes in the nucleus accumbens (NAc) following early life stress underlie the development of AUDs. This review discusses the impact of early life stress on NAc structure and function, how these changes affect cholinergic signaling within the mesolimbic reward pathway and the role nicotinic acetylcholine receptors (nAChRs) play in this process. Understanding the neural pathways and mechanism determining stress resilience or susceptibility will improve our ability to identify individuals susceptible to developing AUDs, formulate cognitive interventions to prevent AUDs in susceptible individuals and to elucidate and enhance potential therapeutic targets, such as the nAChRs, for those struggling to overcome an AUD. PMID:26136145

  6. Cycloxaprid insecticide: nicotinic acetylcholine receptor binding site and metabolism.

    PubMed

    Shao, Xusheng; Swenson, Tami L; Casida, John E

    2013-08-21

    Cycloxaprid (CYC) is a novel neonicotinoid prepared from the (nitromethylene)imidazole (NMI) analogue of imidacloprid. In this study we consider whether CYC is active per se or only as a proinsecticide for NMI. The IC50 values (nM) for displacing [(3)H]NMI binding are 43-49 for CYC and 2.3-3.2 for NMI in house fly and honeybee head membranes and 302 and 7.2, respectively, in mouse brain membranes, potency relationships interpreted as partial conversion of some CYC to NMI under the assay conditions. The 6-8-fold difference in toxicity of injected CYC and NMI to house flies is consistent with their relative potencies as in vivo nicotinic acetylcholine receptor (nAChR) inhibitors in brain measured with [(3)H]NMI binding assays. CYC metabolism in mice largely involves cytochrome P450 pathways without NMI as a major intermediate. Metabolites of CYC tentatively assigned are five monohydroxy derivatives and one each of dihydroxy, nitroso, and amino modifications. CYC appears be a proinsecticide, serving as a slow-release reservoir for NMI with selective activity for insect versus mammalian nAChRs. PMID:23889077

  7. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed

    McCrea, P D; Popot, J L; Engelman, D M

    1987-12-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  8. The twin drug approach for novel nicotinic acetylcholine receptor ligands.

    PubMed

    Tomassoli, Isabelle; Gündisch, Daniela

    2015-08-01

    The association of two pharmacophoric entities generates so-called 'twin drugs' or dimer derivatives. We applied this approach for the design of a small compound library for the interaction with α4β2(∗) nicotinic acetylcholine receptors (nAChRs). In this compound series, the nAChR ligand N,N-dimethyl-2-(pyridin-3-yloxy)ethan-1-amine 9 served as one pharmacological entity and it was initially kept constant as one part of the 'twin' compound. 'Twin' compounds with identical or non-identical entities using the 'no linker mode' or 'overlap' mode were synthesized and evaluated for their nAChR affinities. Compound 17a showed the highest affinity for the α4β2(∗) nAChR subtype (Ki=0.188 nM) and its (di)fluoro analogs could retain nanomolar affinities, when replacing pyridine as the hydrogen bond acceptor system by mono- or difluoro-phenyls. The 'twin drug' approach proved to provide compounds with high affinity and subtype selectivity for α4β2(∗) nAChRs. PMID:26142318

  9. Neuronal Nicotinic Acetylcholine Receptor Modulators Reduce Sugar Intake

    PubMed Central

    Shariff, Masroor; Quik, Maryka; Holgate, Joan; Morgan, Michael; Patkar, Omkar L.; Tam, Vincent; Belmer, Arnauld; Bartlett, Selena E.

    2016-01-01

    Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases α4β2 * and decreases α6β2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption. PMID:27028298

  10. Early Life Stress, Nicotinic Acetylcholine Receptors and Alcohol Use Disorders

    PubMed Central

    Holgate, Joan Y.; Bartlett, Selena E.

    2015-01-01

    Stress is a major driving force in alcohol use disorders (AUDs). It influences how much one consumes, craving intensity and whether an abstinent individual will return to harmful alcohol consumption. We are most vulnerable to the effects of stress during early development, and exposure to multiple traumatic early life events dramatically increases the risk for AUDs. However, not everyone exposed to early life stress will develop an AUD. The mechanisms determining whether an individual’s brain adapts and becomes resilient to the effects of stress or succumbs and is unable to cope with stress remain elusive. Emerging evidence suggests that neuroplastic changes in the nucleus accumbens (NAc) following early life stress underlie the development of AUDs. This review discusses the impact of early life stress on NAc structure and function, how these changes affect cholinergic signaling within the mesolimbic reward pathway and the role nicotinic acetylcholine receptors (nAChRs) play in this process. Understanding the neural pathways and mechanism determining stress resilience or susceptibility will improve our ability to identify individuals susceptible to developing AUDs, formulate cognitive interventions to prevent AUDs in susceptible individuals and to elucidate and enhance potential therapeutic targets, such as the nAChRs, for those struggling to overcome an AUD. PMID:26136145

  11. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed Central

    McCrea, P D; Popot, J L; Engelman, D M

    1987-01-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  12. Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus

    PubMed Central

    Dennis, Siobhan H.; Pasqui, Francesca; Colvin, Ellen M.; Sanger, Helen; Mogg, Adrian J.; Felder, Christian C.; Broad, Lisa M.; Fitzjohn, Steve M.; Isaac, John T.R.; Mellor, Jack R.

    2016-01-01

    Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans. PMID:26472558

  13. Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus.

    PubMed

    Dennis, Siobhan H; Pasqui, Francesca; Colvin, Ellen M; Sanger, Helen; Mogg, Adrian J; Felder, Christian C; Broad, Lisa M; Fitzjohn, Steve M; Isaac, John T R; Mellor, Jack R

    2016-01-01

    Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans. PMID:26472558

  14. Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations.

    PubMed

    Mathé-Allainmat, Monique; Swale, Daniel; Leray, Xavier; Benzidane, Yassine; Lebreton, Jacques; Bloomquist, Jeffrey R; Thany, Steeve H

    2013-12-01

    We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors. PMID:23884575

  15. Muscarinic acetylcholine receptor modulation of mu (mu) opioid receptors in adult rat sphenopalatine ganglion neurons.

    PubMed

    Margas, Wojciech; Mahmoud, Saifeldin; Ruiz-Velasco, Victor

    2010-01-01

    The sphenopalatine ganglion (SPG) neurons represent the parasympathetic branch of the autonomic nervous system involved in controlling cerebral blood flow. In the present study, we examined the coupling mechanism between mu (mu) opioid receptors (MOR) and muscarinic acetylcholine receptors (mAChR) with Ca(2+) channels in acutely dissociated adult rat SPG neurons. Successful MOR activation was recorded in approximately 40-45% of SPG neurons employing the whole cell variant of the patch-clamp technique. In addition, immunofluorescence assays indicated that MOR are not expressed in all SPG neurons while M(2) mAChR staining was evident in all neurons. The concentration-response relationships generated with morphine and [d-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) showed IC(50) values of 15.2 and 56.1 nM and maximal Ca(2+) current inhibition of 26.0 and 38.7%, respectively. Activation of MOR or M(2) mAChR with morphine or oxotremorine-methiodide (Oxo-M), respectively, resulted in voltage-dependent inhibition of Ca(2+) currents via coupling with Galpha(i/o) protein subunits. The acute prolonged exposure (10 min) of neurons to morphine or Oxo-M led to the homologous desensitization of MOR and M(2) mAChR, respectively. The prolonged stimulation of M(2) mAChR with Oxo-M resulted in heterologous desensitization of morphine-mediated Ca(2+) current inhibition, and was sensitive to the M(2) mAChR blocker methoctramine. On the other hand, when the neurons were exposed to morphine or DAMGO for 10 min, heterologous desensitization of M(2) mAChR was not observed. These results suggest that in rat SPG neurons activation of M(2) mAChR likely modulates opioid transmission in the brain vasculature to adequately maintain cerebral blood flow. PMID:19889856

  16. Mechanism of phencyclidine binding to the acetylcholine receptor from Torpedo electroplaque.

    PubMed

    Oswald, R E; Bamberger, M J; McLaughlin, J T

    1984-05-01

    The mechanism of phencyclidine binding to Torpedo acetylcholine receptor-rich membranes was investigated. The rate of [3H]phencyclidine association is 10(3)- to 10(4)-fold more rapid when phencyclidine and carbamoylcholine are added simultaneously to acetylcholine receptor-rich membranes than when phencyclidine is added to membranes previously equilibrated with carbamoylcholine or membranes in the absence of carbamoylcholine. The mechanism of binding under conditions in which the slower rate was observed was studied with thermodynamic, viscosity, and kinetic experiments. Association and dissociation rates were highly dependent on temperature with activation energies of 26-30 kcal/mole. Viscosity had no effect on the association rate but increased the dissociation rate. These studies suggest that the binding is not diffusion-controlled but rather is limited by a significant energy barrier. The association rate was determined as a function of the concentration of acetylcholine receptor-rich membranes and the concentration of phencyclidine. In the presence of carbamoylcholine, the association rate was highly dependent upon the concentration of acetylcholine receptor but virtually insensitive to the concentration of phencyclidine. In the absence of carbamoylcholine, the association rate seemed to be a hyperbolic function of both the phencyclidine and the acetylcholine receptor concentration. The minimal model capable of explaining the data is a mechanism by which phencyclidine binds to two conformations of the acetylcholine receptor, one conformation having a higher affinity and constituting a lower percentage of receptors and the other having a lower affinity and constituting a higher percentage. The data are consistent with the possibility that the high-affinity conformation is the open-channel state of the acetylcholine receptor. PMID:6727862

  17. Inhibition of cortical acetylcholine release and cognitive performance by histamine H3 receptor activation in rats.

    PubMed Central

    Blandina, P.; Giorgetti, M.; Bartolini, L.; Cecchi, M.; Timmerman, H.; Leurs, R.; Pepeu, G.; Giovannini, M. G.

    1996-01-01

    1. The effects of histamine and agents at histamine receptors on spontaneous and 100 mM K(+)-evoked release of acetylcholine, measured by microdialysis from the cortex of freely moving, rats, and on cognitive tests are described. 2. Local administration of histamine (0.1-100 microM) failed to affect spontaneous but inhibited 100 mM K(+)-stimulated release of acetylcholine up to about 50%. The H3 receptor agonists (R)-alpha-methylhistamine (RAMH) (0.1-10 microM), imetit (0.01-10 microM) and immepip (0.01-10 microM) mimicked the effect of histamine. 3. Neither 2-thiazolylethylamine (TEA), an agonist showing some selectivity for H1 receptors, nor the H2 receptor agonist, dimaprit, modified 100 mM K(+)-evoked release of acetylcholine. 4. The inhibitory effect of 100 microM histamine was completely prevented by the highly selective histamine H3 receptor antagonist, clobenpropit but was resistant to antagonism by triprolidine and cimetidine, antagonists at histamine H1 and H2 but not H3 receptors. 5. The H3 receptor-induced inhibition of K(+)-evoked release of acetylcholine was fully sensitive to tetrodotoxin (TTX). 6. The effects of intraperitoneal (i.p.) injection of imetit (5 mg kg-1) and RAMH (5 mg kg-1) were tested on acetylcholine release and short term memory paradigms. Both drugs reduced 100 mM K(+)-evoked release of cortical acetylcholine, and impaired object recognition and a passive avoidance response. 7. These observations provide the first evidence of a regulatory role of histamine H3 receptors on cortical acetylcholine release in vivo. Moreover, they suggest a role for histamine in learning and memory and may have implications for the treatment of degenerative disorders associated with impaired cholinergic function. PMID:8982515

  18. Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

    PubMed Central

    Nayak, Tapan K.; Bruhova, Iva; Chakraborty, Srirupa; Gupta, Shaweta; Zheng, Wenjun; Auerbach, Anthony

    2014-01-01

    A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αε (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔGB1). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔGB1 is ∼−2 kcal/mol more favorable at αγ compared with at αε and αδ. Only three of the aromatics contribute significantly to ΔGB1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔGB1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αε and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αε and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse. PMID:25422413

  19. Functional interaction between Lypd6 and nicotinic acetylcholine receptors.

    PubMed

    Arvaniti, Maria; Jensen, Majbrit M; Soni, Neeraj; Wang, Hong; Klein, Anders B; Thiriet, Nathalie; Pinborg, Lars H; Muldoon, Pretal P; Wienecke, Jacob; Imad Damaj, M; Kohlmeier, Kristi A; Gondré-Lewis, Marjorie C; Mikkelsen, Jens D; Thomsen, Morten S

    2016-09-01

    Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain-containing 6 (Lypd6) with nAChRs in human brain extracts, identifying Lypd6 as a novel regulator of nAChR function. Using protein cross-linking and affinity purification from human temporal cortical extracts, we demonstrate that Lypd6 is a synaptically enriched membrane-bound protein that binds to multiple nAChR subtypes in the human brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine-induced hippocampal inward currents in rat brain slices and decreases nicotine-induced extracellular signal-regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit nAChR-mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post-natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx protein Lypd6 binds to nAChRs in human brain extracts, and that recombinant Lypd6 decreases nicotine-induced ERK phosphorylation and attenuates nicotine-induced hippocampal inward currents. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain. PMID:27344019

  20. Nicotine evokes kinetic tremor by activating the inferior olive via α7 nicotinic acetylcholine receptors.

    PubMed

    Kunisawa, Naofumi; Iha, Higor A; Shimizu, Saki; Tokudome, Kentaro; Mukai, Takahiro; Kinboshi, Masato; Serikawa, Tadao; Ohno, Yukihiro

    2016-11-01

    Nicotinic acetylcholine (nACh) receptors are implicated in the pathogenesis of movement disorders (e.g., tremor) and epilepsy. Here, we performed behavioral and immunohistochemical studies using mice and rats to elucidate the mechanisms underlying nicotine-induced tremor. Treatments of animals with nicotine (0.5-2mg/kg, i.p.) elicited kinetic tremor, which was completely suppressed by the nACh receptor antagonist mecamylamine (MEC). The specific α7 nACh receptor antagonist methyllycaconitine (MLA) also inhibited nicotine-induced tremor, whereas the α4β2 nACh antagonist dihydro-β-erythroidine (DHβE) or the peripheral α3β4 nACh antagonist hexamethonium showed no effects. Mapping analysis of Fos protein expression, a biological marker of neural excitation, revealed that a tremorgenic dose (1mg/kg) of nicotine region-specifically elevated Fos expression in the piriform cortex (PirC), medial habenula, solitary nucleus and inferior olive (IO) among 44 brain regions examined. In addition, similarly to the tremor responses, nicotine-induced Fos expression in the PirC and IO was selectively antagonized by MLA, but not by DHβE. Furthermore, an electrical lesioning of the IO, but not the PirC, significantly suppressed the induction of nicotine tremor. The present results suggest that nicotine elicits kinetic tremor in rodents by activating the IO neurons via α7 nACh receptors. PMID:27506652

  1. Rab11a and myosin Vb regulate recycling of the M4 muscarinic acetylcholine receptor.

    PubMed

    Volpicelli, Laura A; Lah, James J; Fang, Guofu; Goldenring, James R; Levey, Allan I

    2002-11-15

    Agonist-induced internalization followed by subsequent return to the cell surface regulates G-protein-coupled receptor (GPCR) activity. Because the cellular responsiveness to ligand depends on the balance between receptor degradation and recycling, it is crucial to identify the molecules involved in GPCR recovery to the cell surface. In this study, we identify mechanisms involved in the recycling of the M4 subtype of muscarinic acetylcholine receptor. M4 is highly expressed in the CNS, plays a role in locomotor activity, and is a novel therapeutic target for neurologic and psychiatric disorders. Previous studies show that, after cholinergic stimulation, M4 internalizes from the cell surface to endosomes in cell culture and the rat brain. Here, we show that, after activation, M4 traffics to transferrin receptor- and Rab11a-positive perinuclear endosomes. Expression of the constitutively GDP-bound, inactive mutant Rab11aS25N inhibits M4 trafficking to recycling endosomes. Expression of the C-terminal tail of myosin Vb, a Rab11a effector, enhances M4 accumulation in perinuclear endosomes. Both Rab11aS25N and the myosin Vb tail impair M4 recycling. The results demonstrate that GPCR recycling is mediated through a discrete pathway using both Rab11a and myosin Vb. PMID:12427833

  2. The ligand binding domain of the nicotinic acetylcholine receptor. Immunological analysis.

    PubMed

    Kachalsky, S G; Aladjem, M; Barchan, D; Fuchs, S

    1993-03-01

    The interaction of the acetylcholine receptor (AChR) binding site domain with specific antibodies and with alpha-bungarotoxin (alpha-BTX) has been compared. The cloned and expressed ligand binding domain of the mouse AChR alpha-subunit binds alpha-BTX, whereas the mongoose-expressed domain is not recognized by alpha-BTX. On the other hand, both the mouse and mongoose domains bind to the site-specific monoclonal antibody 5.5. These results demonstrate that the structural requirements for binding of alpha-BTX and mcAb 5.5, both of which interact with the AChR binding site, are distinct from each other. PMID:8440381

  3. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  4. The α6 nicotinic acetylcholine receptor subunit influences ethanol-induced sedation

    PubMed Central

    Kamens, Helen M.; Hoft, Nicole R.; Cox, Ryan J.; Miyamoto, Jill; Ehringer, Marissa A.

    2012-01-01

    Alcohol and nicotine are often co-used and data from human and animals studies have demonstrated that common genes underlie responses to these two drugs. Recently, the genes that code for the subunits of the nicotinic acetylcholine receptors have been implicated as a common genetic mediator for alcohol and nicotine responses. The mammalian genes that code for the α6 and β3 subunits of the nicotinic acetylcholine receptor (Chrna6 and Chrnb3, respectively) are located adjacent to each other on human and mouse chromosome 8. These subunits have gained attention as potential regulators of drug behaviors because of their expression in the striatum where they have been shown to modulate dopamine release. Human genetic studies have shown that variation in these genes is associated with alcohol phenotypes. In the current experiments, mice lacking the Chrna6 or Chrnb3 gene were tested for three ethanol behaviors: choice ethanol consumption, ataxia, and sedation. Wildtype (WT), heterozygous (HET), and knockout (KO) mice of each strain went through a standard 2-bottle choice drinking paradigm, the balance beam, and the Loss of Righting Reflex (LORR) paradigm. No genotypic effects on any of the 3 behavioral tasks were observed in Chrnb3 animals. While the Chrna6 gene did not significantly influence ethanol consumption (g/kg) or ataxia, mice lacking the α6 subunit took significantly longer to recover their righting reflex than WT animals. These data provide evidence that receptors containing this subunit modulate the sedative effects of ethanol. Further work examining other models of ethanol consumption and behavioral responses to ethanol is needed to fully characterize the role of these receptor subunits in modulating ethanol responses. PMID:22572056

  5. The α6 nicotinic acetylcholine receptor subunit influences ethanol-induced sedation.

    PubMed

    Kamens, Helen M; Hoft, Nicole R; Cox, Ryan J; Miyamoto, Jill H; Ehringer, Marissa A

    2012-08-01

    Alcohol and nicotine are often co-used and data from human and animals studies have demonstrated that common genes underlie responses to these two drugs. Recently, the genes that code for the subunits of the nicotinic acetylcholine receptors have been implicated as a common genetic mediator for alcohol and nicotine responses. The mammalian genes that code for the α6 and β3 subunits of the nicotinic acetylcholine receptor (Chrna6 and Chrnb3, respectively) are located adjacent to each other on human and mouse chromosome 8. These subunits have gained attention as potential regulators of drug behaviors because of their expression in the striatum where they have been shown to modulate dopamine release. Human genetic studies have shown that variation in these genes is associated with alcohol phenotypes. In the current experiments, mice lacking the Chrna6 or Chrnb3 gene were tested for three ethanol behaviors: choice ethanol consumption, ataxia, and sedation. Wildtype (WT), heterozygous (HET), and knockout (KO) mice of each strain went through a standard 2-bottle choice drinking paradigm, the balance beam, and the Loss of Righting Reflex (LORR) paradigm. No genotypic effects on any of the 3 behavioral tasks were observed in Chrnb3 animals. While the Chrna6 gene did not significantly influence ethanol consumption (g/kg) or ataxia, mice lacking the α6 subunit took significantly longer to recover their righting reflex than WT animals. These data provide evidence that receptors containing this subunit modulate the sedative effects of ethanol. Further work examining other models of ethanol consumption and behavioral responses to ethanol is needed to fully characterize the role of these receptor subunits in modulating ethanol responses. PMID:22572056

  6. Survivin blockade sensitizes rhabdomyosarcoma cells for lysis by fetal acetylcholine receptor-redirected T cells.

    PubMed

    Simon-Keller, Katja; Paschen, Annette; Hombach, Andreas A; Ströbel, Philipp; Coindre, Jean-Michel; Eichmüller, Stefan B; Vincent, Angela; Gattenlöhner, Stefan; Hoppe, Florian; Leuschner, Ivo; Stegmaier, Sabine; Koscielniak, Ewa; Leverkus, Martin; Altieri, Dario C; Abken, Hinrich; Marx, Alexander

    2013-06-01

    Cellular immunotherapy may provide a strategy to overcome the poor prognosis of metastatic and recurrent rhabdomyosarcoma (RMS) under the current regimen of polychemotherapy. Because little is known about resistance mechanisms of RMS to cytotoxic T cells, we investigated RMS cell lines and biopsy specimens for expression and function of immune costimulatory receptors and anti-apoptotic molecules by RT-PCR, Western blot analysis, IHC, and cytotoxicity assays using siRNA or transfection-modified RMS cell lines, together with engineered RMS-directed cytotoxic T cells specific for the fetal acetylcholine receptor. We found that costimulatory CD80 and CD86 were consistently absent from all RMSs tested, whereas inducible T-cell co-stimulator ligand (ICOS-L; alias B7H2) was expressed by a subset of RMSs and was inducible by tumor necrosis factor α in two of five RMS cell lines. Anti-apoptotic survivin, along with other inhibitor of apoptosis (IAP) family members (cIAP1, cIAP2, and X-linked inhibitor of apoptosis protein), was overexpressed by RMS cell lines and biopsy specimens. Down-regulation of survivin by siRNA or pharmacologically in RMS cells increased their susceptibility toward a T-cell attack, whereas induction of ICOS-L did not. Treatment of RMS-bearing Rag(-/-) mice with fetal acetylcholine receptor-specific chimeric T cells delayed xenograft growth; however, this happened without definitive tumor eradication. Combined blockade of survivin and application of chimeric T cells in vivo suppressed tumor proliferation during survivin inhibition. In conclusion, survivin blockade provides a strategy to sensitize RMS cells for T-cell-based therapy. PMID:23562272

  7. Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus.

    PubMed

    Endo, Toshiaki; Yanagawa, Yuchio; Obata, Kunihiko; Isa, Tadashi

    2005-12-01

    The superficial superior colliculus (sSC) is a key station in the sensory processing related to visual salience. The sSC receives cholinergic projections from the parabigeminal nucleus, and previous studies have revealed the presence of several different nicotinic acetylcholine receptor (nAChR) subunits in the sSC. In this study, to clarify the role of the cholinergic inputs to the sSC, we examined current responses induced by ACh in GABAergic and non-GABAergic sSC neurons using in vitro slice preparations obtained from glutamate decarboxylase 67-green fluorescent protein (GFP) knock-in mice in which GFP is specifically expressed in GABAergic neurons. Brief air pressure application of acetylcholine (ACh) elicited nicotinic inward current responses in both GABAergic and non-GABAergic neurons. The inward current responses in the GABAergic neurons were highly sensitive to a selective antagonist for alpha3beta2- and alpha6beta2-containing receptors, alpha-conotoxin MII (alphaCtxMII). A subset of these neurons exhibited a faster alpha-bungarotoxin-sensitive inward current component, indicating the expression of alpha7-containing nAChRs. We also found that the activation of presynaptic nAChRs induced release of GABA, which elicited a burst of miniature inhibitory postsynaptic currents mediated by GABA(A) receptors in non-GABAergic neurons. This ACh-induced GABA release was mediated mainly by alphaCtxMII-sensitive nAChRs and resulted from the activation of voltage-dependent calcium channels. Morphological analysis revealed that recorded GFP-positive neurons are interneurons and GFP-negative neurons include projection neurons. These findings suggest that nAChRs are involved in the regulation of GABAergic inhibition and modulate visual processing in the sSC. PMID:16107532

  8. Muscarinic Acetylcholine Receptor M3 Modulates Odorant Receptor Activity via Inhibition of β-Arrestin-2 Recruitment

    PubMed Central

    Jiang, Yue; Li, Yun Rose; Tian, Huikai; Ma, Minghong; Matsunami, Hiroaki

    2015-01-01

    The olfactory system in rodents serves a critical function in social, reproductive, and survival behaviors. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. Here, we show that an M3-R antagonist attenuates odor-induced responses in OSNs from wild-type, but not M3-R-null mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system. PMID:25800153

  9. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    SciTech Connect

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  10. Multiple binding sites in the nicotinic acetylcholine receptors: An opportunity for polypharmacolgy.

    PubMed

    Iturriaga-Vásquez, Patricio; Alzate-Morales, Jans; Bermudez, Isabel; Varas, Rodrigo; Reyes-Parada, Miguel

    2015-11-01

    For decades, the development of selective compounds has been the main goal for chemists and biologists involved in drug discovery. However, diverse lines of evidence indicate that polypharmacological agents, i.e. those that act simultaneously at various protein targets, might show better profiles than selective ligands, regarding both efficacy and side effects. On the other hand, the availability of the crystal structure of different receptors allows a detailed analysis of the main interactions between drugs and receptors in a specific binding site. Neuronal nicotinic acetylcholine receptors (nAChRs) constitute a large and diverse family of ligand-gated ion channels (LGICs) that, as a product of its modulation, regulate neurotransmitter release, which in turns produce a global neuromodulation of the central nervous system. nAChRs are pentameric protein complexes in such a way that expression of compatible subunits can lead to various receptor assemblies or subtypes. The agonist binding site, located at the extracellular region, exhibits different properties depending on the subunits that conform the receptor. In the last years, it has been recognized that nAChRs could also contain one or more allosteric sites which could bind non-classical nicotinic ligands including several therapeutically useful drugs. The presence of multiple binding sites in nAChRs offers an interesting possibility for the development of novel polypharmacological agents with a wide spectrum of actions. PMID:26318763

  11. Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

    PubMed Central

    Tank, D W; Huganir, R L; Greengard, P; Webb, W W

    1983-01-01

    Small unilamellar vesicles containing purified and reconstituted nicotinic acetylcholine receptors from Torpedo electroplax have been fused by a simple freeze-thaw procedure to form large liposomes. Giga-seal patch-recording techniques were used to form isolated patches of liposome-membrane and to measure single-channel properties of the reconstituted receptor-ion channel complex. The observed properties are quantitatively similar to those reported for vertebrate muscle nicotinic acetylcholine receptor species recorded in situ. The results demonstrate that the pentameric complex consisting of the alpha 2 beta gamma delta subunits is fully functional. The methods used in these experiments should be useful in studying the effects of chemical alterations on the properties of acetylcholine receptor channels as well as other types of purified and reconstituted ion channels. PMID:6308673

  12. (/sup 14/C)chloroacetylcholine as an advantageous affinity label of the acetylcholine receptor

    SciTech Connect

    Bodmer, D.M.; Sin-Ren, A.C.; Waser, P.G.

    1987-01-01

    The alkylating agent (/sup 14/C)chloroacetylcholine perchlorate ((/sup 14/C) 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 (/sup 14/C) ClACh to the cholinergic receptor proved to be specific and saturable, and occurred exclusively to the alpha-subunit. Halogen substitution of acetylcholine by chlorine and insertion of a /sup 14/C-isotope instead of the widely used /sup 3/H resulted in favorable properties of the affinity label.

  13. Ineffectiveness of nicotinic acetylcholine receptor antagonists for treatment-resistant depression: a meta-analysis.

    PubMed

    Wang, Hee Ryung; Woo, Young Sup; Bahk, Won-Myong

    2016-09-01

    Emerging preclinical and clinical evidences suggest a potential role of nicotinic acetylcholine receptors in the pathophysiology of depression. Several clinical trials have investigated the efficacy of nicotinic acetylcholine receptor antagonists in treatment-resistant depression. We carried out this meta-analysis to investigate whether nicotinic acetylcholine receptor antagonists significantly improve symptoms in patients with major depressive disorder who have an inadequate response to standard antidepressant therapy. A comprehensive literature search identified six randomized-controlled trials. These six trials, which included 2067 participants, were pooled for this meta-analysis using a random-effects model. Nicotinic acetylcholine receptor antagonists failed to show superior efficacy compared with placebo in terms of the mean change in the Montgomery-Asberg Depression Rating Scale score [mean difference=-0.12 (95% confidence interval (CI)=-0.96 to 0.71]; response rate [risk ratio=0.92 (95% CI=0.83-1.02)]; and remission rate [risk ratio=1.01 (95% CI=0.83-1.23)]. This meta-analysis failed to confirm preliminary positive evidence for the efficacy of nicotinic acetylcholine receptor antagonists in treatment-resistant depression. Further studies investigating the efficacy of various alternative treatment strategies for treatment-resistant depression will help clinicians to better understand and choose better treatment options for these populations. PMID:26982579

  14. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  15. Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

    PubMed Central

    Hanham, C A; Zhao, F; Tignor, G H

    1993-01-01

    We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR. Images PMID:7677960

  16. Allosteric modifiers of neuronal nicotinic acetylcholine receptors: new methods, new opportunities.

    PubMed

    Moaddel, Ruin; Jozwiak, Krzysztof; Wainer, Irving W

    2007-09-01

    Allosteric, non-competitive inhibitors (NCIs) of neuronal nicotinic acetylcholine receptors (nAChRs) have been shown to produce a wide variety of clinically relevant responses. Many of the observed effects are desired as the nAChR is the therapeutic target, while others are undesired consequences due to off-target binding at the nAChR. Thus, the determination of whether or not a lead drug candidate is an NCI should play an important role in drug discovery programs. However, the current experimental techniques used to identify NCIs are challenging, expensive, and time consuming. This review focuses on an alternative approach to the investigation of interactions between test compounds and nAChRs based upon liquid chromatographic stationary phases containing cellular fragments from cell lines expressing nAChRs. The development and validation of these phases as well as their use in drug discovery and pharmacophore modeling are discussed. PMID:17238157

  17. Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors

    PubMed Central

    Kirsch, Glenn E.; Fedorov, Nikolai B.; Kuryshev, Yuri A.; Liu, Zhiqi; Orr, Michael S.

    2016-01-01

    Abstract The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and β-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3β4, α3β4α5, α4β2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity. PMID:27505073

  18. Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors.

    PubMed

    Kirsch, Glenn E; Fedorov, Nikolai B; Kuryshev, Yuri A; Liu, Zhiqi; Armstrong, Lucas C; Orr, Michael S

    2016-08-01

    The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and β-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3β4, α3β4α5, α4β2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity. PMID:27505073

  19. Monoclonal antibodies against the native or denatured forms of muscarinic acetylcholine receptors.

    PubMed Central

    André, C; Guillet, J G; De Backer, J P; Vanderheyden, P; Hoebeke, J; Strosberg, A D

    1984-01-01

    BALB/c mice were immunized with affinity-purified muscarinic acetylcholine receptors from calf brain and their splenocytes fused with NS1 myeloma cells. Hybrid cultures were grown and selected for production of antibodies on the basis of enzyme immunoassays on calf and rat forebrain membrane preparations. Thirty-four clones were retained and six of them further subcloned. Two of these subclones produced antibodies that selectively recognized muscarinic acetylcholine receptor-bearing membranes. The M-35b antibodies interacted only with native digitonin-solubilized receptors, and not with denatured receptors. The M-23c antibodies did not react with active digitonin-solubilized receptors but recognized the denatured form. The M-23c antibodies should thus be useful in the purification of the receptor and its precursor translation products, while the M-35b antibodies could be used for the immunocytochemical localization of the receptor in cells and tissues of different species. Images Fig. 2. Fig. 3. PMID:6200320

  20. Theoretical investigation of interaction between the set of ligands and α7 nicotinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Glukhova, O. E.; Prytkova, T. R.; Shmygin, D. S.

    2016-03-01

    Nicotinic acetylcholine receptors (nAChRs) are neuron receptor proteins that provide a transmission of nerve impulse through the synapses. They are composed of a pentametric assembly of five homologous subunits (5 α7 subunits for α7nAChR, for example), oriented around the central pore. These receptors might be found in the chemical synapses of central and peripheral nervous system, and also in the neuromuscular synapses. Transmembrane domain of the one of such receptors constitutes ion channel. The conductive properties of ion channel strongly depend on the receptor conformation changes in the response of binding with some molecule, f.e. acetylcholine. Investigation of interaction between ligands and acetylcholine receptor is important for drug design. In this work we investigate theoretically the interaction between the set of different ligands (such as vanillin, thymoquinone, etc.) and the nicotinic acetylcholine receptor (primarily with subunit of the α7nAChR) by different methods and packages (AutodockVina, GROMACS, KVAZAR, HARLEM, VMD). We calculate interaction energy between different ligands in the subunit using molecular dynamics. On the base of obtained calculation results and using molecular docking we found an optimal location of different ligands in the subunit.

  1. Effect of tissue-specific acetylcholinesterase inhibitor C-547 on α3β4 and αβεδ acetylcholine receptors in COS cells.

    PubMed

    Lindovský, Jiří; Petrov, Konstantin; Krůšek, Jan; Reznik, Vladimir S; Nikolsky, Eugeny E; Vyskočil, František

    2012-08-01

    The C-547 is the most effective muscle and tissue-specific anticholinesterase among alkylammonium derivatives of 6-methyluracil (ADEMS) acting in nanomolar concentrations on locomotor muscles but not on respiratory muscles, smooth muscles and heart and brain acetylcholine esterases (AChE). When applied systematically it could influence peripheral acetylcholine receptors. The aim of the present study was to investigate the effect of C-547 on rat α3β4 (ganglionic type) and αβεδ (muscle type) nicotinic receptors expressed in COS cells. Currents evoked by rapid application of acetylcholine or nicotine were recorded in whole-cell mode by electrophysiological patch-clamp technique 2-4 days after cell transfection by plasmids coding the α3β4 or αβεδ combination of receptor subunits. In cells sensitive to acetylcholine, the application of C-547 evoked no responses. When acetylcholine was applied during an already running application of C-547, acetylcholine responses were only inhibited at concentrations higher than 10(-7)M. This inhibition is not voltage-dependent, but is accompanied by an increased rate of desensitization. Thus in both types of receptors, effective doses are approximately 100 times higher than those inhibiting AChE in leg muscles and similar to those inhibiting respiratory diaphragm muscles and external intercostal muscles. These observations show that C-547 can be considered for symptomatic treatment of myasthenia gravis and other congenital myasthenic syndromes as an inhibitor of AChE in leg muscles at concentrations much lower than those inhibiting muscle and ganglion types of acetylcholine receptors. PMID:22634638

  2. Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

    PubMed

    Hartig, P R; Raftery, M A

    1979-04-01

    Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes. PMID:427105

  3. Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus☆

    PubMed Central

    Lees, Kristin; Jones, Andrew K.; Matsuda, Kazuhiko; Akamatsu, Miki; Sattelle, David B.; Woods, Debra J.; Bowman, Alan S.

    2014-01-01

    Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect α1 nAChR group and has been named Rsanα1. Rsanα1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsanα1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken β2 nAChR, Rsanα1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 μM) and choline (100 μM). Rsanα1/β2 was insensitive to both imidacloprid (100 μM) and spinosad (100 μM). The unreliable expression of Rsanα1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides. PMID:24291321

  4. Selective actions of Lynx proteins on different nicotinic acetylcholine receptors in the locust, Locusta migratoria manilensis.

    PubMed

    Wang, Xin; Bao, Haibo; Sun, Huahua; Zhang, Yixi; Fang, Jichao; Liu, Qinghong; Liu, Zewen

    2015-08-01

    Nicotinic acetylcholine receptors (nAChRs) are major neurotransmitter receptors and targets of neonicotinoid insecticides in the insect nervous system. The full function of nAChRs is often dependent on associated proteins, such as chaperones, regulators and modulators. Here, three Lynx (Ly-6/neurotoxin) proteins, Loc-lynx1, Loc-lynx2 and Loc-lynx3, were identified in the locust, Locusta migratoria manilensis. Co-expression with Lynx resulted in a dramatic increase in agonist-evoked macroscopic currents on nAChRs Locα1/β2 and Locα2/β2 in Xenopus oocytes, but no changes in agonist sensitivity. Loc-lynx1 and Loc-lynx3 only modulated nAChRs Locα1/β2 while Loc-lynx2 modulated Locα2/β2 specifically. Meanwhile, Loc-lynx1 induced a more significant increase in currents evoked by imidacloprid and epibatidine than Loc-lynx3, and the effects of Loc-lynx1 on imidacloprid and epibatidine were significantly higher than those on acetylcholine. Among three lynx proteins, only Loc-lynx1 significantly increased [(3) H]epibatidine binding on Locα1/β2. The results indicated that Loc-lynx1 had different modulation patterns in nAChRs compared to Loc-lynx2 and Loc-lynx3. Taken together, these findings indicated that three Lynx proteins were nAChR modulators and had selective activities in different nAChRs. Lynx proteins might display their selectivities from three aspects: nAChR subtypes, various agonists and different modulation patterns. Insect Lynx (Ly-6/neurotoxin) proteins act as the allosteric modulators on insect nicotinic acetylcholine receptors (nAChRs), the important targets of insecticides. We found that insect lynx proteins showed their selectivities from at least three aspects: nAChR subtypes, various agonists and different modulation patterns. PMID:25951893

  5. Roles of nicotinic acetylcholine receptor β subunits in function of human α4-containing nicotinic receptors

    PubMed Central

    Wu, Jie; Liu, Qiang; Yu, Kewei; Hu, Jun; Kuo, Yen-Ping; Segerberg, Marsha; St John, Paul A; Lukas, Ronald J

    2006-01-01

    Naturally expressed nicotinic acetylcholine receptors (nAChR) containing α4 subunits (α4*-nAChR) in combination with β2 subunits (α4β2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. β4 subunits are also richly expressed and colocalize with α4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, α4β4-nAChR also may exist and play important functional roles. In this study, properties were determined of human α4β2- and α4β4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human α4β4-nAChR have ∼4-fold higher amplitude than those mediated via human α4β2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at α4β4-nAChR than at α4β2-nAChR. Cytisine and lobeline serve as full agonists at α4β4-nAChR but are only partial agonists at α4β2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional α4β2- and α4β4-nAChR. Whole-cell current responses show stronger inward rectification for α4β2-nAChR than for α4β4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR β2 or β4 subunits can combine with α4 subunits to generate two forms of α4*-nAChR with distinctive physiological and pharmacological features. Diversity in α4*-nAChR is of potential relevance to nervous system function, disease, and nicotine dependence. PMID:16825297

  6. Catestatin attenuates endoplasmic reticulum induced cell apoptosis by activation type 2 muscarinic acetylcholine receptor in cardiac ischemia/reperfusion

    PubMed Central

    Liao, Feng; Zheng, Yang; Cai, Junyan; Fan, Jinghui; Wang, Jing; Yang, Jichun; Cui, Qinghua; Xu, Guoheng; Tang, Chaoshu; Geng, Bin

    2015-01-01

    Catestatin (CST) is a catecholamine secretion inhibiting peptide as non-competitive inhibitor of nicotinic acetylcholine receptor. CST play a protective role in cardiac ischemia/reperfusion (I/R) but the molecular mechanism remains unclear. Cardiomyocytes endogenously produced CST and its expression was reduced after I/R. CST pretreatment decreased apoptosis especially endoplasmic reticulum (ER) stress response during I/R. The protection of CST was confirmed in H9c2 cardiomyoblasts under Anoxia/reoxygenation (A/R). In contrast, siRNA-mediated knockdown of CST exaggerated ER stress induced apoptosis. The protective effects of CST were blocked by extracellular signal-regulated kinases 1/2 (ERK1/2) inhibitor PD90895 and phosphoinositide 3-kinase (PI3 K) inhibitor wortmannin. CST also increased ERK1/2 and protein kinase B (Akt) phosphorylation and which was blocked by atropine and selective type 2 muscarinic acetylcholine (M2) receptor, but not type 1 muscarinic acetylcholine (M1) receptor antagonist. Receptor binding assay revealed that CST competitively bound to the M2 receptor with a 50% inhibitory concentration of 25.7 nM. Accordingly, CST inhibited cellular cAMP stimulated by isoproterenol or forskolin, and which was blocked by selective M2 receptor antagonist. Our findings revealed that CST binds to M2 receptor, then activates ERK1/2 and PI3 K/Akt pathway to inhibit ER stress-induced cell apoptosis resulting in attenuation cardiac I/R injury. PMID:26567709

  7. Different patterns of nicotinic acetylcholine receptor subunit transcription in human thymus.

    PubMed

    Bruno, Roxana; Sabater, Lidia; Tolosa, Eva; Sospedra, Mireia; Ferrer-Francesch, Xavier; Coll, Jaume; Foz, Marius; Melms, Arthur; Pujol-Borrell, Ricardo

    2004-04-01

    Clinical observations suggest that the thymus is strongly implicated in the pathogenesis of myasthenia gravis (MG), but questions such as the level and location of nicotinic acetylcholine receptor (AChR) subunit expression that are fundamental to postulate any pathogenic mechanism, remain controversial. We have re-examined this question by combining calibrated RT-PCR and real-time PCR to study nicotinic AChR subunit mRNA expression in a panel of normal and myasthenic thymi. The results suggest that the expression of the different AChR subunits follows three distinct patterns: constitutive for, neonatal for gamma and individually variable for alpha1, beta1 and delta. Experiments using confocal laser microdissection suggest that AChR is mainly expressed in the medullary compartment of the thymus but there is not a clear compartmentalization of subunit expression. The different patterns of subunit expression may influence decisively the level of central tolerance to the subunits and explain the focusing of the T cell response to the alpha and gamma subunits. PMID:15020075

  8. Computer modeling of the neurotoxin binding site of acetylcholine receptor spanning residues 185 through 196

    NASA Technical Reports Server (NTRS)

    Garduno-Juarez, R.; Shibata, M.; Zielinski, T. J.; Rein, R.

    1987-01-01

    A model of the complex between the acetylcholine receptor and the snake neurotoxin, cobratoxin, was built by molecular model building and energy optimization techniques. The experimentally identified functionally important residues of cobratoxin and the dodecapeptide corresponding to the residues 185-196 of acetylcholine receptor alpha subunit were used to build the model. Both cis and trans conformers of cyclic L-cystine portion of the dodecapeptide were examined. Binding residues independently identified on cobratoxin are shown to interact with the dodecapeptide AChR model.

  9. Comparative study of muscarinic acetylcholine receptors of human and rat cortical glial cells

    SciTech Connect

    Demushkin, V.P.; Burbaeva, G.S.; Dzhaliashvili, T.A.; Plyashkevich, Y.G.

    1985-04-01

    The aim of the present investigation was a comparative studyof muscarinic acetylcholine receptors in human and rat glial cells. (/sup 3/H)Quinuclidinyl-benzylate ((/sup 3/H)-QB), atropine, platiphylline, decamethonium, carbamylcholine, tubocurarine, and nicotine were used. The glial cell fraction was obtained from the cerebral cortex of rats weighing 130-140 g and from the frontal pole of the postmortem brain from men aged 60-70 years. The use of the method of radioimmune binding of (/sup 3/H)-QB with human and rat glial cell membranes demonstrated the presence of a muscarinic acetylcholine receptor in the glial cells.

  10. Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain.

    PubMed

    Wu, Jie; Liu, Qiang; Tang, Pei; Mikkelsen, Jens D; Shen, Jianxin; Whiteaker, Paul; Yakel, Jerrel L

    2016-07-01

    The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co-assembled with β2 subunits to form a novel type of α7β2 nAChR. Interestingly, the α7β2 nAChR exhibits distinctive function and pharmacology from traditional homomeric α7 nAChRs. We review recent advances in probing the distribution, function, pharmacology, pathophysiology, and stoichiometry of the heteromeric α7β2 nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling. PMID:27179601

  11. Glutamatergic synapse formation is promoted by α7-containing nicotinic acetylcholine receptors.

    PubMed

    Lozada, Adrian F; Wang, Xulong; Gounko, Natalia V; Massey, Kerri A; Duan, Jingjing; Liu, Zhaoping; Berg, Darwin K

    2012-05-30

    Glutamate is the primary excitatory transmitter in adult brain, acting through synapses on dendritic spines and shafts. Early in development, however, when glutamatergic synapses are only beginning to form, nicotinic cholinergic excitation is already widespread; it is mediated by acetylcholine activating nicotinic acetylcholine receptors (nAChRs) that generate waves of activity across brain regions. A major class of nAChRs contributing at this time is a species containing α7 subunits (α7-nAChRs). These receptors are highly permeable to calcium, influence a variety of calcium-dependent events, and are diversely distributed throughout the developing CNS. Here we show that α7-nAChRs unexpectedly promote formation of glutamatergic synapses during development. The dependence on α7-nAChRs becomes clear when comparing wild-type (WT) mice with mice constitutively lacking the α7-nAChR gene. Ultrastructural analysis, immunostaining, and patch-clamp recording all reveal synaptic deficits when α7-nAChR input is absent. Similarly, nicotinic activation of α7-nAChRs in WT organotypic culture, as well as cell culture, increases the number of glutamatergic synapses. RNA interference demonstrates that the α7-nAChRs must be expressed in the neuron being innervated for normal innervation to occur. Moreover, the deficits persist throughout the developmental period of major de novo synapse formation and are still fully apparent in the adult. GABAergic synapses, in contrast, are undiminished in number under such conditions. As a result, mice lacking α7-nAChRs have an altered balance in the excitatory/inhibitory input they receive. This ratio represents a fundamental feature of neural networks and shows for the first time that endogenous nicotinic cholinergic signaling plays a key role in network construction. PMID:22649244

  12. Habenula cholinergic neurons regulate anxiety during nicotine withdrawal via nicotinic acetylcholine receptors.

    PubMed

    Pang, Xueyan; Liu, Liwang; Ngolab, Jennifer; Zhao-Shea, Rubing; McIntosh, J Michael; Gardner, Paul D; Tapper, Andrew R

    2016-08-01

    Cholinergic neurons in the medial habenula (MHb) modulate anxiety during nicotine withdrawal although the molecular neuroadaptation(s) within the MHb that induce affective behaviors during nicotine cessation is largely unknown. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their behavioral role as autoreceptors in these neurons has not been described. To test the hypothesis that nAChR signaling in MHb cholinergic neurons could modulate anxiety, we expressed novel "gain of function" nAChR subunits selectively in MHb cholinergic neurons of adult mice. Mice expressing these mutant nAChRs exhibited increased anxiety-like behavior that was alleviated by blockade with a nAChR antagonist. To test the hypothesis that anxiety induced by nicotine withdrawal may be mediated by increased MHb nicotinic receptor signaling, we infused nAChR subtype selective antagonists into the MHb of nicotine naïve and withdrawn mice. While antagonists had little effect on nicotine naïve mice, blocking α4β2 or α6β2, but not α3β4 nAChRs in the MHb alleviated anxiety in mice undergoing nicotine withdrawal. Consistent with behavioral results, there was increased functional expression of nAChRs containing the α6 subunit in MHb neurons that also expressed the α4 subunit. Together, these data indicate that MHb cholinergic neurons regulate nicotine withdrawal-induced anxiety via increased signaling through nicotinic receptors containing the α6 subunit and point toward nAChRs in MHb cholinergic neurons as molecular targets for smoking cessation therapeutics. PMID:27020042

  13. Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.

    PubMed

    Tillman, Tommy S; Alvarez, Frances J D; Reinert, Nathan J; Liu, Chuang; Wang, Dawei; Xu, Yan; Xiao, Kunhong; Zhang, Peijun; Tang, Pei

    2016-08-26

    Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human α7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design. PMID:27385587

  14. Chemical modification and reactivity of sulfhydryls and disulfides of rat brain nicotinic-like acetylcholine receptors

    SciTech Connect

    Lukas, R.J.; Bennett, E.L.

    1980-06-25

    Rat central nervous system binding sites for ..cap alpha..-bungarotoxin display considerable biochemical homology with characterized nicotinic acetylcholine receptors from the periphery. They possess a critical disulfide residue(s), which is susceptible to chemical modification and consequent specific alteration in the affinity of the binding site for cholinergic agonists. After reaction with Na/sub 2/S/sub 2/O/sub 5/, as with reaction with dithiothreitol and 5,5'-dithiobis(2-nitrobenzoic acid), the binding site is frozen in a high affinity state toward acetylcholine. After reduction with dithiothreitol and alkylation with a variety of compounds of different molecular configuration or electrical charge, or both, the binding site is frozen in a low affinity state toward acetylcholine. Thus, effects of disulfide/sulfhydryl modification on agonist binding affinity appear to be attributable to the nature of the covalent modification rather than charge or steric alteration at the receptor active site brought about by chemical modification.

  15. A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans.

    PubMed

    Gally, Christelle; Eimer, Stefan; Richmond, Janet E; Bessereau, Jean-Louis

    2004-09-30

    Clustering neurotransmitter receptors at the synapse is crucial for efficient neurotransmission. Here we identify a Caenorhabditis elegans locus, lev-10, required for postsynaptic aggregation of ionotropic acetylcholine receptors (AChRs). lev-10 mutants were identified on the basis of weak resistance to the anthelminthic drug levamisole, a nematode-specific cholinergic agonist that activates AChRs present at neuromuscular junctions (NMJs) resulting in muscle hypercontraction and death at high concentrations. In lev-10 mutants, the density of levamisole-sensitive AChRs at NMJs is markedly reduced, yet the number of functional AChRs present at the muscle cell surface remains unchanged. LEV-10 is a transmembrane protein localized to cholinergic NMJs and required in body-wall muscles for AChR clustering. We also show that the LEV-10 extracellular region, containing five predicted CUB domains and one LDLa domain, is sufficient to rescue AChR aggregation in lev-10 mutants. This suggests a mechanism for AChR clustering that relies on extracellular protein-protein interactions. Such a mechanism is likely to be evolutionarily conserved because CUB/LDL transmembrane proteins similar to LEV-10, but lacking any assigned function, are expressed in the mammalian nervous system and might be used to cluster ionotropic receptors in vertebrates. PMID:15457263

  16. Identification of an Ascaris G protein-coupled acetylcholine receptor with atypical muscarinic pharmacology.

    PubMed

    Kimber, Michael J; Sayegh, Laura; El-Shehabi, Fouad; Song, Chuanzhe; Zamanian, Mostafa; Woods, Debra J; Day, Tim A; Ribeiro, Paula

    2009-09-01

    Acetylcholine (ACh) is a neurotransmitter/neuromodulator in the nematode nervous system and induces its effects through interaction with both ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs). The structure, pharmacology and physiological importance of LGICs have been appreciably elucidated in model nematodes, including parasitic species where they are targets for anthelmintic drugs. Significantly less, however, is understood about nematode ACh GPCRs, termed GARs (G protein-linked ACh receptors). What is known comes from the free-living Caenorhabditis elegans as no GARs have been characterized from parasitic species. Here we clone a putative GAR from the pig gastrointestinal nematode Ascaris suum with high structural homology to the C. elegans receptor GAR-1. Our GPCR, dubbed AsGAR-1, is alternatively spliced and expressed in the head and tail of adult worms but not in dorsal or ventral body wall muscle, or the ovijector. ACh activated AsGAR-1 in a concentration-dependent manner but the receptor was not activated by other small neurotransmitters. The classical muscarinic agonists carbachol, arecoline, oxotremorine M and bethanechol were also AsGAR-1 agonists but pilocarpine was ineffective. AsGAR-1 activation by ACh was partially antagonized by the muscarinic blocker atropine but pirenzepine and scopolamine were largely ineffective. Certain biogenic amine GPCR antagonists were also found to block AsGAR-1. Our conclusion is that Ascaris possesses G protein-coupled ACh receptors that are homologous in structure to those present in C. elegans, and that although they have some sequence homology to vertebrate muscarinic receptors, their pharmacology is atypically muscarinic. PMID:19327362

  17. Identification of an Ascaris G protein-coupled acetylcholine receptor with atypical muscarinic pharmacology★

    PubMed Central

    Kimber, Michael J.; Sayegh, Laura; El-Shehabi, Fouad; Song, Chuanzhe; Zamanian, Mostafa; Woods, Debra J.; Day, Tim A.; Ribeiro, Paula

    2009-01-01

    Acetylcholine (ACh) is a neurotransmitter/neuromodulator in the nematode nervous system and induces its effects through interaction with both ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs). The structure, pharmacology and physiological importance of LGICs have been appreciably elucidated in model nematodes, including parasitic species where they are targets for anthelmintic drugs. Significantly less, however, is understood about nematode ACh GPCRs, termed GARs (G protein-linked ACh receptors). What is known comes from the free-living Caenorhabditis elegans as no GARs have been characterized from parasitic species. Here we clone a putative GAR from the pig gastrointestinal nematode Ascaris suum with high structural homology to the C. elegans receptor GAR-1. Our GPCR, dubbed AsGAR-1, isalternatively spliced and expressed in the head and tail of adult worms but not in dorsal or ventralbody wall muscle, or the ovijector. ACh activated AsGAR-1 in a concentration-dependent manner but the receptor was not activated by other small neurotransmitters. The classical muscarinic agonists carbachol, arecoline, oxotremorine M and bethanechol were also AsGAR-1 agonists but pilocarpine was ineffective. AsGAR-1 activation by ACh was partially antagonized by the muscarinic blocker atropine but pirenzepine and scopolamine were largely ineffective. Certain biogenic amine GPCR antagonists were also found to block AsGAR-1. Our conclusion is that Ascaris possesses G protein-coupled ACh receptors that are homologous in structure to thosepresent in C. elegans, and that although they have some sequence homology to vertebrate muscarinic receptors, their pharmacology is atypically muscarinic. PMID:19327362

  18. Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

    SciTech Connect

    Lee, J.H.; El-Fakahany, E.E.

    1985-08-12

    Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent (/sup 3/H)N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37/sup 0/C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system. 20 references, 3 tables.

  19. Cholinergic ligand interactions with acetylcholine receptor proteins and solvent interactions with N,N-dialkylnicotinamides

    SciTech Connect

    Bean, J.W.

    1987-01-01

    A dual-chambered flow dialysis nuclear counting apparatus was used to monitor cholinergic ligand induced displacement of {sup 155}Eu{sup 3+} from acetylcholine receptor proteins. Acetylcholine, nicotine and carbamylcholine induced similar rates of displacement of {sup 155}Eu{sup 3+} probes of calcium binding sites in receptor proteins from wild type Drosophila melanogaster and Torpedo californica. The receptor isolated from a nicotine resistant strain of Drosophila melanogaster displayed an altered dependency of cholinergic ligand induced cation displacement with respect to the other two receptor proteins. Both Drosophila strains' solubilized receptor proteins migrated as three bands of molecular weights 68,000, 66,000, and 60,000 on denaturing polyacrylamide gels. Carbon-13 NMR techniques were employed to examine the effects of solvent environment on rotational energy barriers in a series of molecules related to the analeptic, nikethamide: N,N-dimethylnicotinamide, 1-nicotinoyl piperidine, and N,N-dipropylnicotinamide.

  20. Inhibition of Nicotinic Acetylcholine Receptors, a Novel Facet in the Pleiotropic Activities of Snake Venom Phospholipases A2

    PubMed Central

    Vulfius, Catherine A.; Kasheverov, Igor E.; Starkov, Vladislav G.; Osipov, Alexey V.; Andreeva, Tatyana V.; Filkin, Sergey Yu.; Gorbacheva, Elena V.; Astashev, Maxim E.; Tsetlin, Victor I.; Utkin, Yuri N.

    2014-01-01

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes. PMID:25522251

  1. Agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action

    SciTech Connect

    Forman, S.A.; Firestone, L.L.; Miller, K.W.

    1987-05-19

    Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring /sup 86/Rb/sup +/ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: (1) integrated net ion efflux (in 10 s) from untreated vesicles, (2) integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with ..cap alpha..-bungarotoxin, and (3) initial rates of efflux (5-100 ms) from vesicles that were partially blocked with ..cap alpha..-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10/sup 8/-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum /sup 86/Rb/sup +/ efflux by 50% (K/sub B/ values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, K/sub B/ values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times K/sub B/ did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. The authors conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.

  2. Neuregulin 1 as an endogenous regulator of nicotinic acetylcholine receptors in adult major pelvic ganglion neurons.

    PubMed

    Kim, Han-Gyu; Cho, Sung-Min; Lee, Choong-Ku; Jeong, Seong-Woo

    2015-08-01

    We investigated whether endogenous neuregulin 1 (NRG1) is released in a soluble form (called sNRG1) and upregulates expression of nicotinic acetylcholine receptor (nAChR) in autonomic major pelvic ganglion (MPG) neurons of adult rats. To elicit the release of sNRG1, either the hypogastric nerve or the pelvic nerve was electrically stimulated. Then, the MPG-conditioned medium (CM) was subjected to western blotting using an antibody directed against the N-terminal ectodomain of NRG1. Both sympathetic and parasympathetic nerve activation elicited the release of sNRG1 from MPG neurons in a frequency-dependent manner. The sNRG1 release was also induced by treatment of MPG neurons with either high KCl or neurotrophic factors. The biological activity of the released sNRG1 was detected by tyrosine phosphorylation (p185) of the ErbB2 receptors in MPG neurons. When MPG neurons were incubated for 6 h in the CM, the protein level of the nAChR α3 subunit and ACh-induced current (IACh) density were significantly increased. The CM-induced changes in IACh was abolished by a selective ErbB2 tyrosine kinase inhibitor. Taken together, these data suggest that NRG1 functions as an endogenous regulator of nAChR expression in adult MPG neurons. PMID:26043693

  3. Phosphocholine – an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors

    PubMed Central

    Richter, K.; Mathes, V.; Fronius, M.; Althaus, M.; Hecker, A.; Krasteva-Christ, G.; Padberg, W.; Hone, A. J.; McIntosh, J. M.; Zakrzewicz, A.; Grau, V.

    2016-01-01

    We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions. PMID:27349288

  4. Phosphocholine - an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors.

    PubMed

    Richter, K; Mathes, V; Fronius, M; Althaus, M; Hecker, A; Krasteva-Christ, G; Padberg, W; Hone, A J; McIntosh, J M; Zakrzewicz, A; Grau, V

    2016-01-01

    We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions. PMID:27349288

  5. Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

    PubMed

    Hoffmann, Carsten; Nuber, Susanne; Zabel, Ulrike; Ziegler, Nicole; Winkler, Christiane; Hein, Peter; Berlot, Catherine H; Bünemann, Moritz; Lohse, Martin J

    2012-08-01

    Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3

  6. What is the effect of nicotinic acetylcholine receptor stimulation on osteoarthritis in a rodent animal model?

    PubMed Central

    Bock, Kilian; Plaass, Christian; Coger, Vincent; Peck, Claas-Tido; Reimers, Kerstin; Stukenborg-Colsman, Christina; Claassen, Leif

    2016-01-01

    Objectives: Despite the rising number of patients with osteoarthritis, no sufficient chondroprotective and prophylactic therapy for osteoarthritis has been established yet. The purpose of this study was to verify whether stimulation of the nicotinic acetylcholine receptor via nicotine has a beneficial effect on cartilage degeneration in the development of osteoarthritis and is capable of reducing the expression of proinflammatory cytokines and cartilage degrading enzymes in synovial membranes after osteoarthritis induction. Methods: Experimental osteoarthritis was induced in Lewis rats using a standardized osteoarthritis model with monoiodoacetate. A total of 16 Lewis rats were randomized into four groups: control, sham + nicotine application, osteoarthritis, and osteoarthritis + nicotine application. Nicotine (0.625 mg/kg twice daily) was administered intraperitoneally for 42 days. We analyzed histological sections, radiological images and the expression of the proinflammatory cytokines, such as interleukin-1β, tumor necrosis factor-α and interleukin-6, and of matrix metalloproteases 3, 9 and 13 and tissue inhibitors of metalloprotease-1 in synovial membranes via quantitative polymerase chain reaction. Results: Histological and x-ray examination revealed cartilage degeneration in the osteoarthritis group compared to control or sham + nicotine groups (histological control vs osteoarthritis: p = 0.002 and x-ray control vs osteoarthritis: p = 0.004). Nicotine treatment reduced the cartilage degeneration without significant differences. Osteoarthritis induction led to a higher expression of proinflammatory cytokines and matrix metalloproteases as compared to control groups. This effect was attenuated after nicotine administration. The differences of proinflammatory cytokines and matrix metalloproteases did not reach statistical significance. Conclusion: With the present small-scale study, we could not prove a positive effect of nicotinic

  7. Acetylcholine receptor alpha-subunit and myogenin mRNAs in thymus and thymomas.

    PubMed Central

    Kornstein, M. J.; Asher, O.; Fuchs, S.

    1995-01-01

    Myasthenia gravis is an autoimmune disorder characterized in most cases by serological antibody against the acetylcholine receptor (AChR). Evidence for intrathymic localization of AChR suggests that the thymus has an important role in the pathogenesis of this disorder. Using reverse transcription followed by the polymerase chain reaction, we have demonstrated AChR alpha-subunit mRNA in thymuses and thymomas from patients with and without myasthenia gravis. We have also studied the expression of myogenin which is known to be involved in the regulation of AChR expression. By using the reverse transcription polymerase chain reaction, we found myogenin mRNAs in all of the thymuses and thymomas. Thus, both AChR alpha-subunit and myogenin mRNA are present in all of these specimens. By immunohistochemistry myoid cells (desmin and myoglobin positive) were present in all (four of four) thymuses studied and in two of five thymomas. Thus, in thymomas, nonmyoid cells might express both AChR and myogenin. These results indicate that cells within the thymus and thymoma express AChR and its regulatory protein myogenin and that such cells, under certain conditions, might play a role in the triggering of myasthenia gravis. Images Figure 2 Figure 3 PMID:7778671

  8. The Conserved RIC-3 Coiled-Coil Domain Mediates Receptor-specific Interactions with Nicotinic Acetylcholine Receptors

    PubMed Central

    Biala, Yoav; Liewald, Jana F.; Ben-Ami, Hagit Cohen; Gottschalk, Alexander

    2009-01-01

    RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation. PMID:19116311

  9. Effect of a nicotinic acetylcholine receptor agonists and antagonists on motor function in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nicotinic acetylcholine receptors (nAChR) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChR located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The...

  10. Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor.

    PubMed Central

    Poulter, L; Earnest, J P; Stroud, R M; Burlingame, A L

    1989-01-01

    Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the alpha subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the gamma subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up approximately equal to 22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide. Images PMID:2771948

  11. Role of the nicotinic acetylcholine receptor in Alzheimer's disease pathology and treatment.

    PubMed

    Lombardo, Sylvia; Maskos, Uwe

    2015-09-01

    Alzheimer's Disease (AD) is the major form of senile dementia, characterized by neuronal loss, extracellular deposits, and neurofibrillary tangles. It is accompanied by a loss of cholinergic tone, and acetylcholine (ACh) levels in the brain, which were hypothesized to be responsible for the cognitive decline observed in AD. Current medication is restricted to enhancing cholinergic signalling for symptomatic treatment of AD patients. The nicotinic acetylcholine receptor family (nAChR) and the muscarinic acetylcholine receptor family (mAChR) are the target of ACh in the brain. Both families of receptors are affected in AD. It was demonstrated that amyloid beta (Aβ) interacts with nAChRs. Here we discuss how Aβ activates or inhibits nAChRs, and how this interaction contributes to AD pathology. We will discuss the potential role of nAChRs as therapeutic targets. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25514383

  12. INHIBITORY EFFECTS OF VOLATILE ORGANIC COMPOUNDS ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS.

    EPA Science Inventory

    INHIBITORY EFFECTS OF VOLATILE ORGANIC COMPOUNDS ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS.
    A.S. Bale*; P.J. Bushnell; C.A. Meacham; T.J. Shafer
    Neurotoxicology Division, NHEERL, ORD, US Environmental Protection Agency, Research Triangle Park, NC, USA
    Toluene (TOL...

  13. α4 nicotinic acetylcholine receptor modulated by galantamine on nigrostriatal terminals regulates dopamine receptor-mediated rotational behavior.

    PubMed

    Inden, Masatoshi; Takata, Kazuyuki; Yanagisawa, Daijiro; Ashihara, Eishi; Tooyama, Ikuo; Shimohama, Shun; Kitamura, Yoshihisa

    2016-03-01

    Galantamine, an acetylcholine esterase (AChE) inhibitor used to treat dementia symptoms, also acts as an allosteric potentiating ligand (APL) at nicotinic acetylcholine receptors (nAChRs). This study was designed to evaluate the allosteric effect of galantamine on nAChR regulation of nigrostrial dopaminergic neuronal function in the hemiparkinsonian rat model established by unilateral nigral 6-hydroxydopamine (6-OHDA) injection. Methamphetamine, a dopamine releaser, induced ipsilateral rotation, whereas dopamine agonists apomorphine (a non-selective dopamine receptor agonist), SKF38393 (a selective dopamine D1 receptor agonist), and quinpirole (a selective dopamine D2 receptor agonist) induced contralateral rotation. When 6-OHDA-injected rats were co-treated with nomifensine, a dopamine transporter inhibitor, a more pronounced and a remarkable effect of nicotine and galantamine was observed. Under these conditions, the combination of nomifensine with nicotine or galantamine induced the ipsilateral rotation similar to the methamphetamine-induced rotational behavior, indicating that nicotine and galantamine also induce dopamine release from striatal terminals. Both nicotine- and galantamine-induced rotations were significantly blocked by flupenthixol (an antagonist of both D1 and D2 dopamine receptors) and mecamylamine (an antagonist of nAChRs), suggesting that galantamine modulation of nAChRs on striatal dopaminergic terminals regulates dopamine receptor-mediated movement. Immunohistochemical staining showed that α4 nAChRs were highly expressed on striatal dopaminergic terminals, while no α7 nAChRs were detected. Pretreatment with the α4 nAChR antagonist dihydroxy-β-erythroidine significantly inhibited nicotine- and galantamine-induced rotational behaviors, whereas pretreatment with the α7 nAChR antagonist methyllycaconitine was ineffective. Moreover, the α4 nAChR agonist ABT-418 induced ipsilateral rotation, while the α7 nAChR agonist PNU282987 had no

  14. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    SciTech Connect

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  15. Tracking the molecular evolution of calcium permeability in a nicotinic acetylcholine receptor.

    PubMed

    Lipovsek, Marcela; Fierro, Angélica; Pérez, Edwin G; Boffi, Juan C; Millar, Neil S; Fuchs, Paul A; Katz, Eleonora; Elgoyhen, Ana Belén

    2014-12-01

    Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels. PMID:25193338

  16. Tracking the Molecular Evolution of Calcium Permeability in a Nicotinic Acetylcholine Receptor

    PubMed Central

    Lipovsek, Marcela; Fierro, Angélica; Pérez, Edwin G.; Boffi, Juan C.; Millar, Neil S.; Fuchs, Paul A.; Katz, Eleonora; Elgoyhen, Ana Belén

    2014-01-01

    Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels. PMID:25193338

  17. How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

    PubMed Central

    Barchan, D; Kachalsky, S; Neumann, D; Vogel, Z; Ovadia, M; Kochva, E; Fuchs, S

    1992-01-01

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well. Images PMID:1380164

  18. How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

    PubMed

    Barchan, D; Kachalsky, S; Neumann, D; Vogel, Z; Ovadia, M; Kochva, E; Fuchs, S

    1992-08-15

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well. PMID:1380164

  19. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    PubMed Central

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs. PMID:27485575

  20. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    NASA Astrophysics Data System (ADS)

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-08-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

  1. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors.

    PubMed

    Lyukmanova, E N; Shulepko, M A; Shenkarev, Z O; Bychkov, M L; Paramonov, A S; Chugunov, A O; Kulbatskii, D S; Arvaniti, M; Dolejsi, Eva; Schaer, T; Arseniev, A S; Efremov, R G; Thomsen, M S; Dolezal, V; Bertrand, D; Dolgikh, D A; Kirpichnikov, M P

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs. PMID:27485575

  2. Mechanism of nasal tolerance induced by a recombinant fragment of acetylcholine receptor for treatment of experimental myasthenia gravis.

    PubMed

    Im, S H; Barchan, D; Fuchs, S; Souroujon, M C

    2000-11-01

    Acetylcholine receptor (AChR) is the major autoantigen in myasthenia gravis (MG) and experimental autoimmune MG (EAMG). Here we analyze the mechanisms involved in suppression of ongoing EAMG in rats by nasal administration of a recombinant fragment from the human AChR alpha-subunit. We demonstrate that such a fragment, expressed without a fusion partner, confers nasal tolerance that can be adoptively transferred. Our observations suggest that the underlying mechanism of this nasal tolerance is active suppression involving a shift from a Th1 to a Th2/Th3-regulated AChR-specific response which may be mediated by down regulation of costimulatory factors. PMID:11063834

  3. Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors.

    PubMed

    Ton, Hoai T; Smart, Amanda E; Aguilar, Brittany L; Olson, Thao T; Kellar, Kenneth J; Ahern, Gerard P

    2015-08-01

    The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action. PMID:25964258

  4. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  5. Mode of action of triflumezopyrim: A novel mesoionic insecticide which inhibits the nicotinic acetylcholine receptor.

    PubMed

    Cordova, Daniel; Benner, Eric A; Schroeder, Mark E; Holyoke, Caleb W; Zhang, Wenming; Pahutski, Thomas F; Leighty, Robert M; Vincent, Daniel R; Hamm, Jason C

    2016-07-01

    Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace (3)H-imidacloprid with a Ki value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC50 of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 μM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick β2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism. Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid. PMID:27130855

  6. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  7. M1-M3 muscarinic acetylcholine receptor-deficient mice: novel phenotypes.

    PubMed

    Gautam, Dinesh; Duttaroy, Alokesh; Cui, Yinghong; Han, Sung-Jun; Deng, Chuxia; Seeger, Thomas; Alzheimer, Christian; Wess, Jürgen

    2006-01-01

    The five muscarinic acetylcholine receptors (M1-M5 mAChRs) mediate a very large number of important physiological functions (Caulfield, 1993; Caulfield and Birdsall, 1998; Wess, 2004). Because of the lack of small molecule ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proved to be a challenging task. To overcome these difficulties, we recently generated mutant mouse lines deficient in each of the five mAChR genes (M1R-/- mice, M2R-/- mice, M3R-/- mice, etc. [Wess, 2004]). Phenotyping studies showed that each of the five mutant mouse lines displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits (Wess, 2004). This chapter summarizes recent findings dealing with the importance of the M2mAChR for cognitive processes and the roles of the M1 and M3 mAChRs in mediating stimulation of glandular secretion. PMID:17192665

  8. A prion-like protein from chicken brain copurifies with an acetylcholine receptor-inducing activity.

    PubMed Central

    Harris, D A; Falls, D L; Johnson, F A; Fischbach, G D

    1991-01-01

    The mammalian prion protein (PrPC) is a cellular protein of unknown function, an altered isoform of which (PrPSc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. We report here the isolation of a cDNA that encodes a chicken protein that is homologous to PrPC. This chicken prion-like protein (ch-PrLP) is identical to the mouse PrP at 33% of its amino acid positions, including an uninterrupted stretch of 24 identical residues, and it displays the same structural domains. In addition, ch-PrLP, like its mammalian counterpart, is attached to the cell surface by a glycosyl-phosphatidylinositol anchor. We find that ch-PrLP is the major protein in preparations of an acetylcholine receptor-inducing activity that has been purified greater than 10(6)-fold from brain on the basis of its ability to stimulate synthesis of nicotinic receptors by cultured myotubes. The ch-PrLP gene is expressed in the spinal cord and brain as early as embryonic day 6; and in the spinal cord, the protein appears to be concentrated in motor neurons. Our results therefore raise the possibility that prion proteins serve normally to regulate the chemoreceptor number at the neuromuscular junction and perhaps in the central nervous system as well. Images PMID:1715573

  9. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by GABAB receptors in the rat hippocampus

    PubMed Central

    Morton, Robin A; Manuel, Nick A; Bulters, Diederick O; Cobb, Stuart R; Davies, Ceri H

    2001-01-01

    Both GABAB and muscarinic acetylcholine receptors (mAChRs) influence hippocampal-dependent mnemonic processing. Here the possibility of a direct interaction between GABAB receptors and mAChR-mediated synaptic responses has been studied using intracellular recording in rat hippocampal slices. The GABAB receptor agonist(−)-baclofen (5–10 μm) depressed an atropine-sensitive slow EPSP (EPSPM) and occluded the GABAB-receptor-mediated IPSP (IPSPB) which preceded it. These inhibitory effects were accompanied by postsynaptic hyperpolarization (9 ± 2 mV) and a reduction in cell input resistance (12 ± 3 %). The selective GABAB receptor antagonist CGP 55845A (1 μm) fully reversed the depressant effects of (−)-baclofen (5–10 μm) such that in the combined presence of (−)-baclofen and CGP 55845A the EPSPM was 134 ± 21 % of control. (−)-Baclofen (5–10 μm) caused a small (28 ± 11 %) inhibition of carbachol-induced (3.0 μm) postsynaptic depolarizations and increases in input resistance. CGP 55845A (1 μm) alone caused an increase in the amplitude of the EPSPM (253 ± 74 % of control) and blocked the IPSPB that preceded it. In contrast, the selective GABA uptake inhibitor NNC 05–0711 (10 μm) increased the amplitude of the IPSPB by 141 ± 38 % and depressed the amplitude of the EPSPM by 58 ± 10 %. This inhibition was abolished by CGP 55845A (1 μm). Taken together these data provide good evidence that synaptically released GABA activates GABAB receptors that inhibit mAChR-mediated EPSPs in hippocampal CA1 pyramidal neurones. The mechanism of inhibition may involve both pre- and postsynaptic elements. PMID:11559773

  10. Effects of cannabidiol on the function of α7-nicotinic acetylcholine receptors.

    PubMed

    Mahgoub, Mohamed; Keun-Hang, Susan Yang; Sydorenko, Vadym; Ashoor, Abrar; Kabbani, Nadine; Al Kury, Lina; Sadek, Bassem; Howarth, Christopher F; Isaev, Dmytro; Galadari, Sehamuddin; Oz, Murat

    2013-11-15

    The effects of cannabidiol (CBD), a non-psychoactive ingredient of cannabis plant, on the function of the cloned α7 subunit of the human nicotinic acetylcholine (α7 nACh) receptor expressed in Xenopus oocytes were tested using the two-electrode voltage-clamp technique. CBD reversibly inhibited ACh (100 μM)-induced currents with an IC50 value of 11.3 µM. Other phytocannabinoids such as cannabinol and Δ(9)-tetrahydrocannabinol did not affect ACh-induced currents. CBD inhibition was not altered by pertussis toxin treatment. In addition, CBD did not change GTP-γ-S binding to the membranes of oocytes injected with α7 nACh receptor cRNA. The effect of CBD was not dependent on the membrane potential. CBD (10 µM) did not affect the activity of endogenous Ca(2+)-dependent Cl(-) channels, since the extent of inhibition by CBD was unaltered by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing 2mM Ba(2+). Inhibition by CBD was not reversed by increasing ACh concentrations. Furthermore, specific binding of [(125)I] α-bungarotoxin was not inhibited by CBD (10 µM) in oocytes membranes. Using whole cell patch clamp technique in CA1 stratum radiatum interneurons of rat hippocampal slices, currents induced by choline, a selective-agonist of α7-receptor induced currents were also recoded. Bath application of CBD (10 µM) for 10 min caused a significant inhibition of choline induced currents. Finally, in hippocampal slices, [(3)H] norepinephrine release evoked by nicotine (30 µM) was also inhibited by 10 µM CBD. Our results indicate that CBD inhibits the function of the α7-nACh receptor. PMID:24140434

  11. Mutations of Cytosolic Loop Residues Impair Assembly and Maturation of α7 Nicotinic Acetylcholine Receptors

    PubMed Central

    Mukherjee, Jayanta; Kuryatov, Alexander; Moss, Stephen J.; Lindstrom, Jon M.; Anand, Rene

    2009-01-01

    Mechanisms that regulate early events in the biogenesis of the α7 nicotinic acetylcholine receptor (α7 AChR) are not well understood. Data presented here show that single amino acid mutations in the cytoplasmic loop of the α7 AChR, between position 335 and 343, abolish or attenuate expression of mature pentameric α7 AChRs in both human embryonic kidney tsA201 (HEK) and neuronal SH-SY5Y cells. Although the number of mature α7 AChRs is increased significantly in the presence of the chaperone protein RIC-3 in HEK cells, sucrose gradient sedimentation reveals that the vast majority of α7 subunits are aggregated or improperly assembled. Transfection of α7 AChRs in SH-SY5Y cells, which endogenously express the α7 AChR, results in a much larger fraction of subunits assembled into mature AChRs. Thus, efficient assembly of α7 AChRs is influenced by several regions of the large cytoplasmic domain, as well perhaps by other parts of its structure, and requires as yet unknown factors not required by other AChR subtypes. PMID:19627445

  12. Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor.

    PubMed

    Kamerbeek, Constanza B; Mateos, Melina V; Vallés, Ana S; Pediconi, María F; Barrantes, Francisco J; Borroni, Virginia

    2016-05-01

    Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity. PMID:26898898

  13. Acetylcholine enhances excitability by lowering the threshold of spike generation in olfactory receptor cells.

    PubMed

    Ohkuma, Mahito; Kawai, Fusao; Miyachi, Ei-ichi

    2013-11-01

    Olfactory perception is influenced by behavioral states, presumably via efferent regulation. Using the whole cell version of patch-clamp recording technique, we discovered that acetylcholine, which is released from efferent fibers in the olfactory mucosa, can directly affect the signal encoding in newt olfactory receptor cells (ORCs). Under current-clamp conditions, application of carbachol, an acetylcholine receptor agonist, increased the spike frequency of ORCs and lowered their spike threshold. When a 3-pA current to induce near-threshold depolarization was injected into ORCs, 0.0 spikes/s were generated in control solution and 0.5 spikes/s in the presence of carbachol. By strong stimuli of injection of a 13-pA current into ORCs, 9.1 and 11.0 spikes/s were generated in control and carbachol solutions, respectively. A similar result was observed by bath application of 50 μM acetylcholine. Under voltage-clamp conditions, carbachol increased the peak amplitude of a voltage-gated sodium current by 32% and T-type calcium current by 39%. Atropine, the specific muscarinic receptor antagonist, blocked the enhancement by carbachol of the voltage-gated sodium current and T-type calcium current, suggesting that carbachol increases those currents via the muscarinic receptor rather than via the nicotinic receptor. In contrast, carbachol did not significantly change the amplitude of the L-type calcium current or the delayed rectifier potassium current in the ORCs. Because T-type calcium current is known to lower the threshold in ORCs, we suggest that acetylcholine enhance excitability by lowering the threshold of spike generation in ORCs via the muscarinic receptor. PMID:23926039

  14. Molecular Mechanisms of Cross-inhibition Between Nicotinic Acetylcholine Receptors and P2X Receptors in Myenteric Neurons and HEK-293 cells

    PubMed Central

    Decker, Dima A.; Galligan, James J.

    2010-01-01

    Background P2X2 and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and α3β4subunit-containing nAChRs. Methods The function of P2X2 and α3β4 nAChRs expressed by HEK-293 cells and guinea pig ileum myenteric neurons in culture was studied using whole-cell patch clamp techniques. Results In HEK-293 cells expressing α3β4 nAChRs and P2X2 receptors, co-application of ATP and ACh caused inward currents that were 56 ± 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs and a C-terminal tail truncated P2X2 receptor (P2X2TR)(P >0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2 receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK-293 cells expressing both receptors. Constitutive inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs transfected with P2X2TR. Currents caused by low (≤30 μM), but not high (≥100 μM) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co-expression with α3β4 nAChRs. Conclusions The C-terminal tail of P2X2 receptors mediates cross-inhibition between α3β4 nAChR-P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co-transmitters. PMID:20426799

  15. α7 Nicotinic Acetylcholine Receptor Signaling Inhibits Inflammasome Activation by Preventing Mitochondrial DNA Release

    PubMed Central

    Lu, Ben; Kwan, Kevin; Levine, Yaakov A; Olofsson, Peder S; Yang, Huan; Li, Jianhua; Joshi, Sonia; Wang, Haichao; Andersson, Ulf; Chavan, Sangeeta S; Tracey, Kevin J

    2014-01-01

    The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide–induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release. PMID:24849809

  16. The channel opening rate of adult- and fetal-type mouse muscle nicotinic receptors activated by acetylcholine

    PubMed Central

    Maconochie, David J; Steinbach, Joe Henry

    1998-01-01

    In this paper, we examine acetylcholine (ACh)-induced currents in quail fibroblast cell lines expressing either the fetal (Q-F18) or the adult (Q-A33) complement of nicotinic acetylcholine receptor subunits derived from mouse skeletal muscle. Pulses of ACh were applied to outside-out patches of cell membrane by means of a fast perfusion system, at concentrations from 100 nM to 10 mM. We obtained current records with intracellular potentials of -60 and +40 mV. The goal of this study was to estimate the channel opening rate.By fitting sums of exponentials to averaged responses, we estimated the rate of development of the current on the application of acetylcholine. The rate constant of the predominant exponential component (the on-rate) ranges over 3 orders of magnitude, from around 100 s−1 (fetal) at low concentrations of ACh to over 100 000 s−1 (fetal and adult) at the highest concentrations.We establish that our measurement of the on-rate is not limited by technical constraints, and can therefore be related to the rate constants of a kinetic scheme. Our observations are consistent with a model having a rate-limiting channel opening step with a forwards rate constant (β) of 80 000 s−1 on average for adult receptors and 60 000 s−1 for fetal receptors, and a minimum opening to closing ratio (β/α) of around 33 (adult) or 50 (fetal). The channel opening rate, β, varies from around 30 000 s−1 to well over 100 000 s−1 for different patches. The large variation cannot all be ascribed to errors of measurement, but indicates patch to patch variation. PMID:9481672

  17. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors.

    PubMed

    Thal, David M; Sun, Bingfa; Feng, Dan; Nawaratne, Vindhya; Leach, Katie; Felder, Christian C; Bures, Mark G; Evans, David A; Weis, William I; Bachhawat, Priti; Kobilka, Tong Sun; Sexton, Patrick M; Kobilka, Brian K; Christopoulos, Arthur

    2016-03-17

    Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains. PMID:26958838

  18. Evaluation of Ca2+ permeability of nicotinic acetylcholine receptors in hypothalamic histaminergic neurons

    PubMed Central

    Uteshev, Victor V.

    2010-01-01

    Hypothalamic histaminergic tuberomammillary (TM) neurons express nicotinic acetylcholine receptors (nAChRs) with kinetic and pharmacological properties resembling those of highly Ca2+ permeable α7 nAChRs. However, the Ca2+ permeability of TM nAChR channels has not been determined. To directly evaluate the Ca2+ permeability of TM nAChRs, patch-clamp recordings were conducted using non-cultured acutely dissociated TM neurons and external solutions containing low (2 mM) and high (20 mM) concentrations of Ca2+. A shift in the reversal potentials was determined from the current–voltage relationships and the permeability ratio, PCa/PNa, was estimated within the Goldman-Hodgkin-Katz constant field approximation. TM nAChRs were found to be highly Ca2+ permeable with the permeability ratio, PCa/PNa(nAChR) being ∼5.9 and the fractional Ca2+ current, Pf(nAChR) being ∼10.1% at −60 mV. As a positive control for the applied methods and analysis, the permeability ratio, PCa/PNa(NMDAR) being ∼8.3 and the fractional Ca2+ current, Pf(NMDAR) being ∼13.6% at −60 mV for NMDA receptors were determined using non-cultured acutely dissociated hippocampal pyramidal neurons and found similar to previously reported values. Therefore, these results demonstrate that native TM nAChRs are highly Ca2+ permeable, but ∼1.4 fold less permeable to Ca2+ than native hippocampal pyramidal NMDA receptors. PMID:20043042

  19. Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation.

    PubMed

    Ma, Lei; Seager, Matthew A; Seager, Matthew; Wittmann, Marion; Jacobson, Marlene; Bickel, Denise; Burno, Maryann; Jones, Keith; Graufelds, Valerie Kuzmick; Xu, Guangping; Pearson, Michelle; McCampbell, Alexander; Gaspar, Renee; Shughrue, Paul; Danziger, Andrew; Regan, Christopher; Flick, Rose; Pascarella, Danette; Garson, Susan; Doran, Scott; Kreatsoulas, Constantine; Veng, Lone; Lindsley, Craig W; Shipe, William; Kuduk, Scott; Sur, Cyrille; Kinney, Gene; Seabrook, Guy R; Ray, William J

    2009-09-15

    The forebrain cholinergic system promotes higher brain function in part by signaling through the M(1) muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M(1) receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M(1) mAChR. BQCA reduces the concentration of ACh required to activate M(1) up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 microM. Furthermore studies in M(1)(-/-) mice demonstrates that BQCA requires M(1) to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M(1) allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces beta-arrestin recruitment to M(1), suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M(1) receptor and represents a promising therapeutic strategy for cognitive disorders. PMID:19717450

  20. Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation

    PubMed Central

    Ma, Lei; Seager, Matthew A.; Wittmann, Marion; Jacobson, Marlene; Bickel, Denise; Burno, Maryann; Jones, Keith; Graufelds, Valerie Kuzmick; Xu, Guangping; Pearson, Michelle; McCampbell, Alexander; Gaspar, Renee; Shughrue, Paul; Danziger, Andrew; Regan, Christopher; Flick, Rose; Pascarella, Danette; Garson, Susan; Doran, Scott; Kreatsoulas, Constantine; Veng, Lone; Lindsley, Craig W.; Shipe, William; Kuduk, Scott; Sur, Cyrille; Kinney, Gene; Seabrook, Guy R.; Ray, William J.

    2009-01-01

    The forebrain cholinergic system promotes higher brain function in part by signaling through the M1 muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M1 receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M1 mAChR. BQCA reduces the concentration of ACh required to activate M1 up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M1−/− mice demonstrates that BQCA requires M1 to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M1, suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M1 receptor and represents a promising therapeutic strategy for cognitive disorders. PMID:19717450

  1. Selectivity optimization of substituted 1,2,3-triazoles as α7 nicotinic acetylcholine receptor agonists.

    PubMed

    Arunrungvichian, Kuntarat; Fokin, Valery V; Vajragupta, Opa; Taylor, Palmer

    2015-08-19

    Three series of substituted anti-1,2,3-triazoles (IND, PPRD, and QND), synthesized by cycloaddition from azide and alkyne building blocks, were designed to enhance selectivity and potency profiles of a lead α7 nicotinic acetylcholine receptor (α7-nAChR) agonist, TTIn-1. Designed compounds were synthesized and screened for affinity by a radioligand binding assay. Their functional characterization as agonists and antagonists was performed by fluorescence resonance energy transfer assay using cell lines expressing transfected cDNAs, α7-nAChRs, α4β2-nAChRs, and 5HT3A receptors, and a fluorescence cell reporter. In the IND series, a tropane ring of TTIn-1, substituted at N1, was replaced by mono- and bicyclic amines to vary length and conformational flexibility of a carbon linker between nitrogen atom and N1 of the triazole. Compounds with a two-carbon atom linker optimized binding with Kd's at the submicromolar level. Further modification at the hydrophobic indole of TTIn-1 was made in PPRD and QND series by fixing the amine center with the highest affinity building blocks in the IND series. Compounds from IND and PPRD series are selective as agonists for the α7-nAChRs over α4β2-nAChRs and 5HT3A receptors. Lead compounds in the three series have EC50's between 28 and 260 nM. Based on the EC50, affinity, and selectivity determined from the binding and cellular responses, two of the leads have been advanced to behavioral studies described in the companion article (DOI: 10.1021/acschemneuro.5b00059). PMID:25932897

  2. Role of α5 Nicotinic Acetylcholine Receptors in Pharmacological and Behavioral Effects of Nicotine in Mice

    PubMed Central

    Marks, M. J.; Vann, R. E.; Chen, X.; Gamage, T. F.; Warner, J. A.; Damaj, M. I.

    2010-01-01

    Incorporation of the α5 nicotinic acetylcholine receptor (nAChR) subunit can greatly influence nAChR function without altering receptor number. Although few animal studies have assessed the role of the α5 nAChR in nicotine-mediated behaviors, recent evidence suggests an association between polymorphisms in the α5 nAChR gene and nicotine dependence phenotypes in humans. Thus, additional studies are imperative to elucidate the role and function of the α5 nAChR subunit in nicotine dependence. Using α5(−/−) mice, the current study aimed to examine the role of α5 nAChRs in the initial pharmacological effects of nicotine, nicotine reward using the conditioned place preference model, and the discriminative effects of nicotine using a two-lever drug discrimination model. 86Rb+ efflux and 125I-epibatidine binding assays were conducted to examine the effect of α5 nAChR subunit deletion on expression and activity of functional nAChRs. Results show that α5(−/−) mice are less sensitive to the initial effects of nicotine in antinociception, locomotor activity, and hypothermia measures and that the α5 nAChR is involved in nicotine reward. Alternatively, α5(−/−) mice did not differ from wild-type littermates in sensitivity to the discriminative stimulus effects of nicotine. Furthermore, deletion of the α5 nAChR subunit resulted in a statistically significant decrease in function in the thalamus and hindbrain, but the decreases noted in spinal cord were not statistically significant. Receptor number was unaltered in all areas tested. Taken together, results of the study suggest that α5 nAChRs are involved in nicotine-mediated behaviors relevant to development of nicotine dependence. PMID:20400469

  3. Bladder outlet obstruction causes up-regulation of nicotinic acetylcholine receptors in bladder-projecting pelvic ganglion neurons.

    PubMed

    Chung, Hyun-Chul; Lee, Choong-Ku; Park, Kwang-Hwa; Jeong, Seong-Woo

    2015-03-30

    Pelvic ganglion (PG) neurons relay sympathetic and parasympathetic signals to the lower urinary tract, comprising the urinary bladder and bladder outlet, and are thus essential for both storage and voiding reflexes. Autonomic transmission is mediated by activation of the nicotinic acetylcholine receptor (nAChR) in PG neurons. Previously, bladder outlet obstruction (BOO), secondary to benign prostatic hyperplasia, was found to increase soma sizes of bladder-projecting PG neurons. To date, however, it remains unknown whether these morphological changes are accompanied by functional plasticity in PG neurons. In the present study, we investigated whether BOO alters acetylcholine receptor (nAChR) transcript expression and current density in bladder PG neurons. Partial ligation of the rat urethra for six weeks induced detrusor overactivity (DO), as observed during cystometrical measurement. In rats exhibiting DO, membrane capacitance of parasympathetic bladder PG neurons was selectively increased. Real-time PCR analysis revealed that BOO enhanced the expression of the transcripts encoding the nAChR α3 and β4 subunits in PG neurons. Notably, BOO significantly increased ACh-evoked current density in parasympathetic bladder PG neurons, whereas no changes were observed in sympathetic bladder and parasympathetic penile PG neurons. In addition, other ligand-gated ionic currents were immune to BOO in bladder PG neurons. Taken together, these data suggest that BOO causes upregulation of nAChR in parasympathetic bladder PG neurons, which in turn may potentiate ganglionic transmission and contribute to the development of DO. PMID:25625357

  4. Nicotinic acetylcholine receptor-mediated responses in medial vestibular and prepositus hypoglossi nuclei neurons showing distinct neurotransmitter phenotypes.

    PubMed

    Zhang, Yue; Yanagawa, Yuchio; Saito, Yasuhiko

    2016-06-01

    Cholinergic transmission in both the medial vestibular nucleus (MVN) and prepositus hypoglossi nucleus (PHN) plays an important role in horizontal eye movements. We previously demonstrated that the current responses mediated via nicotinic acetylcholine receptors (nAChRs) were larger than those mediated via muscarinic acetylcholine receptors (mAChRs) in cholinergic MVN and PHN neurons that project to the cerebellum. In this study, to clarify the predominant nAChR responses and the expression patterns of nAChRs in MVN and PHN neurons that exhibit distinct neurotransmitter phenotypes, we identified cholinergic, inhibitory, and glutamatergic neurons using specific transgenic rats and investigated current responses to the application of acetylcholine (ACh) using whole cell recordings in brain stem slices. ACh application induced larger nAChR-mediated currents than mAChR-mediated currents in every neuronal phenotype. In the presence of an mAChR antagonist, we found three types of nAChR-mediated currents that exhibited different rise and decay times and designated these as fast (F)-, slow (S)-, and fast and slow (FS)-type currents. F-type currents were the predominant response in inhibitory MVN neurons, whereas S-type currents were observed in the majority of glutamatergic MVN and PHN neurons. No dominant response type was observed in cholinergic neurons. Pharmacological analyses revealed that the F-, S-, and FS-type currents were mainly mediated by α7, non-α7, and both α7 and non-α7 nAChRs, respectively. These findings suggest that cholinergic responses in the major neuronal populations of the MVN and PHN are predominantly mediated by nAChRs and that the expression of α7 and non-α7 nAChRs differ among the neuronal phenotypes. PMID:26936981

  5. Subtype-selective positive cooperative interactions between brucine analogs and acetylcholine at muscarinic receptors: functional studies.

    PubMed

    Birdsall, N J; Farries, T; Gharagozloo, P; Kobayashi, S; Lazareno, S; Sugimoto, M

    1999-04-01

    In radioligand binding studies, it has been reported that brucine, N-chloromethyl brucine, and brucine N-oxide increased the affinity of acetylcholine for M1, M3, and M4 muscarinic receptors, respectively, in a manner consistent with the predictions of the ternary complex allosteric model. We now demonstrate an equivalent ability of these three allosteric agents to modulate the actions of acetylcholine in functional studies in membranes and in whole cells. The enhancing actions of brucine and brucine N-oxide on acetylcholine (ACh) potency at M1 and M4 receptors respectively have been confirmed in guanosine-5'-O-(3-[35S]thio)triphosphate, GTPase, cAMP, and intracellular Ca2+ mobilization assays of function. In general, neither the basal nor the maximally stimulated response to ACh is affected. The subtype-selective allosteric effects of N-chloromethyl brucine on M2 and M3 receptors were shown to be qualitatively and quantitatively the same in guanosine-5'-O-(3-[35S]thio)triphosphate functional assays, in terms of both its affinity and cooperativity with ACh, as those found in binding assays. Neutral cooperativity of N-chloromethyl brucine with ACh on M4 receptor function was also observed, thereby demonstrating its "absolute subtype selectivity": a lack of action at any concentration at M4 receptors and an action at M2 and M3 receptors. The enhancing action of N-chloromethyl brucine on neurogenically released ACh binding at M3 receptors was also detected in whole tissue as an increased contraction of the isolated guinea pig ileum to submaximal electrical stimulation. In conclusion, these functional studies confirm that brucine analogs are allosteric enhancers of ACh affinity at certain muscarinic receptor subtypes. PMID:10101037

  6. Turnover of acetylcholine receptors: Mechanisms of regulation. Final report, 1 August 1985-30 November 1990

    SciTech Connect

    Drachman, D.B.

    1990-12-31

    The synthesis, insertion and degradation of acetylcholine receptors (AChRs) of skeletal muscle cells as closely regulated both by the muscle cells and by the motor nerves that supply them. The goal of this project is to elucidate the mechanisms of regulation of the AChRs, both at the neuromuscular junctional and at extrajunctional regions. The results of our studies on junctional AChRs have shown that: Both stable and rapidly turned over (RTO) AChRs are present at normally innervated neuromuscular junctions` Synthesis and insertion of AChRs at neuromuscular junctions occurs rapidly, at a rate consistent with the rapid rate of turnover of RTOs. RTOs serve as precursors of stable AChRs. Acetylcholine receptors, RA5 Neuromuscular junctions, Motor nerves.

  7. Anomalous Interaction of the Acetylcholine Receptor Protein with the Nonionic Detergent Triton X-114

    NASA Astrophysics Data System (ADS)

    Maher, Pamela A.; Singer, S. J.

    1985-02-01

    Integral membrane proteins that form water-filled channels through membranes often exist as aggregates of similar or identical subunits spanning the membrane. It has been suggested that the insertion into the membrane of the channel-forming domains of the subunits may impart unusual structural features to the membrane-intercalated portions of the protein. To test this proposal, we have investigated the interaction of a multisubunit channel-forming integral membrane protein, the acetylcholine receptor protein, with the nonionic detergent Triton X-114. Whereas non-channel-forming integral membrane proteins that have heretofore been studied from mixed micelles with the detergent, the acetylcholine receptor was excluded from the Triton X-114 micelles. The structural implications of this result are discussed.

  8. Successive openings of the same acetylcholine receptor channel are correlated in open time.

    PubMed Central

    Jackson, M B; Wong, B S; Morris, C E; Lecar, H; Christian, C N

    1983-01-01

    Previous analysis of single-channel current records has shown that both the opening and closing transitions of chemically activated ion channels are operated by fast and slow kinetic processes. The fast component in the kinetics of channel opening has been interpreted as the reopening of a channel that has just closed. The fast component in the kinetics of channel closure has many possible explanations and is therefore more difficult to interpret. We can gain insight into the closing process by asking whether the lifetimes of successive openings of an acetylcholine receptor channel are correlated in open-state lifetime. Five kinetic models of channel closure are considered. Two of these models predict uncorrelated open-state lifetimes, one predicts correlated open-state lifetimes, and for two others a range of behavior is possible. Acetylcholine receptor channel data from cultured rat muscle are analyzed to show that open-state lifetimes are correlated, eliminating two models of channel gating. PMID:6301575

  9. Molecular cloning and characterization of two nicotinic acetylcholine receptor β subunit genes from Apis cerana cerana.

    PubMed

    Yu, Xiaoli; Wang, Mian; Kang, Mingjiang; Liu, Li; Guo, Xingqi; Xu, Baohua

    2011-08-01

    Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect nervous system and are important targets for insecticides. In this study, we identified and characterized two novel β subunit genes (Accβ1 and Accβ2) from Apis cerana cerana. Homology analysis indicated that Accβ1 and Accβ2 possess characteristics that are typical of nAChR subunits although Accβ2 was distinct from Accβ1 and the other nAChR subunits, due to its unusual transmembrane structure and uncommon exon-intron boundary within the genomic region encoding the TM1 transmembrane domain. Analysis of the 5' flanking regions indicated that Accβ1 and Accβ2 possess different regulatory elements, suggesting that the genes might exhibit various expression and regulatory patterns. RT-PCR analysis demonstrated that Accβ2 was expressed at a much higher level than Accβ1 in the tissues of adult bees. During development, Accβ1 was highly expressed at the pupal stages, whereas Accβ2 was abundantly expressed at the larval stages. Furthermore, Accβ1 and Accβ2 were both induced by exposure to various insecticides and environmental stresses although Accβ2 was more responsive than Accβ1. These results indicate that Accβ1 and Accβ2 may have distinct roles in insect growth and development and that they may belong to separate regulatory pathways involved in the response to insecticides and environmental stresses. This report is the first description of the differences between the nAChR β subunit genes in the Chinese honey bee and establishes an initial foundation for further study. PMID:21618599

  10. Conservation of neural nicotinic acetylcholine receptors from Drosophila to vertebrate central nervous systems.

    PubMed Central

    Bossy, B; Ballivet, M; Spierer, P

    1988-01-01

    Nicotinic acetylcholine receptors (nAChR) are found both in vertebrate and insect central nervous systems. We have isolated a Drosophila gene by crosshybridization with a vertebrate probe. Structural conservation of domains of the deduced protein and of intron/exon boundaries indicate that the Drosophila gene encodes an nAChR alpha-like subunit (ALS). That the Drosophila gene product most resembles the neuronal set of vertebrate nAChRs alpha-subunits is also indicated by the failure of an ALS-beta-galactosidase fusion protein to bind alpha-bungarotoxin on blots in contrast to vertebrate endplate alpha-subunit constructions. The ALS encoding gene exceeds 54 kb in length and the transcript has a very long and unusual 5' leader. As we found previously for a gene whose product is also involved in cholinergic synapses, acetylcholinesterase, the leader encodes short open reading frames, which might be involved in translation control. We also note the presence of opa repeats in the gene, as has been found for various Drosophila genes expressed in the nervous system. Images PMID:2840281

  11. Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen).

    PubMed

    Bao, Haibo; Sun, Huahua; Xiao, Youxin; Zhang, Yixi; Wang, Xin; Xu, Xiaoyong; Liu, Zewen; Fang, Jichao; Li, Zhong

    2015-01-01

    Associated proteins are important for the correct functioning of nicotinic acetylcholine receptors (nAChRs). In the present study, a neonicotinoid-agarose affinity column was used to isolate related proteins from a solubilized membrane preparation from the nervous system of Locusta migratoria manilensis (Meyen). 1530 peptides were identified and most of them were involved in the membranous structure, molecular interaction and cellular communication. Among these peptides, Na(+)/K(+) ATPase had the highest MASCOT score and were involved in the molecular interaction, which suggested that Na(+)/K(+) ATPase and nAChRs might have strong and stable interactions in insect central nervous system. In the present study, functional interactions between nAChRs and Na(+)/K(+) ATPase were examined by heterologous expression in Xenopus oocytes. The results showed that the activated nAChRs increased pump currents of Na(+)/K(+) ATPase, which did not require current flow through open nAChRs. In turn, Na(+)/K(+) ATPase significantly increased agonist sensitivities of nAChRs in a pump activity-independent manner and reduced the maximum current (Imax) of nAChRs. These findings provide novel insights concerning the functional interactions between insect nAChRs and Na(+)/K(+) ATPase. PMID:25743085

  12. Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen)

    PubMed Central

    Bao, Haibo; Sun, Huahua; Xiao, Youxin; Zhang, Yixi; Wang, Xin; Xu, Xiaoyong; Liu, Zewen; Fang, Jichao; Li, Zhong

    2015-01-01

    Associated proteins are important for the correct functioning of nicotinic acetylcholine receptors (nAChRs). In the present study, a neonicotinoid-agarose affinity column was used to isolate related proteins from a solubilized membrane preparation from the nervous system of Locusta migratoria manilensis (Meyen). 1530 peptides were identified and most of them were involved in the membranous structure, molecular interaction and cellular communication. Among these peptides, Na+/K+ ATPase had the highest MASCOT score and were involved in the molecular interaction, which suggested that Na+/K+ ATPase and nAChRs might have strong and stable interactions in insect central nervous system. In the present study, functional interactions between nAChRs and Na+/K+ ATPase were examined by heterologous expression in Xenopus oocytes. The results showed that the activated nAChRs increased pump currents of Na+/K+ ATPase, which did not require current flow through open nAChRs. In turn, Na+/K+ ATPase significantly increased agonist sensitivities of nAChRs in a pump activity-independent manner and reduced the maximum current (Imax) of nAChRs. These findings provide novel insights concerning the functional interactions between insect nAChRs and Na+/K+ ATPase. PMID:25743085

  13. Acetylcholine receptors enable the transport of rapsyn from the Golgi complex to the plasma membrane

    PubMed Central

    Park, Jee-Young; Ikeda, Hiromi; Ikenaga, Takanori; Ono, Fumihito

    2012-01-01

    The accumulation of acetylcholine receptors (AChRs) at nerve terminals is critical for signal transmission at the neuromuscular junction, and rapsyn is essential for this process. Previous studies suggest that AChRs might direct rapsyn self-clusters to the synapse. In vivo experiments with fluorescently tagged AChR or rapsyn in zebrafish larvae revealed that rapsyn self-clusters separate from AChRs did not exist before synapse formation. Examination of rapsyn in the AChR-less mutant sofa potato revealed that rapsyn in the absence of AChR was localized in the Golgi complex. Expression of muscle-type AChR in sofa potato restored synaptic clustering of rapsyn, while neuronal type AChR had no effect. To determine if this requirement of protein interaction is reciprocal, we examined the mutant twitch once, which has a missense mutation in rapsyn. While the AChRs distributed non-synaptically on the plasma membrane in twitch once, mutant rapsyn was retained in the Golgi complex. We conclude that AChRs enable the transport of rapsyn from the Golgi complex to the plasma membrane through a molecule-specific interaction. PMID:22623681

  14. In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor

    NASA Astrophysics Data System (ADS)

    Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence

    1983-05-01

    A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.

  15. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal ventral horn neurons.

    PubMed

    Mine, N; Taniguchi, W; Nishio, N; Izumi, N; Miyazaki, N; Yamada, H; Nakatsuka, T; Yoshida, M

    2015-04-01

    Nicotinic acetylcholine receptors (nAChRs) are distributed widely in the central nervous system and play important roles in higher brain functions, including learning, memory, and recognition. However, functions of the cholinergic system in spinal motoneurons remain poorly understood. In this study, we investigated the actions of presynaptic and postsynaptic nAChRs in spinal ventral horn neurons by performing whole-cell patch-clamp recordings on lumbar slices from male rats. The application of nicotine or acetylcholine generated slow inward currents and increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Slow inward currents by acetylcholine or nicotine were not inhibited by tetrodotoxin (TTX) or glutamate receptor antagonists. In the presence of TTX, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were also increased by acetylcholine or nicotine. A selective α4β2 nicotinic receptor antagonist, dihydro-β-erythroidine hydrobromide (DhβE), significantly decreased nicotine-induced inward currents without affecting the enhancement of sEPSCs and mEPSCs. In addition, a selective α7 nicotinic receptor antagonist, methyllycaconitine, did not affect either nicotine-induced inward currents or the enhancement of sEPSCs and mEPSCs. These results suggest that α4β2 AChRs are localized at postsynaptic sites in the spinal ventral horn, non-α4β2 and non-α7 nAChRs are located presynaptically, and nAChRs enhance excitatory synaptic transmission in the spinal ventral horn. PMID:25613686

  16. Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

    PubMed Central

    Murray, Teresa A; Liu, Qiang; Whiteaker, Paul; Wu, Jie; Lukas, Ronald J

    2009-01-01

    Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors. Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion. PMID:19498423

  17. Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

    SciTech Connect

    Palma, A.L.; Wang, H.H. )

    1991-06-01

    Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclidine, 4-phenyl-4-(1-piperidinyl)-2,2,6,6-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits 86Rb flux (IC50 = 6.6 microM), and (3H)phencyclidine binding to both resting and desensitized acetylcholine receptor (IC50 = 17 microM and 0.22 microM, respectively). From an indirect Hill plot of the inhibition of (3H)phencyclidine binding by PPT, a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in alpha-bungarotoxin-treated preparations. Taken together, these results indicate that PPT mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2-3 units.

  18. Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

    PubMed Central

    Engle, Staci E.; Broderick, Hilary J.; Drenan, Ryan M.

    2012-01-01

    Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority. Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9′A mice 1 and α6 L9′S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices. In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of

  19. Solubilisation and molecular characterisation of muscarinic acetylcholine receptors.

    PubMed

    Hulme, E C; Berrie, C P; Haga, T; Birdsall, N J; Burgen, A S; Stockton, J

    1983-01-01

    Stable, soluble preparations of rat brain muscarinic receptors can be prepared by extracting membranes with digitonin, or with combinations of sodium cholate and sodium chloride. The stability of the cholate/NaCl extract is enhanced by the addition of egg phosphatidylcholine, which, at the same time, suppresses the considerable dispersity apparent in the hydrodynamic behaviour of the solubilised receptor. The Stokes radius of the brain muscarinic receptor in cholate/NaCl/lecithin extracts is 6.7 nm, with very similar values in other detergents, including digitonin and sodium dodecyl sulphate. Its sedimentation coefficient is 3.78s, and its molecular weight approximately 110,000 after correction for detergent binding. The isoelectric point of the digitonin - solubilised receptor is approximately 4.5. PMID:6854547

  20. Sorbs1 and -2 Interact with CrkL and Are Required for Acetylcholine Receptor Cluster Formation

    PubMed Central

    Hallock, Peter T.; Chin, Sherry; Blais, Steven; Neubert, Thomas A.

    2015-01-01

    Crk and CrkL are noncatalytic adaptor proteins necessary for the formation of neuromuscular synapses which function downstream of muscle-specific kinase (MuSK), a receptor tyrosine kinase expressed in skeletal muscle, and the MuSK binding protein Dok-7. How Crk/CrkL regulate neuromuscular endplate formation is not known. To better understand the roles of Crk/CrkL, we identified CrkL binding proteins using mass spectrometry and have identified Sorbs1 and Sorbs2 as two functionally redundant proteins that associate with the initiating MuSK/Dok-7/Crk/CrkL complex, regulate acetylcholine receptor (AChR) clustering in vitro, and are localized at synapses in vivo. PMID:26527617

  1. Molecular mechanism of acetylcholine receptor-controlled ion translocation across cell membranes

    PubMed Central

    Cash, Derek J.; Hess, George P.

    1980-01-01

    Two molecular processes, the binding of acetylcholine to the membrane-bound acetylcholine receptor protein and the receptor-controlled flux rates of specific inorganic ions, are essential in determining the electrical membrane potential of nerve and muscle cells. The measurements reported establish the relationship between the two processes: the acetylcholine receptor-controlled transmembrane ion flux of 86Rb+ and the concentration of carbamoylcholine, a stable analog of acetylcholine. A 200-fold concentration range of carbamoylcholine was used. The flux was measured in the millisecond-to-minute time region by using a quench flow technique with membrane vesicles prepared from the electric organ of Electrophorus electricus in eel Ringer's solution at pH 7.0 and 1°C. The technique makes possible the study of the transmembrane transport of specific ions, with variable known internal and external ion concentrations, in a system in which a determinable number of receptors is exposed to a known concentration of ligand. The response curve of ion flux to ligand was sigmoidal with an average maximum rate of 84 sec-1. Carbamoylcholine induced inactivation of the receptor with a maximum rate of 2.7 sec-1 and a different ligand dependence so that it was fast relative to ion flux at low ligand concentration but slow relative to ion flux at high ligand concentration. The simplest model that fits the data consists of receptor in the active and inactive states in ligand-controlled equilibria. Receptor inactivation occurs with one or two ligand molecules bound. For channel opening, two ligand molecules bound to the active state are required, and cooperativity results from the channel opening process itself. With carbamoylcholine, apparently, the equilibrium position for the channel opening step is only one-fourth open. The integrated rate equation, based on the model, predicts the time dependence of receptor-controlled ion flux over the concentration range of carbamoylcholine

  2. Serotonin 5-HT4 receptors and forebrain cholinergic system: receptor expression in identified cell populations.

    PubMed

    Peñas-Cazorla, Raúl; Vilaró, M Teresa

    2015-11-01

    Activation of serotonin 5-HT4 receptors has pro-cognitive effects on memory performance. The proposed underlying neurochemical mechanism is the enhancement of acetylcholine release in frontal cortex and hippocampus elicited by 5-HT4 agonists. Although 5-HT4 receptors are present in brain areas related to cognition, e.g., hippocampus and cortex, the cellular localization of the receptors that might modulate acetylcholine release is unknown at present. We have analyzed, using dual label in situ hybridization, the cellular localization of 5-HT4 receptor mRNA in identified neuronal populations of the rat basal forebrain, which is the source of the cholinergic innervation to cortex and hippocampus. 5-HT4 receptor mRNA was visualized with isotopically labeled oligonucleotide probes, whereas cholinergic, glutamatergic, GABAergic and parvalbumin-synthesizing neurons were identified with digoxigenin-labeled oligonucleotide probes. 5-HT4 receptor mRNA was not detected in the basal forebrain cholinergic cell population. In contrast, basal forebrain GABAergic, parvalbumin synthesizing, and glutamatergic cells contained 5-HT4 receptor mRNA. Hippocampal and cortical glutamatergic neurons also express this receptor. These results indicate that 5-HT4 receptors are not synthesized by cholinergic cells, and thus would be absent from cholinergic terminals. In contrast, several non-cholinergic cell populations within the basal forebrain and its target hippocampal and cortical areas express these receptors and are thus likely to mediate the enhancement of acetylcholine release elicited by 5-HT4 agonists. PMID:25183542

  3. Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study.

    PubMed

    Brown, L A; Jones, A K; Buckingham, S D; Mee, C J; Sattelle, D B

    2006-05-31

    Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation. PMID:16620825

  4. Nicotine increases GABAergic input on rat dorsal raphe serotonergic neurons through alpha7 nicotinic acetylcholine receptor.

    PubMed

    Hernández-Vázquez, F; Chavarría, K; Garduño, J; Hernández-López, S; Mihailescu, S P

    2014-12-15

    The dorsal raphe nucleus (DRN) contains large populations of serotonergic (5-HT) neurons. This nucleus receives GABAergic inhibitory afferents from many brain areas and from DRN interneurons. Both GABAergic and 5-HT DRN neurons express functional nicotinic acetylcholine receptors (nAChRs). Previous studies have demonstrated that nicotine increases 5-HT release and 5-HT DRN neuron discharge rate by stimulating postsynaptic nAChRs and by increasing glutamate and norepinephrine release inside DRN. However, the influence of nicotine on the GABAergic input to 5-HT DRN neurons was poorly investigated. Therefore, the aim of this work was to determine the effect of nicotine on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of 5-HT DRN neurons and the subtype of nAChR(s) involved in this response. Experiments were performed in coronal slices obtained from young Wistar rats. GABAergic sIPSCs were recorded from post hoc-identified 5-HT DRN neurons with the whole cell voltage patch-clamp technique. Administration of nicotine (1 μM) increased sIPSC frequency in 72% of identified 5-HT DRN neurons. This effect was not reproduced by the α4β2 nAChR agonist RJR-2403 and was not influenced by TTX (1 μM). It was mimicked by the selective agonist for α7 nAChR, PNU-282987, and exacerbated by the positive allosteric modulator of the same receptor, PNU-120596. The nicotine-induced increase in sIPSC frequency was independent on voltage-gated calcium channels and dependent on Ca(2+)-induced Ca(2+) release (CICR). These results demonstrate that nicotine increases the GABAergic input to most 5-HT DRN neurons, by activating α7 nAChRs and producing CICR in DRN GABAergic terminals. PMID:25231613

  5. Calcium signalling mediated by the 9 acetylcholine receptor in a cochlear cell line from the immortomouse.

    PubMed

    Jagger, D J; Griesinger, C B; Rivolta, M N; Holley, M C; Ashmore, J F

    2000-08-15

    1. We have investigated the characteristics of the alpha9 acetylcholine receptor (alpha9AChR) expressed in hair cell precursors in an immortalized cell line UB/OC-2 developed from the organ of Corti of the transgenic H-2Kb-tsA58 mouse (the Immortomouse) using both calcium imaging and whole-cell recording. 2. Ratiometric measurements of fura-2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 microM ACh. The calcium increase was seen in 66 % of the cells grown at 39 degrees C in differentiated conditions. A sm aller fraction (34%) of cells grown at 33 degrees C in proliferative con ditions responded. 3. Caffeine (10mM) elevated cell calcium. In the ab sence of caffeine, the majority of imaged cells responded only once to A Ch presentations. Pretreatment with caffeine ingibited all calcium respo nses to ACh. 4. In whole-cell tight-seal recordings 10 microM ACh activa ted inward current was dependent on the extracellular calcium concentrat ion with an estimated PCa/PNa of 80 for the alpha9 receptor at physiological calcium levels. 5 . The data indicate that ACh activates a calcium-permeable channel alpha 9AChR in UB/OC-2 cells and that the channel has a significantly higher c alcium permeability than other AChRs. The results indicate that the alp ha9AChR may be able to elevate intracellular calcium levels in hair cell s both directly and via store release. PMID:11011664

  6. Nicotine improves the functional activity of late endothelial progenitor cells via nicotinic acetylcholine receptors.

    PubMed

    Yu, Min; Liu, Qian; Sun, Jing; Yi, Kaihong; Wu, Libiao; Tan, Xuerui

    2011-08-01

    The aim of this study is to investigate whether nicotinic acetylcholine receptors (nAChRs) are involved in the modulation of functional activity of late endothelial progenitor cells (EPCs) induced by nicotine. Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-acLDL) uptake and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1) binding. Expression of von Willbrand factor (vWF), kinase insert domain receptor (KDR), and α7 nAChR was detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 32 h with either vehicle or nicotine with or without pre-incubation of nAChR antagonism, mecamylamine, or α-bungarotoxin. The viability, migration, and in vitro vasculogenesis activity of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with 10 nmol/L nicotine enhanced viable, migratory, adhesive, and in vitro vasculogenesis capacity of late EPCs. The effect of nicotine on late EPCs can be attenuated by mecamylamine or α-bungarotoxin. In conclusion, nicotine improves the functional activity of late EPCs via nAChRs. PMID:21774635

  7. Allosteric interactions of quaternary strychnine and brucine derivatives with muscarinic acetylcholine receptors.

    PubMed

    Gharagozloo, P; Lazareno, S; Popham, A; Birdsall, N J

    1999-02-11

    The affinity and allosteric properties of 22 quaternary derivatives of strychnine and brucine at the m1-m4 subtypes of muscarinic receptors have been analyzed and compared. The subtype selectivity, in terms of affinity, was in general m2 > m4 > m1 > m3. The highest affinities were found for N-benzyl, N-2-naphthylmethyl, and N-4-biphenylylmethyl strychnine (13, 14, and 18, respectively). All the strychnine and brucine derivatives were positively cooperative with the antagonist, N-methylscopolamine, at m2 receptors and, in the case of the strychnine analogues, were positively cooperative with N-methylscopolamine at least at one other subtype. The strychnine analogues were negatively cooperative with the neurotransmitter, acetylcholine, at all subtypes whereas brucine and five of the six derivatives examined were positively cooperative with acetylcholine at one or more subtypes (m1-m5) and exhibited different patterns of subtype selectivity. The ability to generate subtype-selective allosteric enhancers of acetylcholine binding and function may be of use in the development of drugs for the treatment of Alzheimer's disease. PMID:9986715

  8. The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

    PubMed Central

    Walker, J H; Boustead, C M; Witzemann, V

    1984-01-01

    Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non-receptor proteins including the receptor-associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one- and two-dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane-bound acetylcholine receptor and the cytoskeleton. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6389118

  9. Characterization of a putative acetylcholine receptor in chick ciliary ganglion neurons

    SciTech Connect

    Stollberg, J.

    1985-01-01

    Monoclonal antibodies to the main immunogenic region on the alpha subunit of acetylcholine receptors in muscle and electric organ recognize membrane components in chick brain and ciliary ganglia that are candidates for the neuronal receptor. The component in chick brain has been purified by immunoaffinity chromatography. It specifically binds nicotine but not alpha-bungarotoxin, and can be affinity labeled with (/sup 3/H)bromoacetylcholine. The cross-reacting component in ciliary ganglion neurons is concentrated in synaptic membrane, and can be modulated by exposure of the cells to cholinergic ligands in culture. The cross-reacting component in ciliary ganglion neurons is an integral membrane component that binds concanavalin A, and it is distinct from the alpha-bungarotoxin binding component. The acetylcholine receptor function in these neurons can be locked by affinity alkylation with bromoacetylcholine, indicating similarity in this respect to receptors from muscle and electric organ. Antisera raised against the partially purified component from chick brain also block receptor function on ciliary ganglion neurons. The subcellular distribution of the ganglion component in culture is assessed, and it is shown that approximately 2/3 of the cross-reacting components are intracellular; the majority of these seem not to be destined for insertion into the plasma membrane.

  10. The fetal form of the acetylcholine receptor distinguishes rhabdomyosarcomas from other childhood tumors.

    PubMed Central

    Gattenloehner, S.; Vincent, A.; Leuschner, I.; Tzartos, S.; Müller-Hermelink, H. K.; Kirchner, T.; Marx, A.

    1998-01-01

    The fetal nicotinic acetylcholine receptor (AChR) of muscle is an oligomeric membrane protein with subunit composition alpha2betadeltagamma. After birth, the adult form, in which an epsilon-subunit replaces the gamma-subunit, predominates, and expression of the fetal form is limited to thymic myoid cells, extraocular muscles, and denervated striated muscle. We looked for expression of AChR in rhabdomyosarcomas and other childhood tumors by reverse transcription polymerase chain reaction and immunohistochemistry. mRNA for the AChR gamma-subunit was detected in all embryonal and alveolar rhabdomyosarcomas tested (n = 16) and in some tumors with a rhabdomyomatous component (n = 2) but not in other nonrhabdomyomatous tumors of childhood and adults (n = 45). The fetal form of the AChR was detected immunohistochemically in five of eight embryonal and four of eight alveolar rhabdomyosarcomas and in two Wilms' tumors with a rhabdomyomatous component but not in other tumors or in normal muscle. We conclude that reverse transcription polymerase chain reaction for AChR gamma-subunit could be useful for the diagnosis of rhabdomyosarcoma of childhood and for the detection of micrometastases and minimal residual disease. In addition, the fetal AChR protein is the first extracellular tumor marker that can distinguish rhabdomyosarcomas from nonrhabdomyomatous tumors and from normal muscle. Our findings, therefore, imply that the fetal AChR may be a target for in vivo imaging and, as AChR internalization and degradation is increased by antibody-induced cross-linking, may also provide a sensitive and specific target for immunotherapeutic strategies. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9466570

  11. α4β2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief.

    PubMed

    McGranahan, Tresa M; Patzlaff, Natalie E; Grady, Sharon R; Heinemann, Stephen F; Booker, T K

    2011-07-27

    Nicotine is the primary psychoactive substance in tobacco, and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the α4β2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal as well as nicotine-induced behaviors. Although α4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addiction, mental illness, and movement control in humans. We developed a unique model system to examine the role of α4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the α4 subunit from dopaminergic neurons in mice. The loss α4 mRNA and α4β2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of α4β2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. α4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. α4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze, and elimination of α4β2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of α4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression; however, nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine-related behaviors. PMID:21795541

  12. alpha4beta2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief

    PubMed Central

    McGranahan, Tresa M.; Patzlaff, Natalie E.; Grady, Sharon R.; Heinemann, Stephen F.; Booker, T.K.

    2012-01-01

    Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addition, mental illness and movement control in humans. We developed a unique model system to examine the role of alpha4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the alpha4 subunit from dopaminergic neurons in mice. The loss alpha4 mRNA and alpha4beta2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of alpha4beta2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. Alpha4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. Alpha4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze and elimination of alpha4-beta2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of alpha4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression, however nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine behaviors. PMID:21795541

  13. Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival

    PubMed Central

    St-Pierre, Stéphanie; Jiang, Wei; Roy, Patrick; Champigny, Camille; LeBlanc, Éric; Morley, Barbara J.; Hao, Junwei; Simard, Alain R.

    2016-01-01

    It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers. PMID:26925951

  14. Escobar Syndrome Is a Prenatal Myasthenia Caused by Disruption of the Acetylcholine Receptor Fetal γ Subunit

    PubMed Central

    Hoffmann, Katrin; Müller, Juliane S.; Stricker, Sigmar; Megarbane, Andre; Rajab, Anna; Lindner, Tom H.; Cohen, Monika; Chouery, Eliane; Adaimy, Lynn; Ghanem, Ismat; Delague, Valerie; Boltshauser, Eugen; Talim, Beril; Horvath, Rita; Robinson, Peter N.; Lochmüller, Hanns; Hübner, Christoph; Mundlos, Stefan

    2006-01-01

    Escobar syndrome is a form of arthrogryposis multiplex congenita and features joint contractures, pterygia, and respiratory distress. Similar findings occur in newborns exposed to nicotinergic acetylcholine receptor (AChR) antibodies from myasthenic mothers. We performed linkage studies in families with Escobar syndrome and identified eight mutations within the γ-subunit gene (CHRNG) of the AChR. Our functional studies show that γ-subunit mutations prevent the correct localization of the fetal AChR in human embryonic kidney–cell membranes and that the expression pattern in prenatal mice corresponds to the human clinical phenotype. AChRs have five subunits. Two α, one β, and one δ subunit are always present. By switching γ to ɛ subunits in late fetal development, fetal AChRs are gradually replaced by adult AChRs. Fetal and adult AChRs are essential for neuromuscular signal transduction. In addition, the fetal AChRs seem to be the guide for the primary encounter of axon and muscle. Because of this important function in organogenesis, human mutations in the γ subunit were thought to be lethal, as they are in γ-knockout mice. In contrast, many mutations in other subunits have been found to be viable but cause postnatally persisting or beginning myasthenic syndromes. We conclude that Escobar syndrome is an inherited fetal myasthenic disease that also affects neuromuscular organogenesis. Because γ expression is restricted to early development, patients have no myasthenic symptoms later in life. This is the major difference from mutations in the other AChR subunits and the striking parallel to the symptoms found in neonates with arthrogryposis when maternal AChR auto-antibodies crossed the placenta and caused the transient inactivation of the AChR pathway. PMID:16826520

  15. Study of the Peripheral Nerve Fibers Myelin Structure Changes during Activation of Schwann Cell Acetylcholine Receptors

    PubMed Central

    Verdiyan, Ekaterina E.; Allakhverdiev, Elvin S.; Maksimov, Georgy V.

    2016-01-01

    In the present paper we consider a new type of mechanism by which neurotransmitter acetylcholine (ACh) regulates the properties of peripheral nerve fibers myelin. Our data show the importance of the relationship between the changes in the number of Schwann cell (SC) acetylcholine receptors (AChRs) and the axon excitation (different intervals between action potentials (APs)). Using Raman spectroscopy, an effect of activation of SC AChRs on the myelin membrane fluidity was investigated. It was found, that ACh stimulates an increase in lipid ordering degree of the myelin lipids, thus providing evidence for specific role of the “axon-SC” interactions at the axon excitation. It was proposed, that during the axon excitation, the SC membrane K+- depolarization and the Ca2+—influx led to phospholipase activation or exocytosis of intracellular membrane vesicles and myelin structure reorganization. PMID:27455410

  16. Changes in acetylcholine content, release and muscarinic receptors in rat hippocampus under cold stress

    SciTech Connect

    Fatranska, M.; Budai, D.; Gulya, K; Kvetnansky, R.

    1989-01-01

    The aim was to study the mechanism of the previously established decrease in acetylcholine (ACh) concentration in the rat hippocampus under cold stress. Male rats were exposed for 14 days to cold (5/degree/C) or kept (controls) at room temperature (24/degree/C). Acetylcholine content, release and muscarinic receptor binding were investigated in the hippocampus. Cold exposure resulted in a decrease of ACh concentration in the dorsal hippocampus. Moreover, the potassium-evoked release of ACh from hippocampal slices was increased and an increase of maximal binding capacity of (/sup 3/H)(-) quinuclidinyl benzilate in the dorsal hippocampus of cold exposed animals was also observed. Thus the decrease of hippocampal ACh concentration under cold exposure is probably due to its increased release. On balance then, our results demonstrate that cold stress in the rat induces significant activation of the hippocampal cholinergic system.

  17. Muscarinic acetylcholine receptors in the nucleus accumbens core and shell contribute to cocaine priming-induced reinstatement of drug seeking

    PubMed Central

    Yee, Judy; Famous, Katie R.; Hopkins, Thomas J.; McMullen, Michael C.; Pierce, R. Christopher; Schmidt, Heath D.

    2011-01-01

    Muscarinic acetylcholine receptors in the nucleus accumbens play an important role in mediating the reinforcing effects of cocaine. However, there is a paucity of data regarding the role of accumbal muscarinic acetylcholine receptors in the reinstatement of cocaine-seeking behavior. The goal of these experiments was to assess the role of muscarinic acetylcholine receptors in the nucleus accumbens core and shell in cocaine and sucrose priming-induced reinstatement. Rats were initially trained to self-administer cocaine or sucrose on a fixed-ratio schedule of reinforcement. Lever-pressing behavior was then extinguished and followed by a subsequent reinstatement phase during which operant responding was induced by either a systemic injection of cocaine in cocaine-experienced rats or non-contingent delivery of sucrose pellets in subjects with a history of sucrose self-administration. Results indicated that systemic administration of the muscarinic acetylcholine receptor antagonist scopolamine (5.0 mg/kg, i.p.) dose-dependently attenuated cocaine, but not sucrose, reinstatement. Furthermore, administration of scopolamine (36.0 μg) directly into the nucleus accumbens shell or core attenuated cocaine-priming induced reinstatement. In contrast, infusion of scopolamine (36.0 μg) directly into the accumbens core, but not shell, attenuated sucrose reinstatement, which suggests that muscarinic acetylcholine receptors in these two subregions of the nucleus accumbens have differential roles in sucrose seeking. Taken together, these results indicate that cocaine-priming induced reinstatement is mediated, in part, by increased signaling through muscarinic acetylcholine receptors in the shell subregion of the nucleus accumbens. Muscarinic acetylcholine receptors in the core of the accumbens, in contrast, appear to play a more general (i.e. not cocaine specific) role in motivated behaviors. PMID:21034738

  18. Positive allosteric action of eburnamonine on cardiac muscarinic acetylcholine receptors.

    PubMed

    Proska, J; Tucek, S

    1996-06-01

    It was discovered recently that alcuronium and strychnine (which is a precursor of alcuronium) allosterically increase the affinity of cardiac muscarinic receptors for the antagonist, N-methylscopolamine. We have now investigated the effects of l-eburnamonine and vincamine, which are both closely related to strychnine. In experiments on rat heart atria, l-eburnamonine was found to increase the binding of [3H]N-methylscopolamine with Ehlert's cooperativity coefficient alpha = 0.35, which indicates that the strength of its allosteric action is close to that of alcuronium and strychnine (alpha = 0.31 and 0.44, respectively). However, the affinity of l-eburnamonine for the cardiac muscarinic receptors is lower than the affinities of alcuronium and strychnine (KAR = 22.6 microM, 0.15 microM, and 3.4 microM, respectively). In spite of its extremely close similarity to l-eburnamonine, vincamine has a negative allosteric effect on the binding of [3H]N-methylscopolamine (alpha = 4.1; KAR = 22.8 microM). It is likely that a systematic investigation of the allosteric effects of the analogues of strychnine will not only yield new allosteric effectors on muscarinic receptors, but also clarify the structural features responsible for the direction (positive or negative) of their allosteric effect. PMID:8813554

  19. Fractional vesamicol receptor occupancy and acetylcholine active transport inhibition in synaptic vesicles.

    PubMed

    Kaufman, R; Rogers, G A; Fehlmann, C; Parsons, S M

    1989-09-01

    Vesamicol [(-)-(trans)-2-(4-phenylpiperidino)cyclohexanol] receptor binding and inhibition of acetylcholine (AcCh) active transport by cholinergic synaptic vesicles that were isolated from Torpedo electric organ were studied for 23 vesamicol enantiomers, analogues, and other drugs. Use of trace [3H]vesamicol and [14C]AcCh allowed simultaneous determination of the concentrations of enantiomer, analogue, or drug required to half-saturate the vesamicol receptor (Ki) and to half-inhibit transport (IC50), respectively. Throughout a wide range of potencies for different compounds, the Ki/IC50 ratios varied from 1.5 to 24. Compounds representative of the diverse structures studied, namely deoxyvesamicol, chloroquine, and levorphanol, were competitive inhibitors of vesamicol binding. It is concluded that many drugs can bind to the vesamicol receptor and binding to only a small fraction of the receptors can result in AcCh active transport inhibition. Possible mechanisms for this effect are discussed. PMID:2550778

  20. The immunomodulation of nicotinic acetylcholine receptor subunits in Zhikong scallop Chlamys farreri.

    PubMed

    Shi, Xiaowei; Zhou, Zhi; Wang, Lingling; Wang, Mengqiang; Shi, Shaoying; Wang, Zhen; Song, Linsheng

    2015-11-01

    Nicotinic acetylcholine receptor (nAChR), the best-studied ionotropic neuron receptor protein, is a key player in neuronal communication, and it has been reported to play an important role in immunomodulation of vertebrates. Although nAChRs have also been identified in most invertebrates, the knowledge about their immunomodulation is still limited. In the present study, two scallop nAChR genes were identified from Chlamys farreri (designed as CfnAChR1 and CfnAChR2), which encoded 384 and 443 amino acids, respectively. The conserved disulfide-linked cystines, ion selectivity residues and the hydrophobic gating residues (L251, V255 and V259) were identified in CfnAChR1 and CfnAChR2. The immunoreactivities of CfnAChR1 and CfnAChR2 were observed in all the tested scallop tissues, including adductor muscle, mantle, gill, hepatopancreas, kidney and gonad. After LPS (0.5 mg mL(-1)) stimulation, the expression of CfnAChR1 mRNA in haemocytes increased significantly by 9.83-fold (P < 0.05) and 12.93-fold (P < 0.05) at 3 h and 24 h, respectively. While the expression level of CfnAChR2 mRNA increased 43.94% at 12 h after LPS stimulation (P < 0.05). After TNF-α (50 ng mL(-1)) stimulation, the expression levels of CfnAChR1 and CfnAChR2 both increased significantly at 1 h, which were 21.33-fold (P < 0.05) and 2.44-fold (P < 0.05) of that in the PBS group, respectively. The results collectively indicated that the cholinergic nervous system in scallops could be activated by immune stimulations through CfnAChR1 and CfnAChR2, which function as the links between the cholinergic nervous system and immune system. PMID:26455648

  1. Evidence for the extramembranous location of the putative amphipathic helix of acetylcholine receptor

    SciTech Connect

    Dwyer, B.P.

    1988-07-26

    Evidence has been obtained demonstrating that the peptides GVKYIAE and AIKYIAE found in the potential amphipathic helices of the ..cap alpha.. and ..beta.. subunits, respectively, of acetylcholine receptor are not buried in the membrane. The peptide KYIAE was synthesized, and polyclonal antibodies were prepared against a conjugate of bovine serum albumin and synthetic peptide. An immunoadsorbent capable of binding and subsequently releasing peptides ending with the sequence-YIAE was produced by attaching these specific antibodies to agarose. Native acetylcholine receptor was labeled with pyridoxal phosphate and Na(/sup 3/H)BH/sub 4/. The labeled protein was stripped of phospholipid and digested with the protease from Staphylococcus aureus strain V8. The digest was submitted to immunoadsorption to isolate the labeled indigenous peptides. As a control, ..cap alpha.. and ..beta.. polypeptides prepared by gel filtration of a solution of acetylcholine receptor in detergent were stripped of detergent and labeled with pyridoxal phosphate and Na(/sup 3/H)BH/sub 4/ in the presence of 8 M urea. The labeled ..cap alpha.. and ..beta.. polypeptides were digested and submitted to immunoadsorption. The specific radioactivities of the indigenous peptides from the ..cap alpha.. and ..beta.. subunits labeled under native and denaturing conditions were nearly equal. In similar experiments using isethionyl (2',4'-dinitrophenyl)-3-aminopropionimidate as the labeling agent, the indigenous peptides from native and denatured receptor were also labeled to the same extent. Since these peptides are labeled to the same extent whether or not the protein is denatured, they cannot be buried in the membrane.

  2. Structure-function studies of the muscle nicotinic acetylcholine receptor by site-directed mutagenesis in the pore region

    NASA Astrophysics Data System (ADS)

    Zhang, Haiyun

    In nicotinic acetylcholine receptors (nAChRs), as in glycine, GABA A, serotonin 5-HT3, and GluCl glutamate receptors, a leucine residue at the approximate midpoint (the 9' position) of the M2 transmembrane domain is conserved across all known subunits. We expressed the embryonic mouse muscle nAChRs with varying numbers (m* s) of subunits (2 αs, 1 β, 1 γ, and 1 δ) mutated at this position in Xenopus oocytes and discovered that mutations to serine (Leu9'Ser) result in a tenfold higher receptor sensitivity to acetylcholine (ACh) for each subunit mutated. Moreover, increases of side-chain polarity increase the sensitivity to ACh when other natural and unnatural residues are incorporated into this position. The data also indicated an especially strong interaction between the γ and δ subunits in the pore region, suggesting a specific arrangement of subunits within the pentamer. Detailed single-channel kinetic studies reveal that Leu9'Ser AChRs have (1) longer voltage- relaxation time constants, (2) longer ACh-induced openings and bursts, and (3) more frequent spontaneous openings. These effects increase with m* s. Synthesized postsynaptic currents were produced with a piezoelectric micromanipulator that delivered brief ACh pulses to multi-channel patches. Their decay time constants were, as expected, similar to the channel burst duration. Thus, both longer and more frequent openings contribute to the >=104-fold increase in the receptor sensitivity to ACh from the wild-type receptor to the receptor with m*s=4; and the highly conserved 9' leucine is crucial for the brief synaptic events that are normally observed. We also explored the effects of ligand-binding domain mutations: γD174N and δD180N (aspartic acid (D) to asparagine (N)). Macroscopic dose-response relations revealed that these mutations decrease the receptor's sensitivity to ACh. The combined effect with Leu9'Ser, however, differs from that predicted from a linear or independent sum of effects from

  3. Hydroxycinnamic acid amides from Scopolia tangutica inhibit the activity of M1 muscarinic acetylcholine receptor in vitro.

    PubMed

    Zhang, Yan; Long, Zhen; Guo, Zhimou; Wang, Zhiwei; Zhang, Xiuli; Ye, Richard D; Liang, Xinmiao; Civelli, Olivier

    2016-01-01

    Scopolia tangutica Maxim (S. tangutica) extracts have been traditionally used as antispasmodic, sedative, and analgesic agents in Tibet and in the Qinghai province of China. Their active compositions are however poorly understood. We have recently isolated five new hydroxycinnamic acid (HCA) amides along with two known HCA amides, one cinnamic acid amide from these extracts. In this study, we evaluate their abilities to inhibit carbacol-induced activity of M1 muscarinic acetylcholine receptor along with the crude extracts. Chinese hamster ovary cells stably expressing the recombinant human M1 receptor (CHO-M1 cells) were employed to evaluate the anticholinergic potentials. Intracellular Ca(2+) changes were monitored using the FLIPR system. Five HCA amides as well as the crude S. tangutica extract displayed dose-dependent inhibitory effects against M1 receptor. These findings demonstrate that HCA amides are part of the M1 receptor-inhibiting principles of S. tangutica. Since blockade of parasympathetic nerve impulse transmission through the inhibition of the M1 receptor lessens smooth muscle spasms, our findings provided a molecular explanation for the traditional use of S. tangutica against spasm. PMID:26586621

  4. Amino acids of the Torpedo marmorata acetylcholine receptor. cap alpha. subunit labeled by a photoaffinity ligand for the acetylcholine binding site

    SciTech Connect

    Dennis, M.; Giraudat, J.; Kotzyba-Hibert, F.; Goeldner, M.; Hirth, C.; Chang, J.Y.; Lazure, C.; Chretien, M.; Changeux, J.P.

    1988-04-05

    The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent (/sup 3/H)-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The ..cap alpha..-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the ..cap alpha..-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment ..cap alpha.. 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the ..cap alpha..-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the ..cap alpha..-chain that assign the amino-terminal segment ..cap alpha.. 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified (/sup 3/H)DDF-labeled resides, which are conserved in muscle and neuronal ..cap alpha..-chains but not in the other subunits, may be directly involved in agonist binding.

  5. Absence of nicotinic acetylcholine receptor α7 subunit amplifies inflammation and accelerates onset of fibrosis: an inflammatory kidney model

    PubMed Central

    Truong, Luan D.; Trostel, Jessica; Garcia, Gabriela E.

    2015-01-01

    Inflammation is regulated by endogenous mechanisms, including anti-inflammatory cytokines, adenosine, and the nicotinic acetylcholine receptor α7 subunit (α7nAChR). We investigated the role of α7nAChR in protection against the progression of tissue injury in a model of severe, macrophage-mediated, cytokine-dependent anti-glomerular basement membrane (GBM) glomerulonephritis (GN), in α7nAChR-deficient (α7−/−) mice . At d 7 after the injection of anti-GBM antibody, kidneys from α7−/− mice displayed severe glomeruli (P < 0.0001) and tubulointerstitial lesions (P < 0.001) compared to kidneys from WT mice. An important finding was the presence of severe glomerulosclerosis in α7−/− mice in this early phase of the disease. Kidneys of α7−/− mice showed greater accumulation of inflammatory cells and higher expression of chemokines and cytokines than did those of WT mice. In addition, in α7−/− fibrotic kidneys, the expression of fibrin, collagen, TGF-β, and tissue inhibitor of metalloproteinase (TIMP)-2 increased, and the expression of TIMP3 declined. The increase in counterregulatory responses to inflammation in α7−/− nephritic kidneys did not compensate for the lack of α7nAChR. These findings indicate that α7nAChR plays a key role in regulating the inflammatory response in anti-GBM GN and that disruption of the endogenous protective α7nAChR amplifies inflammation to accelerate kidney damage and fibrosis.—Truong, L. D., Trostel. J., Garcia, G. E. Absence of nicotinic acetylcholine receptor α7 subunit amplifies inflammation and accelerates onset of fibrosis: an inflammatory kidney model. PMID:25985801

  6. Glycosylation sites selectively interfere with alpha-toxin binding to the nicotinic acetylcholine receptor.

    PubMed

    Kreienkamp, H J; Sine, S M; Maeda, R K; Taylor, P

    1994-03-18

    Sequence analysis reveals unique features in the alpha-subunit of nicotinic acetylcholine receptors from the alpha-toxin-resistant cobra and mongoose. Included are N-linked glycosylation signals just amino-terminal to the Tyr190, Cys192-Cys193 region of the ligand binding domain, substitution of Trp187 and Phe189 by non-aromatic residues and alteration of the proline sequence Pro194-X-X-Pro197. Glycosylation signals were inserted into the toxin-sensitive mouse alpha-subunit by the mutations F189N and W187N/F189T. The F189N alpha-subunit, when transfected with beta, gamma and delta, showed a 140-fold loss of alpha-bungarotoxin affinity, whereas the W187N/F189T double mutation exhibited a divergence in alpha-toxin affinities at the two sites, one class showing a 600-fold and the other showing an 11-fold reduction. The W187N mutant and the double mutant F189N/S191A lacking the requisite glycosylation signals exhibited little alteration in affinity, as did the P194L and P197H mutations. The glycosylation sites had little or no influence on binding of toxins of intermediate (alpha-conotoxin, 1500 Da) or small mass (lophotoxin, 500 Da) and of the agonist, carbamylcholine. The two sites for the binding of alpha-conotoxin M1 have widely divergent dissociation constants of 2.1 and 14,800 nM. Expression of alpha/gamma- and alpha/delta-subunit pairs indicated that the high and low affinity sites are formed by the alpha/delta and alpha/gamma contacts, respectively. PMID:7907588

  7. Gastrointestinal hypomotility with loss of enteric nicotinic acetylcholine receptors: active immunization model in mice

    PubMed Central

    Meeusen, Jeffrey W.; Haselkorn, Keegan E.; Fryer, James P.; Kryzer, Thomas J.; Gibbons, Simon J.; Xiao, Yingxian; Lennon, Vanda A.

    2012-01-01

    Background Autoimmune gastrointestinal dysmotility (AGID) is a limited form of dysautonomia. The only proven effector to date is IgG specific for ganglionic nicotinic-acetylcholine receptors containing α3 subunits (α3*-nAChR). Rabbits immunized with recombinant α3-polypeptide produce α3*-nAChR autoantibodies, and profound AGID ensues. Human and rabbit α3*-nAChR-specific-IgGs induce transient hypomotility when injected into mice. Here we describe success and problems encountered inducing gastrointestinal hypomotility in mice by active immunization. Methods We repeatedly injected young adult mice of seven different strains susceptible to autoimmunity (spontaneous diabetes or neural antigen immunization-induced myasthenia gravis or encephalomyelitis) with: i) α3-polypeptide, intradermally, or ii) live α3*-nAChR-expressing xenogeneic cells, intraperitoneally. We measured serum α3*-nAChR-IgG twice monthly, and terminally assessed blue dye gastrointestinal transit, total small intestinal α3*-nAChR content (radiochemically) and myenteric plexus neuron numbers (immunohistochemically, ileal-jejunal whole-mount preparations). Key Results Standard cutaneous inoculation with α3-polypeptide was minimally immunogenic, regardless of dose. Intraperitoneally-injected live cells were potently immunogenic. Self-reactive α3*-nAChR-IgG was induced only by rodent immunogen; small intestinal transit slowing and enteric α3*-nAChR loss required high serum levels. Ganglionic neurons were not lost. Conclusions & Inferences AGID is inducible in mice by active immunization. Accompanying enteric α3*-nAChR reduction without neuronal death is consistent with an IgG-mediated rather than T cell-mediated pathogenesis, as is improvement of symptoms in patients receiving antibody-depleting therapies. PMID:23072523

  8. The binding site of the nicotinic acetylcholine receptor in animal species resistant to alpha-bungarotoxin.

    PubMed

    Barchan, D; Ovadia, M; Kochva, E; Fuchs, S

    1995-07-18

    The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the alpha-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193. We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to alpha-bungarotoxin (alpha-BTX). Our earlier work on the snake and mongoose AChR, both of which do not bind alpha-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR alpha-subunit are important in determining the resistance of these AChRs to alpha-BTX. In the present study, we have examined the correlation between alpha-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human. Fragments of the AChR alpha-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli. The hedgehog fragment does not bind alpha-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder. The shrew and cat fragments bind alpha-BTX to a similar extent as the mouse fragment. The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the alpha-subunit, as is seen with the "toxin resistant" snake and mongoose, and in contrast with the "toxin binders", which have aromatic residues at these two positions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7619817

  9. Rapsyn Mutations in Humans Cause Endplate Acetylcholine-Receptor Deficiency and Myasthenic Syndrome

    PubMed Central

    Ohno, Kinji; Engel, Andrew G.; Shen, Xin-Ming; Selcen, Duygu; Brengman, Joan; Harper, C. Michel; Tsujino, Akira; Milone, Margherita

    2002-01-01

    Congenital myasthenic syndromes (CMSs) stem from genetic defects in endplate (EP)-specific presynaptic, synaptic, and postsynaptic proteins. The postsynaptic CMSs identified to date stem from a deficiency or kinetic abnormality of the acetylcholine receptor (AChR). All CMSs with a kinetic abnormality of AChR, as well as many CMSs with a deficiency of AChR, have been traced to mutations in AChR-subunit genes. However, in a subset of patients with EP AChR deficiency, the genetic defect has remained elusive. Rapsyn, a 43-kDa postsynaptic protein, plays an essential role in the clustering of AChR at the EP. Seven tetratricopeptide repeats (TPRs) of rapsyn subserve self-association, a coiled-coil domain binds to AChR, and a RING-H2 domain associates with β-dystroglycan and links rapsyn to the subsynaptic cytoskeleton. Rapsyn self-association precedes recruitment of AChR to rapsyn clusters. In four patients with EP AChR deficiency but with no mutations in AChR subunits, we identify three recessive rapsyn mutations: one patient carries L14P in TPR1 and N88K in TPR3; two are homozygous for N88K; and one carries N88K and 553ins5, which frameshifts in TPR5. EP studies in each case show decreased staining for rapsyn and AChR, as well as impaired postsynaptic morphological development. Expression studies in HEK cells indicate that none of the mutations hinders rapsyn self-association but that all three diminish coclustering of AChR with rapsyn. PMID:11791205

  10. Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: Pharmacological and biochemical properties

    SciTech Connect

    Buck, M.A.; Fraser, C.M. )

    1990-12-14

    The pharmacological and biochemical properties of rat m1 and m3 muscarinic acetylcholine receptors (mAChR) stably transfected into Chinese hamster ovary-K1 (CHO) cells were characterized with ligand binding, affinity labeling and biochemical assays. Both mAChR subtypes display saturable, high affinity binding of (3H)-quinuclidinyl benzilate (QNB) and a rank order of antagonist potency of QNB greater than atropine greater than pirenzepine greater than AF-DX 116. Carbachol displacement of (3H)-QNB binding to the m3 mAChR revealed an approximate 17-fold higher affinity than observed with the m1 mAChR. (3H)-propylbenzilylcholine mustard (PrBCM) labeling of mAChR revealed that m1 and m3 mAChR migrated on SDS-polyacrylamide gels with apparent molecular masses of 80,000 and 94,000 daltons, respectively, consistent with the known differences in their molecular sizes. Both m1 and m3 mAChR elicited dose-dependent increases in the hydrolysis of phosphoinositides; however, the maximal increase in total inositol phosphates elicited with the m1 mAChR was approximately 2-fold greater than that observed in cells expressing similar densities of m3 mAChR. Agonist activation of the m1 mAChR also elicited increases in basal and forskolin-stimulated cAMP, whereas the m3 mAChR had no effect on intracellular cAMP levels. These data suggest that although m1 and m3 mAChR display a considerable degree of structural homology, they exhibit distinct pharmacological and biochemical properties.

  11. The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity.

    PubMed

    Kharrat, Riadh; Servent, Denis; Girard, Emmanuelle; Ouanounou, Gilles; Amar, Muriel; Marrouchi, Riadh; Benoit, Evelyne; Molgó, Jordi

    2008-11-01

    Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples. PMID:18990115

  12. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    PubMed

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists. PMID:27055770

  13. Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids.

    PubMed

    Green, Benedict T; Lee, Stephen T; Welch, Kevin D; Cook, Daniel; Kem, William R

    2016-01-01

    Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic. PMID:27384586

  14. Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids

    PubMed Central

    Green, Benedict T.; Lee, Stephen T.; Welch, Kevin D.; Cook, Daniel; Kem, William R.

    2016-01-01

    Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic. PMID:27384586

  15. Expression of m1-type muscarinic acetylcholine receptors by parvalbumin-immunoreactive neurons in the primary visual cortex: a comparative study of rat, guinea pig, ferret, macaque, and human.

    PubMed

    Disney, Anita A; Reynolds, John H

    2014-04-01

    Cholinergic neuromodulation is a candidate mechanism for aspects of arousal and attention in mammals. We have reported previously that cholinergic modulation in the primary visual cortex (V1) of the macaque monkey is strongly targeted toward GABAergic interneurons, and in particular that the vast majority of parvalbumin-immunoreactive (PV) neurons in macaque V1 express the m1-type (pirenzepine-sensitive, Gq-coupled) muscarinic ACh receptor (m1AChR). In contrast, previous physiological data indicates that PV neurons in rats rarely express pirenzepine-sensitive muscarinic AChRs. To examine further this apparent species difference in the cholinergic effectors for the primary visual cortex, we have conducted a comparative study of the expression of m1AChRs by PV neurons in V1 of rats, guinea pigs, ferrets, macaques, and humans. We visualize PV- and mAChR-immunoreactive somata by dual-immunofluorescence confocal microscopy and find that the species differences are profound; the vast majority (>75%) of PV-ir neurons in macaques, humans, and guinea pigs express m1AChRs. In contrast, in rats only ∼25% of the PV population is immunoreactive for m1AChRs. Our data reveal that while they do so much less frequently than in primates, PV neurons in rats do express Gq-coupled muscarinic AChRs, which appear to have gone undetected in the previous in vitro studies. Data such as these are critical in determining the species that represent adequate models for the capacity of the cholinergic system to modulate inhibition in the primate cortex. PMID:23983014

  16. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-01-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. Images PMID:3458258

  17. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-05-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. PMID:3458258

  18. Neuronal specificity of the alpha 7 nicotinic acetylcholine receptor promoter develops during morphogenesis of the central nervous system.

    PubMed Central

    Matter-Sadzinski, L; Hernandez, M C; Roztocil, T; Ballivet, M; Matter, J M

    1992-01-01

    A transient transfection assay has been developed to analyse promoter activity in neuronal cells freshly dissociated from the chick central nervous system. The assay enabled us to identify cis-acting regulatory elements within the 5'-flanking region of the alpha 7 nicotinic acetylcholine receptor gene. In differentiated retina, regulatory elements direct reporter gene expression to a small subset of neurons which has been identified as ganglion cells, i.e. to the population of neurons in which alpha 7 transcripts were localized by in situ hybridization. However, these promoter elements exhibit ubiquitous activity in undifferentiated neural cells and in mesodermal stem cells. Our study supports the idea that alpha 7 regulatory elements acquire their neuronal specificity in the course of embryogenesis. Images PMID:1425587

  19. The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation.

    PubMed

    Cecchini, Marco; Changeux, Jean-Pierre

    2015-09-01

    Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger - a neurotransmitter - into an ion flux through the postsynaptic membrane. Here, we present an overview of the most recent advances on the signal transduction mechanism boosted by X-ray crystallography of both prokaryotic and eukaryotic homologues of the nicotinic acetylcholine receptor (nAChR) in conjunction with time-resolved analyses based on single-channel electrophysiology and Molecular Dynamics simulations. The available data consistently point to a global mechanism of gating that involves a large reorganization of the receptor mediated by two distinct quaternary transitions: a global twisting and a radial expansion/contraction of the extracellular domain. These transitions profoundly modify the organization of the interface between subunits, which host several sites for orthosteric and allosteric modulatory ligands. The same mechanism may thus mediate both positive and negative allosteric modulations of pLGICs ligand binding at topographically distinct sites. The emerging picture of signal transduction is expected to pave the way to new pharmacological strategies for the development of allosteric modulators of nAChR and pLGICs in general. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25529272

  20. Statistical methods for model discrimination. Applications to gating kinetics and permeation of the acetylcholine receptor channel.

    PubMed Central

    Horn, R

    1987-01-01

    Methods are described for discrimination of models of the gating kinetics and permeation of single ionic channels. Both maximum likelihood and regression procedures are discussed. In simple situations, where models are nested, standard hypothesis tests can be used. More commonly, however, non-nested models are of interest, and several procedures are described for model discrimination in these cases, including Monte Carlo methods, which allow the comparison of models at significance levels of choice. As an illustration, the methods are applied to single-channel data from acetylcholine receptor channels. PMID:2435330

  1. Vector-averaged gravity does not alter acetylcholine receptor single channel properties

    NASA Technical Reports Server (NTRS)

    Reitstetter, R.; Gruener, R.

    1994-01-01

    To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.

  2. Structure-activity relationships of alpha-conotoxins targeting neuronal nicotinic acetylcholine receptors.

    PubMed

    Millard, Emma L; Daly, Norelle L; Craik, David J

    2004-06-01

    alpha-Conotoxins that target the neuronal nicotinic acetylcholine receptor have a range of potential therapeutic applications and are valuable probes for examining receptor subtype selectivity. The three-dimensional structures of about half of the known neuronal specific alpha-conotoxins have now been determined and have a consensus fold containing a helical region braced by two conserved disulfide bonds. These disulfide bonds define the two-loop framework characteristic for alpha-conotoxins, CCX(m)CX(n)C, where loop 1 comprises four residues (m = 4) and loop 2 between three and seven residues (n = 3, 6 or 7). Structural studies, particularly using NMR spectroscopy have provided an insight into the role and spatial location of residues implicated in receptor binding and biological activity. PMID:15182347

  3. Effects of extracellular acetylcholine on muscarinic receptor binding assessed by [125I]dexetimide and a simple probe.

    PubMed

    Sánchez-Roa, P M; Wagner, H N; Villemagne, V L; London, E D; Lever, J R

    1998-10-01

    New pharmacologic approaches to enhance brain cholinergic function focus on increasing intrasynaptic acetylcholine. We examined the usefulness of a simple probe and [125I]dexetimide to evaluate in vivo the effects of extracellular acetylcholine on muscarinic receptor binding in the mouse brain. After radiotracer injection continuous time/activity curves were generated over 330 min. [125I]Dexetimide reached a plateau at 90 min post-injection. To increase extracellular acetylcholine, the anticholinesterase physostigmine was administered at 120 min, producing a reversible decrease in [125I]dexetimide specific binding (23%) for 30 min. These findings demonstrate that dynamic changes in extracellular acetylcholine can be evaluated by displacement of [125I]dexetimide binding in vivo using a simple probe system. PMID:9822886

  4. Thiochrome enhances acetylcholine affinity at muscarinic M4 receptors: receptor subtype selectivity via cooperativity rather than affinity.

    PubMed

    Lazareno, S; Dolezal, V; Popham, A; Birdsall, N J M

    2004-01-01

    Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of l-[3H]N-methyl scopolamine ([3H]NMS) to the five human muscarinic receptor subtypes (M1-M5) at concentrations up to 0.3 mM. In contrast, it inhibits [3H]NMS dissociation from M1 to M4 receptors at submillimolar concentrations and from M5 receptors at 1 mM. These results suggest that thiochrome binds allosterically to muscarinic receptors and has approximately neutral cooperativity with [3H]NMS at M1 to M4 and possibly M5 receptors. Thiochrome increases the affinity of acetylcholine (ACh) 3- to 5-fold for inhibiting [3H]NMS binding to M4 receptors but has no effect on ACh affinity at M1 to M3 or M5 receptors. Thiochrome (0.1 mM) also increases the direct binding of [3H]ACh to M4 receptors but decreases it slightly at M2 receptors. In agreement with the binding data, thiochrome does not affect the potency of ACh for stimulating the binding of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) to membranes containing M1 to M3 receptors, but it increases ACh potency 3.5-fold at M4 receptors. It also selectively reduces the release of [3H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M4 receptors, but not from hippocampal slices, which contain presynaptic M2 receptors. We conclude that thiochrome is a selective M4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M1 to M3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity. PMID:14722259

  5. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-12-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  6. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-01-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  7. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  8. Rapid synthesis of acetylcholine receptors at neuromuscular junctions. (Reannouncement with new availability information)

    SciTech Connect

    Ramsay, D.A.; Drachman, D.B.; Pestronk, A.

    1988-12-31

    The rate of acetylcholine receptor (AChR) degradation in mature, innervated mammalian neuromuscular junctions has recently been shown to be biphasic; up to 20% are rapidly turned over whereas the remainder are lost more slowly. In order to maintain normal junctional receptor density, synthesis and insertion of AChRs should presumably be sufficiently rapid to replace both the RTOs and the stable receptors. The authors have tested this prediction by blocking pre-existing AChRs in the mouse sternomastoid muscle with alpha bungarotoxin and monitoring the subsequent appearance of new junctional AChRs at intervals of 3 h to 20 days by labelling them. The results show that new receptors were initially inserted rapidly. The rate of increase of new binding sites gradually slowed down during the remainder of the time period studied. Control observations excluded possible artifacts of the experimental procedure including incomplete blockade of AChRs, dissociation of toxin receptor complexes, or experimentally induced alteration of receptor synthesis. The present demonstration of rapid synthesis and incorporation of AChRs at innervated neuromuscular junctions provides support for the concept of a subpopulation of rapidly turned over AChRs. The RTOs may serve as precursors for the large population of stable receptors and have an important role in the metabolism of the neuromuscular synapse.

  9. Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein

    PubMed Central

    Rondinelli, Sergio; Näreoja, Katja; Näsman, Johnny

    2011-01-01

    Muscarinic toxin 7 (MT7) is a mamba venom peptide that binds selectively to the M1 muscarinic acetylcholine receptor. We have previously shown that the second (ECL2) and third (ECL3) extracellular loops of the M1 receptor are critically involved in binding the peptide. In this study we used a mutagenesis approach on the M5 subtype of the receptor family to find out if this possesses a similar structural architecture in terms of toxin binding as the M1 receptor. An M5 receptor construct (M5-E175Y184E474), mutated at the formerly deciphered critical residues on ECL2 and 3, gained the ability to bind MT7, but with rather low affinity as determined in a functional assay (apparent Ki = 24 nM; apparent Ki for M1 = 0.5 nM). After screening for different domains and residues, we found a specific residue (P179 to L in M5) in the middle portion of ECL2 that was necessary for high affinity binding of MT7 (M5-EL179YE, apparent Ki = 0.5 nM). Mutation of P179 to A confirmed a role for the leucine side chain in the binding of MT7. Together the results reveal new binding interactions between receptors and the MT7 peptide and strengthen the hypothesis that ECL2 sequence is of utmost importance for MT binding to muscarinic receptors. PMID:22174976

  10. Characterization of the retina in the alpha7 nicotinic acetylcholine receptor knockout mouse

    NASA Astrophysics Data System (ADS)

    Smith, Marci L.

    Acetylcholine receptors (AChRs) are involved in visual processing and are expressed by inner retinal neurons in all species studied to date (Keyser et al., 2000; Dmitrieva et al., 2007; Liu et al., 2009), but their distribution in the mouse retina remains unknown. Reductions in alpha7 nicotinic AChRs (nAChRs) are thought to contribute to memory and visual deficits observed in Alzheimer's and schizophrenia (Coyle et al., 1983; Nordberg et al., 1999; Leonard et al., 2006). However, the alpha7 nAChR knockout (KO) mouse has a mild phenotype (Paylor et al., 1998; Fernandes et al., 2006; Young et al., 2007; Origlia et al., 2012). The purpose of this study was to determine the expression of AChRs in wildtype (WT) mouse retina and to assess whether up-regulation of other AChRs in the alpha7 nAChR KO retina may explain the minimal deficits described in the KO mouse. Reverse-transcriptase PCR (RT-PCR) showed that mRNA transcripts for alpha2-7, alpha 9, alpha10, beta2-4 nAChR subunits and m1-m5 muscarinic AChR (mAChR) subtypes were present in WT murine retina. Western blot analysis confirmed the presence of alpha3-5, alpha9, and m1-m5 AChR proteins and immunohistochemical analysis demonstrated nAChR and mAChR proteins expressed by subsets of bipolar, amacrine and ganglion cells. This is the first reported expression of alpha9 and alpha10 nAChR transcripts and alpha9 nAChR proteins in the retina of any species. Quantitative RT-PCR (qPCR) showed changes in AChR transcript expression in the alpha7 nAChR KO mouse retina relative to WT. Within whole retina alpha2, alpha9, alpha10, beta4, m1 and m4 AChR transcripts were up-regulated, while alpha5 nAChR transcripts were down-regulated. However, cell populations showed subtle differences; m4 mAChR transcripts were up-regulated in the ganglion cell layer and outer portion of the inner nuclear layer (oINL),while beta4 nAChR transcript up-regulation was limited to the oINL. Surprisingly, alpha2, alpha9, beta4, m2 and m4 transcripts were

  11. CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential responsiveness of human intestinal epithelial cells to LPS

    PubMed Central

    Dang, Xitong; Eliceiri, Brian P.; Baird, Andrew; Costantini, Todd W.

    2015-01-01

    The human genome contains a unique, distinct, and human-specific α7-nicotinic acetylcholine receptor (α7nAChR) gene [CHRNA7 (gene-encoding α7-nicotinic acetylcholine receptor)] called CHRFAM7A (gene-encoding dup-α7-nicotinic acetylcholine receptor) on a locus of chromosome 15 associated with mental illness, including schizophrenia. Located 5′ upstream from the “wild-type” CHRNA7 gene that is found in other vertebrates, we demonstrate CHRFAM7A expression in a broad range of epithelial cells and sequenced the CHRFAM7A transcript found in normal human fetal small intestine epithelial (FHs) cells to prove its identity. We then compared its expression to CHRNA7 in 11 gut epithelial cell lines, showed that there is a differential response to LPS when compared to CHRNA7, and characterized the CHRFAM7A promoter. We report that both CHRFAM7A and CHRNA7 gene expression are widely distributed in human epithelial cell lines but that the levels of CHRFAM7A gene expression vary up to 5000-fold between different gut epithelial cells. A 3-hour treatment of epithelial cells with 100 ng/ml LPS increased CHRFAM7A gene expression by almost 1000-fold but had little effect on CHRNA7 gene expression. Mapping the regulatory elements responsible for CHRFAM7A gene expression identifies a 1 kb sequence in the UTR of the CHRFAM7A gene that is modulated by LPS. Taken together, these data establish the presence, identity, and differential regulation of the human-specific CHRFAM7A gene in human gut epithelial cells. In light of the fact that CHRFAM7A expression is reported to modulate ligand binding to, and alter the activity of, the wild-type α7nAChR ligand-gated pentameric ion channel, the findings point to the existence of a species-specific α7nAChR response that might regulate gut epithelial function in a human-specific fashion.—Dang, X., Eliceiri, B. P., Baird, A., Costantini, T. W. CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential

  12. Heterogeneous Inhibition in Macroscopic Current Responses of Four Nicotinic Acetylcholine Receptor Subtypes by Cholesterol Enrichment.

    PubMed

    Báez-Pagán, Carlos A; Del Hoyo-Rivera, Natalie; Quesada, Orestes; Otero-Cruz, José David; Lasalde-Dominicci, José A

    2016-08-01

    The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane. PMID:27116687

  13. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  14. Minimum number of lipids are required to support the functional properties of the nicotinic acetylcholine receptor

    SciTech Connect

    Jones, O.T.; Eubanks, J.H.; Earnest, J.P.; McNamee, M.G.

    1988-05-17

    The detergent sodium cholate was used to both solubilize and partially delipidate the nicotinic acetylcholine receptor from Torpedo californica. Using both native membranes and reconstituted membranes, it is shown that the detergent to lipid molar ratio is the most important parameter in determining the effect of the detergent on the functional properties of the receptor. Receptor-lipid complexes were quantitatively separated from detergent and excess lipids by centrifugation through detergent-free sucrose gradients. The lipid to protein molar ratio of the complexes could be precisely controlled by adjusting the cholate and lipid concentrations of the starting membranes. Analyses of both ion influx activity and ligand binding revealed that a minimum of 45 lipids per receptor was required for stabilization of the receptor in a fully functional state. Progressive irreversible inactivation occurred as the lipid to protein mole ratio was decreased below 45, and complete inactivation occurred below a ratio of 20. The results are consistent with a functional requirement for a single shell of lipids around the perimeter of the receptor.

  15. Embryonic brain extract induces collagen biosynthesis in cultured muscle cells: involvement in acetylcholine receptor aggregation.

    PubMed Central

    Kalcheim, C; Vogel, Z; Duksin, D

    1982-01-01

    The involvement of extracellular matrix components in induction of the aggregation of acetylcholine (AcCho) receptors by factor(s) present in embryonic brain extract was investigated. Embryonic brain extract induced a three-fold increase in the number of AcCho receptor aggregates on the surface of cultured myotubes and a 5- to 10-fold increase in the synthesis of procollagen, which was secreted into the medium and converted to collagen. Adult brain extract, embryonic serum, and embryonic liver extract were less active in stimulating both collagen synthesis and AcCho receptor aggregation. A physiological connection between the two processes is suggested, since the number of AcCho receptor aggregates could be reduced to control levels by treating brain extract-stimulated myotubes with purified bacterial collagenase. In addition, stimulation of collagen secretion by ascorbic acid (50 micrograms/ml) promoted a 1.6-fold increase in AcCho receptor aggregation. When ascorbic acid was added together with the brain extract, further increases in both collagen synthesis and AcCho receptor aggregation were observed. Images PMID:6285338

  16. Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

    SciTech Connect

    Gu, Y.

    1989-01-01

    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.

  17. Steroids induce acetylcholine receptors on cultured human muscle: Implications for myasthenia gravis

    SciTech Connect

    Kaplan, I.; Blakely, B.T.; Pavlath, G.K.; Travis, M.; Blau, H.M. )

    1990-10-01

    Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. The authors show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. The results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also a direct effect on muscle. They propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.

  18. The role of α7 nicotinic acetylcholine receptor in modulation of heart rate dynamics in endotoxemic rats.

    PubMed

    Mazloom, Roham; Eftekhari, Golnar; Rahimi-Balaei, Maryam; Rahimi, Maryam; Khori, Vahid; Hajizadeh, Sohrab; Dehpour, Ahmad R; Mani, Ali R

    2013-01-01

    Previous reports have indicated that artificial stimulation of the vagus nerve reduces systemic inflammation in experimental models of sepsis. This phenomenon is a part of a broader cholinergic anti-inflammatory pathway which activates the vagus nerve to modulate inflammation through activation of alpha7 nicotinic acetylcholine receptors (α7nACHR). Heart rate variability represents the complex interplay between autonomic nervous system and cardiac pacemaker cells. Reduced heart rate variability and increased cardiac cycle regularity is a hallmark of clinical conditions that are associated with systemic inflammation (e.g. endotoxemia and sepsis). The present study was aimed to assess the role of α7nACHR in modulation of heart rate dynamics during systemic inflammation. Systemic inflammation was induced by injection of endotoxin (lipopolysaccharide) in rats. Electrocardiogram and body temperature were recorded in conscious animals using a telemetric system. Linear and non-linear indices of heart rate variability (e.g. sample entropy and fractal-like temporal structure) were assessed. RT-PCR and immunohistochemistry studies showed that α7nACHR is expressed in rat atrium and is mainly localized at the endothelial layer. Systemic administration of an α7nACHR antagonist (methyllycaconitine) did not show a significant effect on body temperature or heart rate dynamics in naïve rats. However, α7nACHR blockade could further reduce heart rate variability and elicit a febrile response in endotoxemic rats. Pre-treatment of endotoxemic animals with an α7nACHR agonist (PHA-543613) was unable to modulate heart rate dynamics in endotoxemic rats but could prevent the effect of endotoxin on body temperature within 24 h experiment. Neither methyllycaconitine nor PHA-543613 could affect cardiac beating variability of isolated perfused hearts taken from control or endotoxemic rats. Based on our observations we suggest a tonic role for nicotinic acetylcholine receptors in

  19. The α4β2 nicotinic acetylcholine receptor modulates autism-like behavioral and motor abnormalities in pentylenetetrazol-kindled mice.

    PubMed

    Takechi, Kenshi; Suemaru, Katsuya; Kiyoi, Takeshi; Tanaka, Akihiro; Araki, Hiroaki

    2016-03-15

    Epilepsy is associated with several psychiatric disorders, including cognitive impairment, autism and attention deficit/hyperactivity disorder (ADHD). However, the psychopathology of epilepsy is frequently unrecognized and untreated in patients. In the present study, we investigated the effects of ABT-418, a neuronal nicotinic acetylcholine receptor agonist, on pentylenetetrazol (PTZ)-kindled mice with behavioral and motor abnormalities. PTZ-kindled mice displayed impaired motor coordination (in the rotarod test), anxiety (in the elevated plus maze test) and social approach impairment (in the three-chamber social test) compared with control mice. ABT-418 treatment (0.05mg/kg, intraperitoneally) alleviated these behavioral abnormalities in PTZ-kindled mice. Immunolabeling of tissue sections demonstrated that expression of the α4 nicotinic acetylcholine receptor subunit in the medial habenula was similar in control and PTZ-kindled mice. However, expression was significantly decreased in the piriform cortex in PTZ-kindled mice. In addition, we examined the expression of the synaptic adhesion molecule neuroligin 3 (NLG3). NLG3 expression in the piriform cortex was significantly higher in PTZ-kindled mice compared with control mice. Collectively, our findings suggest that ADHD-like or autistic-like behavioral abnormalities associated with epilepsy are closely related to the downregulation of the α4 nicotinic receptor and the upregulation of NLG3 in the piriform cortex. In summary, this study indicates that ABT-418 might have therapeutic potential for attentional impairment in epileptic patients with psychiatric disorders such as autism and ADHD. PMID:26868186

  20. Functional characterization of a mutated chicken α7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2

    PubMed Central

    Buckingham, Steven D; Adcock, Charlotte; Sansom, Mark S P; Sattelle, David B; Baylis, Howard A

    1998-01-01

    Site-directed mutagenesis was used to create an altered form of the chicken α7 nicotinic acetylcholine (ACh) receptor subunit (α7x61) in which a leucine residue was inserted between residues Leu9′ and Ser10′ in transmembrane domain 2. The properties of α7x61 receptors are distinct from those of the wild-type receptor.Oocytes expressing wild-type α7 receptors responded to 10 μM nicotine with rapid inward currents that desensitized with a time-constant of 710±409 ms (mean±s.e.mean, n=5). However in α7x61 receptors 10 μM nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684±3403 ms (mean±s.e.mean, n=4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild-type receptors was observed. Dihydro-β-erythroidine (DHβE) acted as an antagonist on both receptors.Molecular modelling of the α7x61 receptor channel pore formed by a bundle of M2 α-helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10′ would be replaced by the leucine insertion, Val13′ and Phe14′ would be replaced, by Thr and Val, respectively.When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild-type and α7x61 receptors. However, block was more dependent on membrane potential for the α7x61 receptors.We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity. PMID:9690867

  1. Adenosine A1 Receptors in Mouse Pontine Reticular Formation Depress Breathing, Increase Anesthesia Recovery Time, and Decrease Acetylcholine Release

    PubMed Central

    Gettys, George C.; Liu, Fang; Kimlin, Ed; Baghdoyan, Helen A.; Lydic, Ralph

    2012-01-01

    Background Clinical and preclinical data demonstrate the analgesic actions of adenosine. Central administration of adenosine agonists, however, suppresses arousal and breathing by poorly understood mechanisms. This study tested the two-tailed hypothesis that adenosine A1 receptors in the pontine reticular formation (PRF) of C57BL/6J mice modulate breathing, behavioral arousal, and PRF acetylcholine release. Methods Three sets of experiments used 51 mice. First, breathing was measured by plethysmography after PRF microinjection of the adenosine A1 receptor agonist N6-sulfophenyl adenosine (SPA) or saline. Second, mice were anesthetized with isoflurane and time to recovery of righting response (RoRR) was quantified after PRF microinjection of SPA or saline. Third, acetylcholine release in the PRF was measured before and during microdialysis delivery of SPA, the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), or SPA and DPCPX. Results First, SPA significantly decreased respiratory rate (−18%), tidal volume (−12%) and minute ventilation (−16%). Second, SPA concentration accounted for 76% of the variance in RoRR. Third, SPA concentration accounted for a significant amount of the variance in acetylcholine release (52%), RoRR (98%), and breathing rate (86%). DPCPX alone caused a concentration-dependent increase in acetylcholine, decrease in RoRR, and decrease in breathing rate. Coadministration of SPA and DPCPX blocked the SPA-induced decrease in acetylcholine and increase in RoRR. Conclusions Endogenous adenosine acting at adenosine A1 receptors in the PRF modulates breathing, behavioral arousal, and acetylcholine release. The results support the interpretation that an adenosinergic-cholinergic interaction within the PRF comprises one neurochemical mechanism underlying the wakefulness stimulus for breathing. PMID:23263018

  2. Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid.

    PubMed

    Tomizawa, M; Wen, Z; Chin, H L; Morimoto, H; Kayser, H; Casida, J E

    2001-09-01

    The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs. PMID:11579144

  3. Rational design of partial agonists for the muscarinic m1 acetylcholine receptor.

    PubMed

    Chen, Xinyu; Klöckner, Jessika; Holze, Janine; Zimmermann, Cornelia; Seemann, Wiebke K; Schrage, Ramona; Bock, Andreas; Mohr, Klaus; Tränkle, Christian; Holzgrabe, Ulrike; Decker, Michael

    2015-01-22

    Aiming to design partial agonists for a G-protein-coupled receptor based on dynamic ligand binding, we synthesized three different series of bipharmacophoric ligands composed of the orthosteric building blocks iperoxo and 1 linked to allosteric modulators (BQCA-derived compounds, BQCAd; TBPB-derived compound, TBPBd). Their interactions were studied with the human muscarinic acetylcholine M1-receptor (hM1) with respect to receptor binding and Gq-protein signaling. Results demonstrate that iperoxo/BQCAd (2, 3) and 1/BQCAd hybrids (4) act as M1 partial agonists, whereas 1/TBPBd hybrids (5) did not activate M1-receptors. Among the iperoxo/BQCAd-hybrids, spacer length in conjunction with the pattern of substitution tuned efficacy. Most interestingly, a model of dynamic ligand binding revealed that the spacer length of 2a and 3a controlled the probability of switch between the inactive purely allosteric and the active bitopic orthosteric/allosteric binding pose. In summary, dynamic ligand binding can be exploited in M1 receptors to design partial agonists with graded efficacy. PMID:25478907

  4. Tramadol state-dependent memory: involvement of dorsal hippocampal muscarinic acetylcholine receptors.

    PubMed

    Jafari-Sabet, Majid; Jafari-Sabet, Ali-Reza; Dizaji-Ghadim, Ali

    2016-08-01

    The effects on tramadol state-dependent memory of bilateral intradorsal hippocampal (intra-CA1) injections of physostigmine, an acetylcholinesterase inhibitor, and atropine, a muscarinic acetylcholine receptor antagonist, were examined in adult male NMRI mice. A single-trial step-down passive avoidance task was used for the assessment of memory retention. Post-training intra-CA1 administration of an atypical μ-opioid receptor agonist, tramadol (0.5 and 1 μg/mouse), dose dependently impaired memory retention. Pretest injection of tramadol (0.5 and 1 μg/mouse, intra-CA1) induced state-dependent retrieval of the memory acquired under the influence of post-training tramadol (1 μg/mouse, intra-CA1). A pretest intra-CA1 injection of physostigmine (1 μg/mouse) reversed the memory impairment induced by post-training administration of tramadol (1 μg/mouse, intra-CA1). Moreover, pretest administration of physostigmine (0.5 and 1 μg/mouse, intra-CA1) with an ineffective dose of tramadol (0.25 μg/mouse, intra-CA1) also significantly restored retrieval. Pretest administration of physostigmine (0.25, 0.5, and 1 μg/mouse, intra-CA1) by itself did not affect memory retention. A pretest intra-CA1 injection of the atropine (1 and 2 μg/mouse) 5 min before the administration of tramadol (1 μg/mouse, intra-CA1) dose dependently inhibited tramadol state-dependent memory. Pretest administration of atropine (0.5, 1, and 2 μg/mouse, intra-CA1) by itself did not affect memory retention. It can be concluded that dorsal hippocampal muscarinic acetylcholine receptor mechanisms play an important role in the modulation of tramadol state-dependent memory. PMID:27089282

  5. The role of the a7 subunit of the nicotinic acetylcholine receptor in the acute toxicosis of methyllycaconitine in mice.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research demonstrated a correlation between the LD50 of MLA and the amount of a7 nAChR in various mouse strains, suggesting that mice...

  6. Activation and desensitization of peripheral muscle and neuronal nicotinic acetylcholine receptors by selected, naturally-occurring pyridine alkaloids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Teratogenic alkaloids can cause developmental defects due to inhibition of fetal movement that results from desensitization of fetal muscletype nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiper...

  7. Potency of nondepolarizing muscle relaxants on muscle-type acetylcholine receptors in denervated mouse skeletal muscle

    PubMed Central

    Wang, Hong; Yang, Bin; Han, Guang-wei; Li, Shi-tong

    2010-01-01

    Aim: To investigate the changing resistance to nondepolarizing muscle relaxants (NDMRs) during the first month after denervation. Methods: The denervated and innervated skeletal muscle cells were examined on days 1, 4, 7, 14, 21, and 28 after denervation. Individual denervated and innervated cells were prepared from the flexor digitorum brevis of the surgically denervated and contralateral hind feet, respectively. Nicotinic acetylcholine receptors (nAChRs) in the cells were activated with 30 μmol/L acetylcholine, either alone or in combination with various concentrations of vecuronium. Currents were recorded using a whole-cell patch-clamp technique. Results: The concentrations of vecuronium resulting in half-maximal inhibitory responses (IC50) increased 1.2- (P>0.05), 1.7-, 3.7-, 2.5-, 1.9-, and 1.8-fold (P<0.05) at Days 1, 4, 7, 14, 21, and 28 after denervation, respectively, compared to the innervated control. Resistance to vecuronium appeared at Day 4, peaked at Day 7, and declined at Day 14 after denervation. Nevertheless, IC50 values at Day 28 remained significantly higher than those for the innervated control, suggesting that the resistance to vecuronium had not disappeared at Day 28. Conclusion: The NDMR doses required to achieve satisfactory clinical effects differ at different times after muscle denervation. PMID:21102480

  8. Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites

    SciTech Connect

    Forman, S.A.; Miller, K.W. )

    1989-02-21

    The relationship between the high-affinity procaine channel inhibition site and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid {sup 86}Rb{sup +} quenched-flux assays at 4 {degree}C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with {alpha}-bungarotoxin ({alpha}-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations alone, AChR undergoes one phase of inactivation in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting ACh concentrations rapid inactivation complete in 30-75 ms is followed by fast desensitization at the same k{sub d} observed without procaine. The dependence of k{sub r} on (procaine) is consistent with a bimolecular association between procaine and its AChR site. Inhibition of AChR function by mixtures of procaine plus self-inhibiting concentrations of ACh or suberyldicholine was studied by reducing the level of {alpha}-BTX block in vesicles. The data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and k{sub r}'s dependence on (ACh) in channel-activating range closely parallels that of {sup 86}Rb{sup +} flux response to ACh.

  9. α-Bungarotoxin Binding to Acetylcholine Receptor Membranes Studied by Low Angle X-Ray Diffraction

    PubMed Central

    Young, Howard S.; Herbette, Leo G.; Skita, Victor

    2003-01-01

    The nicotinic acetylcholine receptor (nAChR) carries two binding sites for snake venom neurotoxins. α-Bungarotoxin from the Southeast Asian banded krait, Bungarus multicinctus, is a long neurotoxin which competitively blocks the nAChR at the acetylcholine binding sites in a relatively irreversible manner. Low angle x-ray diffraction was used to generate electron density profile structures at 14-Å resolution for Torpedo californica nAChR membranes in the absence and presence of α-bungarotoxin. Analysis of the lamellar diffraction data indicated a 452-Å lattice spacing between stacked nAChR membrane pairs. In the presence of α-bungarotoxin, the quality of the diffraction data and the lamellar lattice spacing were unchanged. In the plane of the membrane, the nAChRs packed together with a nearest neighbor distance of 80 Å, and this distance increased to 85 Å in the presence of toxin. Electron density profile structures were calculated in the absence and presence of α-bungarotoxin, revealing a location for the toxin binding sites. In native, fully-hydrated nAChR membranes, α-bungarotoxin binds to the nAChR outer vestibule and contacts the surface of the membrane bilayer. PMID:12885641

  10. Spontaneous opening of the acetylcholine receptor channel in developing muscle cells from normal and dystrophic mice

    SciTech Connect

    Franco-Obregon, A.; Lansman, J.B.

    1995-12-31

    Single-channel activity was recorded from cell-attached patches on skeletal muscle cells isolated from wild-type mice and from mice carrying the dy or mdx mutations. Spontaneous openings of the nicotinic acetylcholine receptor channel (nAChR) were detected in virtually all recordings from either 4v/dy or dyl + myotubes. but only infrequently from wild-type or mdx myotubes. Spontaneous openings were also present in most recordings from undifferentiated myoblasts from all of the mouse strains studied. The biophysical properties of the spontaneous activity were similar to those of the embryonic form of the nAChR in the presence of acetylcholine (ACh). Examination of the single-channel currents evoked by low concentrations of ACh showed a reduced sensitivity to the agonist in the dystrophic dy and mdx myotubes. but not in wild- type myotubes. The results suggest that alterations in nAChR function are associated with the pathogenesis of muscular dystrophy in the dy mouse.

  11. Novel Fused Arylpyrimidinone Based Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor.

    PubMed

    Mistry, Shailesh N; Lim, Herman; Jörg, Manuela; Capuano, Ben; Christopoulos, Arthur; Lane, J Robert; Scammells, Peter J

    2016-05-18

    Benzoquinazolinone 1 is a positive allosteric modulator (PAM) of the M1 muscarinic acetylcholine receptor (mAChR), which is significantly more potent than the prototypical PAM, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA). In this study, we explored the structural determinants that underlie the activity of 1 as a PAM of the M1 mAChR. We paid particular attention to the importance of the tricyclic scaffold of compound 1, for the activity of the molecule. Complete deletion of the peripheral fused benzene ring caused a significant decrease in affinity and binding cooperativity with acetylcholine (ACh). This loss of affinity was rescued with the addition of either one or two methyl groups in the 7- and/or 8-position of the quinazolin-4(3H)-one core. These results demonstrate that the tricyclic benzo[h]quinazolin-4(3H)-one core could be replaced with a quinazolin-4(3H)-one core and maintain functional affinity. As such, the quinazolin-4(3H)-one core represents a novel scaffold to further explore M1 mAChR PAMs with improved physicochemical properties. PMID:26891194

  12. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2014-01-01

    Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation. PMID:25421240

  13. The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

    PubMed Central

    2012-01-01

    Background Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. Results A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular

  14. Agonist mediated conformational changes of solubilized calf forebrain muscarinic acetylcholine receptors.

    PubMed

    Vanderheyden, P; Andre, C; de Backer, J P; Vauquelin, G

    1984-10-01

    Muscarinic receptors in calf forebrain membranes can be identified by the specific binding of the radiolabelled antagonist [3H]dexetimide. These receptors (2.8 pM/mg protein) comprise two non-interconvertible subpopulations with respectively high and low agonist affinity but with the same antagonist affinity. For all the agonists tested the low affinity sites represent 85 +/- 5% of the total receptor population. 0.5% Digitonin solubilized extracts contain 0.8 pM muscarinic receptor/mg protein. In contrast with the membranes, these extracts contain only sites with low agonist affinity. The alkylating reagent N-ethylmaleimide causes an increase of the acetylcholine affinity for the low affinity sites in membranes as well as for the solubilized sites. This effect is time dependent until a maximal 3-fold increase in affinity is attained. The rate of N-ethylmaleimide action is enhanced by the concomitant presence of agonists. In contrast, N-ethylmaleimide does not affect antagonist binding. This suggests that agonists mediate a conformational change of both the membrane bound low affinity muscarinic sites and of the solubilized sites, resulting in their increased susceptibility towards NEM alkylation. PMID:6487351

  15. Brain α7 nicotinic acetylcholine receptors in MPTP-lesioned monkeys and parkinsonian patients.

    PubMed

    Morissette, Marc; Morin, Nicolas; Grégoire, Laurent; Rajput, Alex; Rajput, Ali H; Di Paolo, Thérèse

    2016-06-01

    L-DOPA-induced dyskinesias (LID) appear in the majority of Parkinson's disease (PD) patients. Nicotinic acetylcholine (nACh) receptor-mediated signaling has been implicated in PD and LID and modulation of brain α7 nACh receptors might be a potential therapeutic target for PD. This study used [(125)I]α-Bungarotoxin autoradiography to investigate α7 nACh receptors in LID in post-mortem brains from PD patients (n=14) and control subjects (n=11), and from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys treated with saline (n=5), L-DOPA (n=4) or L-DOPA+2-methyl-6-(phenylethynyl)pyridine (MPEP) (n=5), and control monkeys (n=4). MPEP is the prototypal metabotropic glutamate 5 (mGlu5) receptor antagonist; it reduced the development of LID in these monkeys. [(125)I]α-Bungarotoxin specific binding to striatal and pallidal α7 nACh receptors were only increased in L-DOPA-treated dyskinetic MPTP monkeys as compared to controls, saline and L-DOPA+MPEP MPTP monkeys; dyskinesia scores correlated positively with this binding. The total group of Parkinsonian patients had higher [(125)I]α-Bungarotoxin specific binding compared to controls in the caudate nucleus but not in the putamen. PD patients without motor complications had higher [(125)I]α-Bungarotoxin specific binding compared to controls only in the caudate nucleus. PD patients with LID only had higher [(125)I]α-Bungarotoxin specific binding compared to controls in the caudate nucleus and compared to those without motor complications and controls in the putamen. PD patients with wearing-off only, had [(125)I]α-Bungarotoxin specific binding at control values in the caudate nucleus and lower in the putamen. Reduced motor complications were associated with normal striatal α7 nACh receptors, suggesting the potential of this receptor to manage motor complications in PD. PMID:27038656

  16. Solubilization and hydrodynamic properties of pig atrial muscarinic acetylcholine receptor in dodecyl beta-D-maltoside.

    PubMed Central

    Peterson, G L; Rosenbaum, L C; Schimerlik, M I

    1988-01-01

    The pig atrial muscarinic acetylcholine receptor (mAcChR) has been solubilized from the membrane-bound state in high yield and in stable conformation by the non-ionic detergent dodecyl beta-D-maltoside (DBM). The yield and selectivity for receptor solubilization is dependent on the detergent/protein ratio during extraction. Extraction at 2 mg of DBM/mg of protein gave a 75% yield of solubilized receptor with a 1.5-fold enrichment. A double-extraction procedure, in which non-receptor protein was first extracted at 0.4 mg of DBM/mg of protein and mAcChR was selectively solubilized by a second extraction at 0.35 mg of DBM/mg of protein, gave a 50% overall yield and a 2.8-fold enrichment. Both preparations had a half-life of about 20 days on ice without addition of muscarinic ligands. Receptor stability was decreased by the presence of cations, particularly bivalent cations, and enhanced by the agonist carbachol. Dissociation constants for the interaction of the DBM-solubilized receptor with the antagonist L-quinuclidinyl benzilate (Kd = 223 pM) and the agonist carbachol (Kd = 100 microM) were similar to those for the digitonin/cholate-solubilized receptor. Pig atrial mAcChR purified in digitonin/cholate and exchanged into DBM displayed reliable hydrodynamic behaviour during sucrose density sedimentation in gradients of 2H2O and H2O and during gel filtration in Sephacryl S-300. DBM is thus the first detergent which will solubilize a stable form of the ligand-free mAcChR in yields similar to those with digitonin, and is the only stabilizing detergent thus far suitable for hydrodynamic studies. DBM is also likely to be similarly useful in studying other membrane proteins for which digitonin has been the solubilizing detergent of choice. PMID:3202834

  17. Solubilization and hydrodynamic properties of pig atrial muscarinic acetylcholine receptor in dodecyl beta-D-maltoside.

    PubMed

    Peterson, G L; Rosenbaum, L C; Schimerlik, M I

    1988-10-15

    The pig atrial muscarinic acetylcholine receptor (mAcChR) has been solubilized from the membrane-bound state in high yield and in stable conformation by the non-ionic detergent dodecyl beta-D-maltoside (DBM). The yield and selectivity for receptor solubilization is dependent on the detergent/protein ratio during extraction. Extraction at 2 mg of DBM/mg of protein gave a 75% yield of solubilized receptor with a 1.5-fold enrichment. A double-extraction procedure, in which non-receptor protein was first extracted at 0.4 mg of DBM/mg of protein and mAcChR was selectively solubilized by a second extraction at 0.35 mg of DBM/mg of protein, gave a 50% overall yield and a 2.8-fold enrichment. Both preparations had a half-life of about 20 days on ice without addition of muscarinic ligands. Receptor stability was decreased by the presence of cations, particularly bivalent cations, and enhanced by the agonist carbachol. Dissociation constants for the interaction of the DBM-solubilized receptor with the antagonist L-quinuclidinyl benzilate (Kd = 223 pM) and the agonist carbachol (Kd = 100 microM) were similar to those for the digitonin/cholate-solubilized receptor. Pig atrial mAcChR purified in digitonin/cholate and exchanged into DBM displayed reliable hydrodynamic behaviour during sucrose density sedimentation in gradients of 2H2O and H2O and during gel filtration in Sephacryl S-300. DBM is thus the first detergent which will solubilize a stable form of the ligand-free mAcChR in yields similar to those with digitonin, and is the only stabilizing detergent thus far suitable for hydrodynamic studies. DBM is also likely to be similarly useful in studying other membrane proteins for which digitonin has been the solubilizing detergent of choice. PMID:3202834

  18. Characterization of alpha-conotoxin interactions with the nicotinic acetylcholine receptor and monoclonal antibodies.

    PubMed Central

    Ashcom, J D; Stiles, B G

    1997-01-01

    The venoms of predatory marine cone snails, Conus species, contain numerous peptides and proteins with remarkably diverse pharmacological properties. One group of peptides are the alpha-conotoxins, which consist of 13-19 amino acids constrained by two disulphide bonds. A biologically active fluorescein derivative of Conus geographus alpha-conotoxin GI (FGI) was used in novel solution-phase-binding assays with purified Torpedo californica nicotinic acetylcholine receptor (nAchR) and monoclonal antibodies developed against the toxin. The binding of FGI to nAchR or antibody had apparent dissociation constants of 10-100 nM. Structure-function studies with alpha-conotoxin GI analogues composed of a single disulphide loop revealed that different conformational restraints are necessary for effective toxin interactions with nAchR or antibodies. PMID:9359860

  19. Comparison between ab initio and semiempirical net atomic charges of some nicotinic acetylcholine receptor agonists

    SciTech Connect

    Yadav, J.S.; Hermsmeier, M.; Gund, T. )

    1989-01-01

    We have calculated the net atomic charges and molecular electrostatic potentials of two potent nicotinic acetylcholine receptor agonists, isoarecolone and acetylpiperazine, by three different methods to see how well they correlate and if the simplest method gives the same predictive results. The calculational methods involved calculating net atomic charges by semiempirical (MNDO from MOPAC) and ab initio (Mulliken) and ab initio (potential derived) at STO-3G basis set level. Some deviations were observed when comparisons were made atom by atom, but when group comparisons were made, good correlations were observed. When these partial charges were used to calculate the respective molecular electrostatic potentials on the van der Waals surface, very good correlations were obtained. This study shows that for routine electrostatic calculations, semiempirical MNDO Calculations give similar results and thus lead to similar predictions.

  20. Membrane lipid heterogeneity associated with acetylcholine receptor particle aggregates in Xenopus embryonic muscle cells.

    PubMed Central

    Bridgman, P C; Nakajima, Y

    1981-01-01

    Filipin, digitonin, and saponin react with membrane cholesterol to produce unique membrane alterations (sterol-specific complexes) that are easily discernible in freeze-fracture replicas. We have treated both noninnervated and innervated Xenopus embryonic muscle cells in culture with these agents. Freeze-fracture of these treated muscle cells showed that most areas of the muscle plasma membrane contain sterol-specific complexes (19- to 40-nm protuberances and dimples with filipin, a scalloped appearance with digitonin, or an irregular, rough appearance with saponin). However, these complexes were virtually absent from membrane areas of junctional and nonjunctional aggregates of acetylcholine receptor particles. This result suggests that the membrane matrix of these aggregates is low in cholesterol and that this membrane lipid heterogeneity may be linked to the mechanisms involved in their formation and stabilization on muscle cells in culture. Images PMID:6940140

  1. [Molecular dynamics simulations of migration of ions and molecules through the acetylcholine receptor channel].

    PubMed

    Shaĭtan, K V; Li, A; Tershkina, K B; Kirpichnikov, M P

    2007-01-01

    A dynamic model of the channel of an acetylcholine receptor in a closed state has been proposed. The channel is formed by five a-helices of subunit M2 and stabilized by the cyclic hydrocarbon (CH2)105. The migration of charged and unchanged van der Waals particles with a diameter of 7.72 A equivalent to the diameter of a hydrated sodium ion has been studied. The migration occurred by the action of external force applied to the complex along the channel axis. In the closed state, the inhibition of ions is due to two components: electrostatic interaction and steric constraints. The van der Waals channel gate is formed by residues 13'-A-Val255, B-Val261, C-Val269, D-Val255, and E-Ile264, and the negatively changed residues occurring in the upper part of the channel have a great effect on ion selectivity. PMID:17633536

  2. Exon-intron structure of the human neuronal nicotinic acetylcholine receptor {alpha}4 subunit (CHRNA4)

    SciTech Connect

    Steinlein, O.; Weiland, S.; Stoodt, J.; Propping, P.

    1996-03-01

    The human neuronal nicotinic acetylcholine receptor {alpha}4 subunit gene (CHRNA4) is located in the candidate region for three different phenotypes: benign familial neonatal convulsions, autosomal dominant nocturnal frontal lobe epilepsy, and low-voltage EEG. Recently, a missense mutation in transmembrane domain 2 of CHRNA4 was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy in one extended pedigree. We have determined the genomic organization of CHRNA4, which consists of six exons distributed over approximately 17 kb of genomic DNA. The nucleotide sequence obtained from the genomic regions adjacent to the exon boundaries enabled us to develop a set of primer pairs for PCR amplification of the complete coding region. The sequence analysis provides the basis for a comprehensive mutation screening of CHRNA4 in the above-mentioned phenotypes and possibly in other types of idopathic epilepsies. 29 refs., 3 figs., 1 tab.

  3. Dynamic heterogeneity and non-Gaussian statistics for acetylcholine receptors on live cell membrane.

    PubMed

    He, W; Song, H; Su, Y; Geng, L; Ackerson, B J; Peng, H B; Tong, P

    2016-01-01

    The Brownian motion of molecules at thermal equilibrium usually has a finite correlation time and will eventually be randomized after a long delay time, so that their displacement follows the Gaussian statistics. This is true even when the molecules have experienced a complex environment with a finite correlation time. Here, we report that the lateral motion of the acetylcholine receptors on live muscle cell membranes does not follow the Gaussian statistics for normal Brownian diffusion. From a careful analysis of a large volume of the protein trajectories obtained over a wide range of sampling rates and long durations, we find that the normalized histogram of the protein displacements shows an exponential tail, which is robust and universal for cells under different conditions. The experiment indicates that the observed non-Gaussian statistics and dynamic heterogeneity are inherently linked to the slow-active remodelling of the underlying cortical actin network. PMID:27226072

  4. AzoCholine Enables Optical Control of Alpha 7 Nicotinic Acetylcholine Receptors in Neural Networks.

    PubMed

    Damijonaitis, Arunas; Broichhagen, Johannes; Urushima, Tatsuya; Hüll, Katharina; Nagpal, Jatin; Laprell, Laura; Schönberger, Matthias; Woodmansee, David H; Rafiq, Amir; Sumser, Martin P; Kummer, Wolfgang; Gottschalk, Alexander; Trauner, Dirk

    2015-05-20

    Nicotinic acetylcholine receptors (nAChRs) are essential for cellular communication in higher organisms. Even though a vast pharmacological toolset to study cholinergic systems has been developed, control of endogenous neuronal nAChRs with high spatiotemporal precision has been lacking. To address this issue, we have generated photoswitchable nAChR agonists and re-evaluated the known photochromic ligand, BisQ. Using electrophysiology, we found that one of our new compounds, AzoCholine, is an excellent photoswitchable agonist for neuronal α7 nAChRs, whereas BisQ was confirmed to be an agonist for the muscle-type nAChR. AzoCholine could be used to modulate cholinergic activity in a brain slice and in dorsal root ganglion neurons. In addition, we demonstrate light-dependent perturbation of behavior in the nematode, Caenorhabditis elegans. PMID:25741856

  5. Dynamic heterogeneity and non-Gaussian statistics for acetylcholine receptors on live cell membrane

    PubMed Central

    He, W.; Song, H.; Su, Y.; Geng, L.; Ackerson, B. J.; Peng, H. B.; Tong, P.

    2016-01-01

    The Brownian motion of molecules at thermal equilibrium usually has a finite correlation time and will eventually be randomized after a long delay time, so that their displacement follows the Gaussian statistics. This is true even when the molecules have experienced a complex environment with a finite correlation time. Here, we report that the lateral motion of the acetylcholine receptors on live muscle cell membranes does not follow the Gaussian statistics for normal Brownian diffusion. From a careful analysis of a large volume of the protein trajectories obtained over a wide range of sampling rates and long durations, we find that the normalized histogram of the protein displacements shows an exponential tail, which is robust and universal for cells under different conditions. The experiment indicates that the observed non-Gaussian statistics and dynamic heterogeneity are inherently linked to the slow-active remodelling of the underlying cortical actin network. PMID:27226072

  6. Dynamic heterogeneity and non-Gaussian statistics for acetylcholine receptors on live cell membrane

    NASA Astrophysics Data System (ADS)

    He, W.; Song, H.; Su, Y.; Geng, L.; Ackerson, B. J.; Peng, H. B.; Tong, P.

    2016-05-01

    The Brownian motion of molecules at thermal equilibrium usually has a finite correlation time and will eventually be randomized after a long delay time, so that their displacement follows the Gaussian statistics. This is true even when the molecules have experienced a complex environment with a finite correlation time. Here, we report that the lateral motion of the acetylcholine receptors on live muscle cell membranes does not follow the Gaussian statistics for normal Brownian diffusion. From a careful analysis of a large volume of the protein trajectories obtained over a wide range of sampling rates and long durations, we find that the normalized histogram of the protein displacements shows an exponential tail, which is robust and universal for cells under different conditions. The experiment indicates that the observed non-Gaussian statistics and dynamic heterogeneity are inherently linked to the slow-active remodelling of the underlying cortical actin network.

  7. Differential Effects of Quercetin and Quercetin Glycosides on Human α7 Nicotinic Acetylcholine Receptor-Mediated Ion Currents.

    PubMed

    Lee, Byung-Hwan; Choi, Sun-Hye; Kim, Hyeon-Joong; Jung, Seok-Won; Hwang, Sung-Hee; Pyo, Mi-Kyung; Rhim, Hyewhon; Kim, Hyoung-Chun; Kim, Ho-Kyoung; Lee, Sang-Mok; Nah, Seung-Yeol

    2016-07-01

    Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin- 3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance α7 nicotinic acetylcholine receptor (α7 nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of α7 nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current (IACh) in Xenopus oocytes expressing the α7 nAChR. IACh was measured with a two-electrode voltage clamp technique. In oocytes injected with α7 nAChR copy RNA, quercetin enhanced IACh, whereas quercetin glycosides inhibited IACh. Quercetin glycosides mediated an inhibition of IACh, which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of IACh inhibition by quercetin glycosides was Rutin≥Rham1>Rham2. Quercetin glycosides-mediated IACh enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated IACh inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated α7 nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the α7 nAChR in a differential manner. PMID:27098860

  8. Differential Effects of Quercetin and Quercetin Glycosides on Human α7 Nicotinic Acetylcholine Receptor-Mediated Ion Currents

    PubMed Central

    Lee, Byung-Hwan; Choi, Sun-Hye; Kim, Hyeon-Joong; Jung, Seok-Won; Hwang, Sung-Hee; Pyo, Mi-Kyung; Rhim, Hyewhon; Kim, Hyoung-Chun; Kim, Ho-Kyoung; Lee, Sang-Mok; Nah, Seung-Yeol

    2016-01-01

    Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin-3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance α7 nicotinic acetylcholine receptor (α7 nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of α7 nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current (IACh) in Xenopus oocytes expressing the α7 nAChR. IACh was measured with a two-electrode voltage clamp technique. In oocytes injected with α7 nAChR copy RNA, quercetin enhanced IACh, whereas quercetin glycosides inhibited IACh. Quercetin glycosides mediated an inhibition of IACh, which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of IACh inhibition by quercetin glycosides was Rutin≥Rham1>Rham2. Quercetin glycosides-mediated IACh enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated IACh inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated α7 nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the α7 nAChR in a differential manner. PMID:27098860

  9. Distribution of calcitonin gene-related peptide in vertebrate neuromuscular junctions: relationship to the acetylcholine receptor.

    PubMed

    Csillik, B; Tajti, L; Kovács, T; Kukla, E; Rakic, P; Knyihár-Csillik, E

    1993-10-01

    Calcitonin gene-related peptide (CGRP), regarded by several authors to be involved in maintenance of the acetylcholine receptor, is present in the motor axons of various striated rat muscles. It is present, however, only in motor endplates of several selected striated muscles, where it is located in presynaptic axon terminals of neuromuscular junctions. No immunoreactivity could be seen within synaptic vesicles themselves. In the non-human primate Macaca fasciculata, neuromuscular junctions, including those in the diaphragm, display an intense CGRP reaction. The structure of the simian motor endplates is more elaborate than that of the rat. Amphibian motor nerve endings, both in tetanic and tonic muscles, display CGRP immunoreactivity. In tetanic muscles the CGRP reaction outlines "terminaisons en placque" (true motor end plates) and weakly reacting "terminaisons en grappe" (grape-like endings) in tonic muscles. On supramaximal stimulation of the motor nerve, CGRP is depleted from the affected neuromuscular junctions. Wallerian degeneration of the motor axon results in complete disappearance of CGRP. In most rat muscles in which motor endplates do not normally exhibit CGRP immunoreactivity, e.g., the diaphragm and buccinator muscles, the pre-terminal motor axons are CGRP-positive. After immobilization of such muscles by local bupivacaine injection to rats under brief chloral hydrate anesthesia, CGRP immunoreactivity of the neuromuscular junctions can be elicited because blockade of neuromuscular transmission results in accumulation of CGRP in the endplates. Even more striking is the appearance of CGRP immunoreactivity in normally non-reactive motor endplates during axon regeneration after an experimentally induced Wallerian degeneration of the motor axons. We conclude that CGRP is a regular, genotypically determined component of neuromuscular junctions, present either in a manifest or in a latent form. The latter can be elicited by various experimental approaches

  10. Neuronal Nicotinic Acetylcholine Receptors: Common Molecular Substrates of Nicotine and Alcohol Dependence

    PubMed Central

    Hendrickson, Linzy M.; Guildford, Melissa J.; Tapper, Andrew R.

    2013-01-01

    Alcohol and nicotine are often co-abused. As many as 80–95% of alcoholics are also smokers, suggesting that ethanol and nicotine, the primary addictive component of tobacco smoke, may functionally interact in the central nervous system and/or share a common mechanism of action. While nicotine initiates dependence by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels normally activated by endogenous acetylcholine (ACh), ethanol is much less specific with the ability to modulate multiple gene products including those encoding voltage-gated ion channels, and excitatory/inhibitory neurotransmitter receptors. However, emerging data indicate that ethanol interacts with nAChRs, both directly and indirectly, in the mesocorticolimbic dopaminergic (DAergic) reward circuitry to affect brain reward systems. Like nicotine, ethanol activates DAergic neurons of the ventral tegmental area (VTA) which project to the nucleus accumbens (NAc). Blockade of VTA nAChRs reduces ethanol-mediated activation of DAergic neurons, NAc DA release, consumption, and operant responding for ethanol in rodents. Thus, ethanol may increase ACh release into the VTA driving activation of DAergic neurons through nAChRs. In addition, ethanol potentiates distinct nAChR subtype responses to ACh and nicotine in vitro and in DAergic neurons. The smoking cessation therapeutic and nAChR partial agonist, varenicline, reduces alcohol consumption in heavy drinking smokers and rodent models of alcohol consumption. Finally, single nucleotide polymorphisms in nAChR subunit genes are associated with alcohol dependence phenotypes and smoking behaviors in human populations. Together, results from pre-clinical, clinical, and genetic studies indicate that nAChRs may have an inherent role in the abusive properties of ethanol, as well as in nicotine and alcohol co-dependence. PMID:23641218

  11. ID1 facilitates the growth and metastasis of non-small cell lung cancer in response to nicotinic acetylcholine receptor and epidermal growth factor receptor signaling.

    PubMed

    Pillai, Smitha; Rizwani, Wasia; Li, Xueli; Rawal, Bhupendra; Nair, Sajitha; Schell, Michael J; Bepler, Gerold; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2011-07-01

    Expression of ID1 (inhibitor of differentiation) has been correlated with the progression of a variety of cancers, but little information is available on its role in non-small cell lung cancer (NSCLC). Here we show that ID1 is induced by nicotinic acetylcholine receptor (nAChR) and epidermal growth factor receptor (EGFR) signaling in a panel of NSCLC cell lines and primary cells from the lung. ID1 induction was Src dependent and mediated through the α7 subunit of nAChR; transfection of K-Ras or EGFR to primary cells induced ID1. ID1 depletion prevented nicotine- and EGF-induced proliferation, migration, and invasion of NSCLC cells and angiogenic tubule formation of human microvascular endothelial cells from lungs (HMEC-Ls). ID1 could induce the expression of mesenchymal markers such as vimentin and fibronectin by downregulating ZBP-89, a zinc finger repressor protein. ID1 levels were elevated in tumors from mice that were exposed to nicotine. Further, human lung tissue microarrays (TMAs) showed elevated levels of ID1 in NSCLC samples, with maximal levels in metastatic lung cancers. Quantitative reverse transcription-PCR (RT-PCR) performed on patient lung tumors showed that ID1 levels were elevated in advanced stages of NSCLC and correlated with elevated expression of vimentin and fibronectin, irrespective of smoking history. PMID:21606196

  12. Pharmacological and ionic characterizations of the muscarinic receptors modulating (/sup 3/H)acetylcholine release from rat cortical synaptosomes

    SciTech Connect

    Meyer, E.M.; Otero, D.H.

    1985-05-01

    The muscarinic receptors that modulate acetylcholine release from rat cortical synaptosomes were characterized with respect to sensitivity to drugs that act selectively at M1 or M2 receptor subtypes, as well as to changes in ionic strength and membrane potential. The modulatory receptors appear to be of the M2 type, since they are activated by carbachol, acetylcholine, methacholine, oxotremorine, and bethanechol, but not by pilocarpine, and are blocked by atropine, scopolamine, and gallamine (at high concentrations), but not by pirenzepine or dicyclomine. The ED50S for carbachol, acetylcholine, and oxotremorine are less than 10 microM, suggesting that the high affinity state of the receptor is functional. High ionic strength induced by raising the NaCl concentration has no effect on agonist (oxotremorine) potency, but increases the efficacy of this compound, which disagrees with receptor-binding studies. On the other hand, depolarization with either KCl or with veratridine (20 microM) reduces agonist potencies by approximately an order of magnitude, suggesting a potential mechanism for receptor regulation.

  13. Nicotinic acetylcholine receptors: a comparison of the nAChRs of Caenorhabditis elegans and parasitic nematodes.

    PubMed

    Holden-Dye, Lindy; Joyner, Michelle; O'Connor, Vincent; Walker, Robert J

    2013-12-01

    Nicotinic acetylcholine receptors (nAChRs) play a key role in the normal physiology of nematodes and provide an established target site for anthelmintics. The free-living nematode, Caenorhabditis elegans, has a large number of nAChR subunit genes in its genome and so provides an experimental model for testing novel anthelmintics which act at these sites. However, many parasitic nematodes lack specific genes present in C. elegans, and so care is required in extrapolating from studies using C. elegans to the situation in other nematodes. In this review the properties of C. elegans nAChRs are reviewed and compared to those of parasitic nematodes. This forms the basis for a discussion of the possible subunit composition of nAChRs from different species of parasitic nematodes. Currently our knowledge on this is largely based on studies using heterologous expression and pharmacological analysis of receptor subunits in Xenopus laevis oocytes. It is concluded that more information is required regarding the subunit composition and pharmacology of endogenous nAChRs in parasitic nematodes. PMID:23500392

  14. Highly Selective and Sensitive Detection of Acetylcholine Using Receptor-Modified Single-Walled Carbon Nanotube Sensors

    NASA Astrophysics Data System (ADS)

    Xu, Shihong; Kim, Byeongju; Song, Hyun Seok; Jin, Hye Jun; Park, Eun Jin; Lee, Sang Hun; Lee, Byung Yang; Park, Tai Hyun; Hong, Seunghun

    2015-03-01

    Acetylcholine (ACh) is a neurotransmitter in a human central nervous system and is related to various neural functions such as memory, learning and muscle contractions. Dysfunctional ACh regulations in a brain can induce several neuropsychiatric diseases such as Alzheimer's disease, Parkinson's disease and myasthenia gravis. In researching such diseases, it is important to measure the concentration of ACh in the extracellular fluid of the brain. Herein, we developed a highly sensitive and selective ACh sensor based on single-walled carbon nanotube-field effect transistors (swCNT-FETs). In our work, M1 mAChR protein, an ACh receptor, was expressed in E.coli and coated on swCNT-FETs with lipid membranes. Here, the binding of ACh onto the receptors could be detected by monitoring the change of electrical currents in the underlying swCNT-FETs, allowing the real-time detection of ACh at a 100 pM concentration. Furthermore, our sensor could selectively detect ACh from other neurotransmitters. This is the first report of the real-time sensing of ACh utilizing specific binding between the ACh and M1 mAChR, and it may lead to breakthroughs in various biomedical applications such as drug screening and disease diagnosis.

  15. alpha4/7-conotoxin Lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors.

    PubMed

    Peng, Can; Han, Yuhong; Sanders, Tanya; Chew, Geoffrey; Liu, Jing; Hawrot, Edward; Chi, Chengwu; Wang, Chunguang

    2008-10-01

    Cone snails comprise approximately 700 species of venomous molluscs which have evolved the ability to generate multiple toxins with varied and exquisite selectivity. alpha-Conotoxin is a powerful tool for defining the composition and function of nicotinic acetylcholine receptors which play a crucial role in excitatory neurotransmission and are important targets for drugs and insecticides. An alpha4/7 conotoxin, Lp1.1, originally identified by cDNA and genomic DNA cloning from Conus leopardus, was found devoid of the highly conserved Pro residue in the first intercysteine loop. To further study this toxin, alpha-Lp1.1 was chemically synthesized and refolded into its globular disulfide isomer. The synthetic Lp1.1 induced seizure and paralysis on freshwater goldfish and selectively reversibly inhibited ACh-evoked currents in Xenopus oocytes expressing rat alpha3beta2 and alpha6alpha3beta2 nAChRs. Comparing the distinct primary structure with other functionally related alpha-conotoxins could indicate structural features in Lp1.1 that may be associated with its unique receptor recognition profile. PMID:18588930

  16. Acetylcholinesterase inhibitors used in treatment of Alzheimer's disease prevent glutamate neurotoxicity via nicotinic acetylcholine receptors and phosphatidylinositol 3-kinase cascade.

    PubMed

    Takada-Takatori, Yuki; Kume, Toshiaki; Sugimoto, Mitsuhiro; Katsuki, Hiroshi; Sugimoto, Hachiro; Akaike, Akinori

    2006-09-01

    We show here that donepezil, galanathamine and tacrine, therapeutic acetylcholinesterase inhibitors currently being used for treatment of Alzheimer's disease, protect neuronal cells in a time- and concentration-dependent manner from glutamate neurotoxicity that involves apoptosis. The neuroprotective effects were antagonized by mecamylamine, an inhibitor of nicotinic acetylcholine receptors (nAChRs). Dihydro-beta-erythroidine and methyllycaconitine, antagonists for alpha4-nAChR and alpha7-nAChR, respectively, antagonized the protective effect of donepezil and galanthamine, but not that of tacrine. Previous reports suggest the involvement of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway in the nicotine-induced neuroprotection. Inhibitors for a non-receptor type tyrosine kinase, Fyn, and janus-activated kinase 2, suppressed the neuroprotective effect of donepezil and galanthamine, but not that of tacrine. Furthermore, LY294002, a PI3K inhibitor, also suppressed the neuroprotective effect of donepezil and galanthamine, but not that of tacrine. The phosphorylation of Akt, an effector of PI3K, and the expression level of Bcl-2, an anti-apoptotic protein, increased with donepezil and galanthamine treatment, but not with tacrine treatment. These results suggest that donepezil and galanthamine prevent glutamate neurotoxicity through alpha4- and alpha7-nAChRs, followed by the PI3K-Akt pathway, and that tacrine protects neuronal cells through a different pathway. PMID:16762377

  17. Role of β4* Nicotinic Acetylcholine Receptors in the Habenulo-Interpeduncular Pathway in Nicotine Reinforcement in Mice.

    PubMed

    Harrington, Lauriane; Viñals, Xavier; Herrera-Solís, Andrea; Flores, Africa; Morel, Carole; Tolu, Stefania; Faure, Philippe; Maldonado, Rafael; Maskos, Uwe; Robledo, Patricia

    2016-06-01

    Nicotine exerts its psychopharmacological effects by activating the nicotinic acetylcholine receptor (nAChR), composed of alpha and/or beta subunits, giving rise to a diverse population of receptors with a distinct pharmacology. β4-containing (β4*) nAChRs are located almost exclusively in the habenulo-interpeduncular pathway. We examined the role of β4* nAChRs in the medial habenula (MHb) and the interpeduncular nucleus (IPN) in nicotine reinforcement using behavioral, electrophysiological, and molecular techniques in transgenic mice. Nicotine intravenous self-administration (IVSA) was lower in constitutive β4 knockout (KO) mice at all doses tested (7.5, 15, 30, and 60 μg/kg/infusion) compared with wild-type (WT) mice. In vivo microdialysis showed that β4KO mice have higher extracellular dopamine (DA) levels in the nucleus accumbens than in WT mice, and exhibit a differential sensitivity to nicotine-induced DA outflow. Furthermore, electrophysiological recordings in the ventral tegmental area (VTA) demonstrated that DA neurons of β4KO mice are more sensitive to lower doses of nicotine than that of WT mice. Re-expression of β4* nAChRs in IPN neurons fully restored nicotine IVSA, and attenuated the increased sensitivity of VTA DA neurons to nicotine. These findings suggest that β4* nAChRs in the IPN have a role in maintaining nicotine IVSA. PMID:26585290

  18. Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library.

    PubMed Central

    Balass, M; Heldman, Y; Cabilly, S; Givol, D; Katchalski-Katzir, E; Fuchs, S

    1993-01-01

    Monoclonal antibody (mAb) 5.5 is directed against the ligand-binding site of the nicotinic acetylcholine receptor. The epitope for this antibody is conformation-dependent, and the antibody does not react with synthetic peptides derived from the receptor sequence. We have identified a ligand peptide that mimics this conformation-dependent epitope from a phage-epitope library composed of filamentous phage displaying random hexapeptides. Among 38 positive phage clones, individually selected from the library, 34 positive clones carried the sequence Asp-Leu-Val-Trp-Leu-Leu (DLVWLL), 1 positive clone had the sequence Asp-Ile-Val-Trp-Leu-Leu (DIVWLL), and 3 positive clones expressed the sequence Leu-Ile-Glu-Trp-Leu-Leu (LIEWLL), none of which are significantly homologous with the nicotinic acetylcholine receptor alpha subunit sequence. All of these phages bind specifically to mAb 5.5. The synthetic peptide DLVWLL inhibits binding of mAb 5.5 to the related peptide-presenting phage and to the nicotinic acetylcholine receptor in a concentration-dependent manner; the IC50 value is of the order of 10(-4) M. Bioactivity of the peptide "mimotope" DLVWLL was demonstrated in vivo in hatched chickens by inhibition of the mAb 5.5 effect by the peptide. The neuromuscular block and myasthenia gravis-like symptoms that are induced in chicken by passive transfer of mAb 5.5 were specifically abolished by DLVWLL. This study shows the potential of a random peptide phage-epitope library for selecting a mimotope for an antibody that recognizes a folded form of the protein, where peptides from the linear amino acid sequence of the protein are not applicable. Images Fig. 5 PMID:7504273

  19. Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Dacosta, Corrie J. B.; Free, Chris R.; Sine, Steven M.

    2015-08-01

    α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homopentameric α7-acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state.

  20. Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes.

    PubMed

    Toyoshima, C; Unwin, N

    1988-11-17

    The nicotinic acetylcholine receptor belongs to a class of molecules that respond transiently to chemical stimuli by opening a water-filled channel through the cell membrane for cations to diffuse. This channel lies along the central axis delineated by a ring of five homologous, membrane-spanning subunits and thus has properties, such as conductance and ion selectivity, which depend on the profile created by the encircling subunits. Insight has been gained recently about the amino-acid residues implicated directly in the ion transport, and some information about the subunit configuration around the channel has come from electron microscopy studies of postsynaptic membranes crystallized in the form of flattened tubular vesicles. The resolution along the axis of the channel has, however, been limited by the restricted range of views obtainable. Here we report the structure of the channel at 17 A resolution, determined by three-dimensional image reconstruction from tubular vesicles having receptors organized in helical arrays across their surfaces. The helical symmetry is preserved by suspending the tubes in thin films of ice, and the receptors in such tubes can be seen from all angles, allowing the channel to be revealed clearly in relation to the lipid bilayer and the peripheral protein for the first time. PMID:2461515

  1. Photoaffinity labeling and quaternary structure of the acetylcholine receptor from Torpedo californica.

    PubMed Central

    Hucho, F; Layer, P; Kiefer, H R; Bandini, G

    1976-01-01

    Membrane fragments from electric tissue of Torpedo californica containing nicotinic acetylcholine receptor are composed of four different polypeptide chains with molecular weights of 40,000 (alpha), 48,000 (beta), 62,000 (gamma), and 66,000 (delta). The alpha and beta chains are still present in all and gamma and delta in some of the receptor preparations after Triton X-100 extraction and purification by affinity chromatography. All components of the receptor react covalently with the photoaffinity label 4-azido-2-nitrobenzyltrimethylammonium fluoroborate, the delta chain incorporating less of the reagent as compared to the alpha and beta chains. Agonists and antagonists containing a quaternary ammonium group protect all chains against the label; the principal neurotoxin from Naja naja siamensis protects the alpha chain only. We conclude that the alpha chain binds the neurotoxin from Naja naja, the alpha and beta chains are involved in the binding of ligands with quaternary ammonium groups, and the function of the gamma and delta chains remains to be determined. Images PMID:1066671

  2. Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

    PubMed Central

    daCosta, Corrie J. B.; Free, Chris R.; Sine, Steven M.

    2015-01-01

    α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homopentameric α7-acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state. PMID:26282895

  3. Identification, characterization, and regulation of a nicotinic acetylcholine receptor on bovine adrenal chromaffin cells in culture

    SciTech Connect

    Higgins, L.S.

    1988-01-01

    Synaptic input to bovine adrenal chromaffin cells is mediated by nicotinic acetylcholine receptors (AChRs) and results in secretion of catecholamines. Three probes previously shown to recognize AChRs on neurons were used to identify the AChR on bovine adrenal chromaffin cells in culture: monoclonal antibody mAb 35, a toxin that blocks receptor function, and the agonist nicotine. Competition for {sup 3}H-nicotine binding was used to measure the affinity of cholinergic ligands, and revealed the pharmacological profile expected for a neuronal-type AChR. At steady state the rate both of receptor insertion into and loss from the plasma membrane is about 3%/hour, resulting in a half-life in the surface of about 24 hours. Exposure to the anti-AChR antibody results in a loss of AChRs from the surface of the cells through a process that has the characteristics of antigenic modulation. The number of AChRs on the surface of the chromaffin cells can also be modulated by agonists and hormones, including glucocotricoids. Catecholamines, three peptides that may be secreted by chromaffin cells, and K{sup +}-induced secretion reduce agonist-induced catecholamine release by decreasing the number of AChRs, providing a mechanism for autoregulation.

  4. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  5. Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking

    PubMed Central

    Watty, Anke; Methfessel, Christoph; Hucho, Ferdinand

    1997-01-01

    Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an “unconventional allosteric protein” which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3′-dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly “freezes” the nAChR in a high agonist affinity/closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a high-affinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states. PMID:9223339

  6. The prototoxin LYPD6B modulates heteromeric α3β4-containing nicotinic acetylcholine receptors, but not α7 homomers.

    PubMed

    Ochoa, Vanessa; George, Andrew A; Nishi, Rae; Whiteaker, Paul

    2016-03-01

    Prototoxins are a diverse family of membrane-tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain-containing (LYPD)-6B on α3β4-, α3α5β4-, and α7-containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human α3, β4, and α5 (D and N variants) subunits were expressed in Xenopus laevis oocytes and tested with or without LYPD6B. We used the 2-electrode voltage-clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist-induced current (Imax), and time constant (τ) of desensitization. For β4-α3-α3-β4-α3 and β4-α3-β4-α3-α3, LYPD6B decreased EC50 from 631 to 79 μM, reduced Imax by at least 59%, and decreased τ. For β4-α3-α5D-β4-α3 and β4-α3-β4-α-α5D, LYPD6B decreased Imax by 63 and 32%, respectively. Thus, LYPD6B acted only on (α3)3(β4)2 and (α3)2(α5D)(β4)2 and did not affect the properties of (α3)2(β4)3, α7, or (α3)2(α5N)(β4)2 nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (α3)3(β4)2 nAChRs to ACh while reducing ACh-induced whole-cell currents. LYPD6B also negatively modulates α3β4 nAChRs that include the α5D common human variant, but not the N variant associated with nicotine dependence. PMID:26586467

  7. T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

    PubMed Central

    Manfredi, A A; Protti, M P; Dalton, M W; Howard, J F; Conti-Tronconi, B M

    1993-01-01

    We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected. Images PMID:7688757

  8. Activation of α7 nicotinic acetylcholine receptors protects astrocytes against oxidative stress-induced apoptosis: implications for Parkinson's disease.

    PubMed

    Liu, Yuan; Zeng, Xiaoning; Hui, Yujian; Zhu, Chenlei; Wu, Jie; Taylor, Devin H; Ji, Juan; Fan, Weimin; Huang, Zuhu; Hu, Jun

    2015-04-01

    Astrocytes have been implicated in the immune responses associated with Parkinson's disease (PD). Inhibition of astrocyte apoptosis is a novel strategy for the treatment of PD. Recent studies suggest that α7 nicotinic acetylcholine receptors (α7-nAChRs) expressed in glial cells are critical links between inflammation and neurodegeneration in PD. However, little is known about their contribution to astrocyte apoptosis during the development of this disorder. In the present study, we showed that nicotine exerts a protective effect on H2O2-induced astrocyte apoptosis and glial cell-derived neurotrophic factor (GDNF) downregulation, and this effect was abolished by an α7-nAChR-selective antagonist. The underlying mechanisms might involve alleviation of mitochondrial membrane potential loss, stabilization of the Bax/Bcl-2 balance, and inhibition of cleaved caspase-9 activity through α7-nAChR activation. Systemic administration of nicotine dramatically alleviated MPTP-induced symptoms, protected dopaminergic neurons against degeneration, inhibited astrocytes and microglia activation in the substantia nigra pars compacta (SNpc) and blocked the decrease of GDNF in the striatum by activating α7-nAChRs. Taken together these findings demonstrate, for the first time, that nicotine suppresses H2O2-induced astrocyte apoptosis via the mitochondrial pathway through the stimulation of α7-nAChRs. Targeting α7-nAChRs expressed in astrocytes may be a novel therapeutic strategy for the treatment of neurodegenerative disorders. PMID:25486621

  9. Targeting the Nicotinic Acetylcholine Receptors (nAChRs) in Astrocytes as a Potential Therapeutic Target in Parkinson's Disease.

    PubMed

    Jurado-Coronel, Juan Camilo; Avila-Rodriguez, Marco; Capani, Francisco; Gonzalez, Janneth; Moran, Valentina Echeverria; Barreto, George E

    2016-01-01

    Parkinson's disease (PD) is a relatively common disorder of the Central Nervous System (CNS), whose etiology is characterized by a selective and progressive degeneration of dopaminergic neurons, and the presence of Lewy bodies in the pars compacta of the substantia nigra, and gaping dopamine depletion in the striatum. Patients with this disease suffer from tremors, slowness of movements, gait instability, and rigidity. These patients may also present functional disability, reduced quality of life, and rapid cognitive decline. It has been shown that nicotine exerts beneficial effects in patients with PD and in in-vitro and in-vivo models of this disease. Astrocytes are an important component in the immune response associated with PD, and that nicotine might be able to inhibit the inflammation-related apoptosis of these cells, being this a potential strategy for PD treatment. This action of nicotine could be due mainly to activation of α7 nicotinic acetylcholine receptors (α7-nAChRs) expressed in glial cells. However, nicotine administration can protect dopaminergic neurons against degeneration by inhibiting astrocytes activation in the substantia nigra pars compacta (SNpc) and therefore reduce inflammation. Owing to the toxicity and capacity of nicotine to induce addiction, analogues of this substance have been designed and tested in various experimental paradigms, and targeting α7-nAChRs expressed in glial cells may be a novel therapeutic strategy for PD treatment. PMID:26972289

  10. The cholesterol dependence of activation and fast desensitization of the nicotinic acetylcholine receptor.

    PubMed Central

    Rankin, S E; Addona, G H; Kloczewiak, M A; Bugge, B; Miller, K W

    1997-01-01

    When nicotinic acetylcholine receptors are reconstituted into lipid bilayers lacking cholesterol, agonists no longer stimulate cation flux. The kinetics of this process are difficult to study because variations in vesicle morphology cause errors in flux measurements. We developed a new stopped-flow fluorescence assay to study activation independently of vesicle morphology. When receptors were rapidly mixed with agonist plus ethidium, the earliest fluorescence increase reported the fraction of channels that opened and their apparent rate of fast desensitization. These processes were absent when the receptor was reconstituted into dioleoylphosphatidylcholine or into a mixture of that lipid with dioleoylphosphatidic acid (12 mol%), even though a fluorescent agonist reported that resting-state receptors were still present. The agonist-induced channel opening probability increased with bilayer cholesterol, with a midpoint value of 9 +/- 1.7 mol% and a Hill coefficient of 1.9 +/- 0.69, reaching a plateau above 20-30 mol% cholesterol that was equal to the native value. On the other hand, the observed fast desensitization rate was comparable to that for native membranes from the lowest cholesterol concentration examined (5 mol%). Thus the ability to reach the open state after activation varies with the cholesterol concentration in the bilayer, whereas the rate of the open state to fast desensitized state transition is unaffected. The structural basis for this is unknown, but an interesting corollary is that the channels of newly synthesized receptors are not fully primed by cholesterol until they are inserted into the plasma membrane--a novel form of posttranslational processing. PMID:9370438

  11. 6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

    PubMed Central

    Kasheverov, Igor E.; Shelukhina, Irina V.; Kudryavtsev, Denis S.; Makarieva, Tatyana N.; Spirova, Ekaterina N.; Guzii, Alla G.; Stonik, Valentin A.; Tsetlin, Victor I.

    2015-01-01

    6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes. PMID:25775422

  12. 6-bromohypaphorine from marine nudibranch mollusk Hermissenda crassicornis is an agonist of human α7 nicotinic acetylcholine receptor.

    PubMed

    Kasheverov, Igor E; Shelukhina, Irina V; Kudryavtsev, Denis S; Makarieva, Tatyana N; Spirova, Ekaterina N; Guzii, Alla G; Stonik, Valentin A; Tsetlin, Victor I

    2015-03-01

    6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes. PMID:25775422

  13. Nicotinic Acetylcholine Receptors containing the α6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons

    PubMed Central

    Liu, Liwang; Zhao-Shea, Rubing; McIntosh, J. Michael; Tapper, Andrew R.

    2013-01-01

    Nicotine and alcohol are often co-abused suggesting a common mechanism of action may underlie their reinforcing properties. Both drugs acutely increase activity of ventral tegmental area (VTA) dopaminergic (DAergic) neurons, a phenomenon associated with reward behavior. Recent evidence indicates that nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels activated by ACh and nicotine, may contribute to ethanol-mediated activation of VTA DAergic neurons although the nAChR subtype(s) involved has not been fully elucidated. Here we show that expression and activation of nAChRs containing the α6 subunit contribute to ethanol-induced activation of VTA DAergic neurons. In wild-type (WT) mouse midbrain sections that contain the VTA, ethanol (50 or 100 mM) significantly increased firing frequency of DAergic neurons. In contrast, ethanol did not significantly increase activity of VTA DAergic neurons in mice that do not express CHRNA6, the gene encoding the α6 nAChR subunit (α6 knock-out (KO) mice). Ethanol-induced activity in WT slices was also reduced by pre-application of the α6 subtype-selective nAChR antagonist, α-conotoxin MII[E11A]. When co-applied, ethanol potentiated the response to ACh in WT DAergic neurons; whereas co-application of ACh and ethanol failed to significantly increase activity of DAergic neurons in α6 KO slices. Finally, pre-application of α-conotoxin MII[E11A] in WT slices reduced ethanol potentiation of ACh responses. Together our data indicate that α6-subunit containing nAChRs may contribute to ethanol activation of VTA DAergic neurons. These receptors are predominantly expressed in DAergic neurons and known to be critical for nicotine reinforcement, providing a potential common therapeutic molecular target to reduce nicotine and alcohol co-abuse. PMID:23811312

  14. Molecular-Dynamics Simulations of ELIC a Prokaryotic Homologue of the Nicotinic Acetylcholine Receptor

    SciTech Connect

    Cheng, Xiaolin; Ivanov, Ivaylo N; Wang, Hailong; McCammon, Jonathan

    2009-01-01

    The ligand-gated ion channel from Erwinia chrysanthemi (ELIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor (nAChR) that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. ELIC is similar to the nAChR in its primary sequence and overall subunit organization, but despite their structural similarity, it is not clear whether these two ligand-gated ion channels operate in a similar manner. Further, it is not known to what extent mechanistic insights gleaned from the ELIC structure translate to eukaryotic counterparts such as the nAChR. Here we use molecular-dynamics simulations to probe the conformational dynamics and hydration of the transmembrane pore of ELIC. The results are compared with those from our previous simulation of the human ?7 nAChR. Overall, ELIC displays increased stability compared to the nAChR, whereas the two proteins exhibit remarkable similarity in their global motion and flexibility patterns. The majority of the increased stability of ELIC does not stem from the deficiency of the models used in the simulations, and but rather seems to have a structural basis. Slightly altered dynamical correlation features are also observed among several loops within the membrane region. In sharp contrast to the nAChR, ELIC is completely dehydrated from the pore center to the extracellular end throughout the simulation. Finally, the simulation of an ELIC mutant substantiates the important role of F246 on the stability, hydration and possibly function of the ELIC channel.

  15. The value of acetylcholine receptor antibody in children with postural tachycardia syndrome.

    PubMed

    Li, Jiawei; Zhang, Qingyou; Liao, Ying; Zhang, Chunyu; Hao, Hongjun; Du, Junbao

    2015-01-01

    Postural tachycardia syndrome (POTS) is characterized by symptoms of orthostatic intolerance. Antibodies of acetylcholine receptor (AChR-ab) affect acetylcholine transmission between the ganglia and result in imbalance of the autonomic nervous system in POTS. This study was designed to analyze the clinical characteristics of POTS patients with AChR-ab positive and explore the value of AChR-ab in children with POTS. In 82 children with POTS, twenty patients (24.39%) were found as AChR-ab positive. Their clinical characteristics and hemodynamic responses to orthostatic changes were compared with the remaining 60 patients with negative AChR-ab. Symptoms of POTS children with AChR-ab positive were significantly severe than those of AChR-ab negative patients (p = 0.001). Preceding infection was predominant in patients with AChR-ab positive compared with that of patients with AChR-ab negative (p < 0.001). Syncope and fatigue were more common in the AChR-ab positive patients (p < 0.05). The change of upright heart rate was increased significantly in AChR-ab positive patients compared with AChR-ab negative cases (p = 0.013). Multiple logistic regression analysis revealed that preceding infection (OR 22.356, 95% CI 2.151-34.920), syncope (OR 11.570, 95% CI 2.098-63.810), and fatigue (OR 11.145, 95% CI 1.658-74.911) were independent risk factors for POTS with AChR-ab positive. In conclusion, POTS with positive AChR-ab was a heterogeneous disorder. Preceding infection, syncope and fatigue were their main clinical characteristics. PMID:25087056

  16. Channel properties of the purified acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayer membranes.

    PubMed Central

    Montal, M; Labarca, P; Fredkin, D R; Suarez-Isla, B A

    1984-01-01

    The electrophysiological properties of the cation channel of the purified nicotinic acetylcholine receptor (AChR) reconstituted in planar lipid bilayers were characterized. Single-channel currents were activated by acetylcholine, carbamylcholine and suberyldicholine. The single channel conductance (28 pS in 0.3 M NaCl) was ohmic and independent of the agonist. Single channel currents increased with Na+ concentration to a maximum conductance of 95 pS and showed a half-saturation point of 395 mM. The apparent ion selectivity sequence, derived from single-channel current recordings, is: NH+4 greater than Cs+ greater than Rb+ greater than or equal to Na+ Cl-, F-, SO2-(4). The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of at least two distinct open states. The time constants depend on the choice of agonist, being consistently longer for suberyldicholine than for carbamylcholine. Similar channel properties were recorded in bilayers formed from monolayers at the tip of patch pipets . Single-channel currents occur in paroxysms of channel activity followed by quiescent periods. This pattern is more pronounced as the agonist concentration increases, and is reflected in histograms of channel-opening frequencies. Computer simulations with a three-state model, consisting of two closed (unliganded and liganded) and one open state, do not resemble the recorded pattern of channel activity, especially at high agonist concentration. Inclusion of a desensitized liganded state reproduces the qualitative features of channel recordings. The occurrence of paroxysms of channel activity thus seems to result from the transit of AChR through its active conformation, from which it can open several times before desensitizing. PMID:6324900

  17. Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

    PubMed Central

    Krause, R M; Hamann, M; Bader, C R; Liu, J H; Baroffio, A; Bernheim, L

    1995-01-01

    1. Fusion of myogenic cells is important for muscle growth and repair. The aim of this study was to examine the possible involvement of nicotinic acetylcholine receptors (nAChR) in the fusion process of myoblasts derived from postnatal human satellite cells. 2. Acetylcholine-activated currents (ACh currents) were characterized in pure preparations of freshly isolated satellite cells, proliferating myoblasts, myoblasts triggered to fuse and myotubes, using whole-cell and single-channel voltage clamp recordings. Also, the effect of cholinergic agonists on myoblast fusion was tested. 3. No nAChR were observed in freshly isolated satellite cells. nAChR were first observed in proliferating myoblasts, but ACh current densities increased markedly only just before fusion. At that time most mononucleated myoblasts had ACh current densities similar to those of myotubes. ACh channels had similar properties at all stages of myoblast maturation. 4. The fraction of myoblasts that did not fuse under fusion-promoting conditions had no ACh current and thus resembled freshly isolated satellite cells. 5. The rate of myoblast fusion was increased by carbachol, an effect antagonized by alpha-bungarotoxin, curare and decamethonium, but not by atropine, indicating that nAChR were involved. Even though a prolonged exposure to carbachol led to desensitization, a residual ACh current persisted after several days of exposure to the nicotinic agonist. 6. Our observations suggest that nAChR play a role in myoblast fusion and that part of this role is mediated by the flow of ions through open ACh channels. Images Figure 1 Figure 2 Figure 3 PMID:8788942

  18. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    SciTech Connect

    Middleton, R.E.; Cohen, J.B. )

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.

  19. The novel small molecule α9α10 nicotinic acetylcholine receptor antagonist ZZ-204G is analgesic

    PubMed Central

    Holtman, Joseph R.; Dwoskin, Linda P.; Dowell, Cheryl; Wala, Elzbieta P.; Zhang, Zhenfa; Crooks, Peter A.; McIntosh, J. Michael

    2011-01-01

    Chronic pain is inadequately managed with currently available classes of analgesic drugs. Recently, peptide antagonists of the α9α10 nicotinic acetylcholine receptor were shown to be analgesic. The present study was conducted to characterize a novel small molecule, non-peptide antagonist at nicotinic receptors. The tetrakis-quaternary ammonium compound ZZ-204G was evaluated for functional activity on cloned nicotinic receptors expressed in Xenopus oocytes. In-vivo efficacy was assessed in rat models of tonic inflammatory pain (formalin test), neuropathic pain (chronic constriction nerve injury), and thermal nociception (tail flick test). ZZ-204G was an antagonist at nicotinic receptors inhibiting the α9α10 subtype with an IC50 of 0.51 (0.35–0.72) nM. Antagonist activity at other nicotinic subtypes (α1β1δε, α2β2, α2β4, α3β2, α3β4, α4β2, α4β4, α6/α3β2β3, α6/α3β4 and α7) was 10–1000-fold lower than at the α9α10 subtype. In competition binding assays, the ki of ZZ-204G at γ-aminobutyric acid(A), serotonin(3), γ-aminobutyric acid(B), κ- and μ-opioid receptors was 1000- to >10,000- fold lower than at α9α10 nicotinic receptors. Parenteral administration of ZZ-204G dose-dependently decreased nociceptive behaviors (paw flinches) in the formalin test and mechanical hyperalgesia in the chronic constriction nerve injury model of neuropathic pain. ZZ-204G was not antinociceptive in the tail flick assay. Results from the rotarod assay indicated that lower doses of ZZ-204G that were analgesic did not alter motor function. In summary, ZZ-204G represents a prototype small molecule antagonist for α9α10 nicotinic receptors and provides a novel molecular scaffold for analgesic agents with the potential to treat chronic inflammatory or neuropathic pain. PMID:21944926

  20. Analysis of neuronal nicotinic acetylcholine receptor α4β2 activation at the single-channel level.

    PubMed

    Carignano, Camila; Barila, Esteban Pablo; Spitzmaul, Guillermo

    2016-09-01

    The neuronal nicotinic acetylcholine receptor α4β2 forms pentameric proteins with two alternate stoichiometries. The high-sensitivity receptor is related to (α4)2(β2)3 stoichiometry while the low-sensitivity receptor to (α4)3(β2)2 stoichiometry. Both subtypes share two binding sites at the α4((+))/β2((-)) interface with high affinity for agonists. (α4)3(β2)2 has an additional binding site at the α4((+))/α4((-)) interface with low affinity for agonists. We investigated activation kinetics of both receptor subtypes by patch-clamp recordings of single-channel activity in the presence of several concentrations of acetylcholine (0.5 to 300μM). We used kinetic software to fit these data with kinetic models. We found that the high-sensitivity subtype correlates with the low-conductance channel (g-70=29pS) and does not activate with high efficacy. On the contrary, the low-sensitivity subtype correlated with a high-conductance channel (g-70=44pS) and exhibited higher activation efficacy. Opening events of individual nAChRs at high agonist concentrations occurred in clusters, which allowed us to determine kinetic constants for the activation of the triliganded receptor. Our kinetic modeling identified an intermediate state, between resting and open conformation of the receptor. Binding of the third molecule increases the efficacy of receptor activation by favoring the transition between resting and intermediate state around 18 times. The low rate for this transition in the diliganded receptor explains the action of acetylcholine as partial agonist when it binds to the high-affinity sites. The presence of the third binding site emerges as a potent modulator of nicotinic receptor α4β2 activation which may display different functions depending on agonist concentration. PMID:27233449

  1. Activation of α2A-Containing Nicotinic Acetylcholine Receptors Mediates Nicotine-Induced Motor Output in Embryonic Zebrafish

    PubMed Central

    Menelaou, Evdokia; Udvadia, Ava J.; Tanguay, Robert L.; Svoboda, Kurt R.

    2014-01-01

    It is well established that cholinergic signaling has critical roles during central nervous system development. In physiological and behavioral studies, activation of nicotinic acetylcholine receptors has been implicated in mediating cholinergic signaling. In developing spinal cord, cholinergic transmission is associated with neural circuits responsible for producing locomotor behaviors. In this study, we investigated the expression pattern of the α2A nAChR subunit as evidence from others suggested it could be expressed by spinal neurons. In situ hybridization and immunohistochemistry revealed that the α2A nAChR subunits are expressed in spinal Rohon-Beard (RB) neurons and olfactory sensory neurons in young embryos. In order to examine the functional role of the α2A nAChR subunit during embryogenesis, we blocked its expression using antisense modified oligonucleotides. Blocking the expression of α2A nAChR subunits had no effect on spontaneous motor activity. However, it did alter the embryonic nicotine-induced motor output. This reduction in motor activity was not accompanied by defects in neuronal and muscle elements associated with the motor output. Moreover, the anatomy and functionality of RB neurons was normal even in the absence of the α2A nAChR subunit. Thus, we propose that α2A-containing nAChR are dispensable for normal RB development. However, in the context of nicotine-induced motor output, α2A-containing nAChRs on RB neurons provide the substrate that nicotine acts upon to induce the motor output. These findings also indicate that functional neuronal nAChRs are present within spinal cord at the time when locomotor output in zebrafish first begins to manifest itself. PMID:24738729

  2. Functional Upregulation of α4* Nicotinic Acetylcholine Receptors in VTA GABAergic Neurons Increases Sensitivity to Nicotine Reward

    PubMed Central

    Ngolab, Jennifer; Liu, Liwang; Zhao-Shea, Rubing; Gao, Guangping; Gardner, Paul D.

    2015-01-01

    Chronic nicotine exposure increases sensitivity to nicotine reward during a withdrawal period, which may facilitate relapse in abstinent smokers, yet the molecular neuroadaptation(s) that contribute to this phenomenon are unknown. Interestingly, chronic nicotine use induces functional upregulation of nicotinic acetylcholine receptors (nAChRs) in the mesocorticolimbic reward pathway potentially linking upregulation to increased drug sensitivity. In the ventral tegmental area (VTA), functional upregulation of nAChRs containing the α4 subunit (α4* nAChRs) is restricted to GABAergic neurons. To test the hypothesis that increased functional expression of α4* nAChRs in these neurons modulates nicotine reward behaviors, we engineered a Cre recombinase-dependent gene expression system to selectively express α4 nAChR subunits harboring a “gain-of-function” mutation [a leucine mutated to a serine residue at the 9′ position (Leu9′Ser)] in VTA GABAergic neurons of adult mice. In mice expressing Leu9′Ser α4 nAChR subunits in VTA GABAergic neurons (Gad2VTA:Leu9′Ser mice), subreward threshold doses of nicotine were sufficient to selectively activate VTA GABAergic neurons and elicit acute hypolocomotion, with subsequent nicotine exposures eliciting tolerance to this effect, compared to control animals. In the conditioned place preference procedure, nicotine was sufficient to condition a significant place preference in Gad2VTA:Leu9′Ser mice at low nicotine doses that failed to condition control animals. Together, these data indicate that functional upregulation of α4* nAChRs in VTA GABAergic neurons confers increased sensitivity to nicotine reward and points to nAChR subtypes specifically expressed in GABAergic VTA neurons as molecular targets for smoking cessation therapeutics. PMID:26041923

  3. A novel α-conotoxin MII-sensitive nicotinic acetylcholine receptor modulates [(3) H]-GABA release in the superficial layers of the mouse superior colliculus.

    PubMed

    McClure-Begley, Tristan D; Wageman, Charles R; Grady, Sharon R; Marks, Michael J; McIntosh, J Michael; Collins, Allan C; Whiteaker, Paul

    2012-07-01

    Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [(125) I]-Epibatidine competition-binding studies revealed that the α3β2* and α6β2* nicotinic subtype-selective peptide α-conotoxin MII-blocked binding to 40 ± 5% of SuSC nAChRs. Acetylcholine-evoked [(3) H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [(3) H]-GABA release is inhibited by α-conotoxin MII. However, the highly α6β2*-subtype-selective α-conotoxin PIA did not affect [(3) H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [(3) H]-GABA release is entirely β2 subunit-dependent. α4 subunit deletion decreased total function by >90%, and eliminated α-conotoxin MII-resistant release. ACh-stimulated SuSC [(3) H]-GABA release was unaffected by β3, α5 or α6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, α-conotoxin MII-sensitive α3α4β2 nAChR. The remaining α-conotoxin MII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via α4β2* subtype nAChRs. PMID:22506481

  4. Thrombin action decreases acetylcholine receptor aggregate number and stability in cultured mouse myotubes.

    PubMed

    Davenport, R W; Lanuza, M; Kim, S; Jia, M; Snyder, E; Nelson, P G

    2000-08-30

    Neurons develop and make very stable, long-term synaptic connections with other nerve cells and with muscle. Synaptic stability at the neuromuscular junction changes over development in that a proliferation of synaptic input are made to individual myotubes and synapses from all but one neuron are lost during development. In an established co-culture paradigm in which spinal motoneurons synaptically contact myotubes, thrombin and associated protease inhibitors have been shown to affect the loss of functional synaptic contacts [6]. Evidence has not been provided which clearly demonstrate whether protease/protease inhibitors affect either the pre- or postsynaptic terminal, or both. In an effort to determine whether these reagents directly affect postsynaptic receptors on myotubes, myotubes were cultured in the absence of neurons and the spontaneous presence and stability of aggregates of acetylcholine receptors (AChR) in control and thrombin-containing media were evaluated. In dishes fixed after treatment and in dishes in which individual aggregates were observed live, thrombin action appeared to increase loss of AChR aggregates over time. Hirudin, a specific inhibitor of the thrombin protease, diminished this loss. Neither reagent affected the overall incorporation or degradation of AChR; therefore, it appears these protease/protease inhibitors affect the state of AChR aggregation. PMID:10960680

  5. Cloning and mapping of the mouse {alpha}7-neuronal nicotinic acetylcholine receptor

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1995-03-20

    We report the isolation of cDNA clones for the mouse {alpha}7 neuronal nicotinic acetylcholine receptor subunit (gene symbol Acra7), the only nicotinic receptor subunit known to bind a-bungarotoxin in mammalian brain. This gene may have relevance to nicotine sensitivity and to some electrophysiologic findings in schizophrenia. The mouse {alpha}7 subunit gene encodes a protein of 502 amino acids with substantial identity to the rat (99.6%), human (92.8%), and chicken (87.5%) amino acid sequences. The {alpha}7 gene was mapped to mouse chromosome 7 near the p locus with the following gene order from proximal to distal: Myod1-3.5 {+-}1.7 cM-Gas2-0.9 cM {+-} 0.9 cM-D7Mit70-1.8 {+-} 1.2 cM- Acra7-4.4 {+-}1.0 cM-Hras1-ps11/Igf1r/Snrp2a. The human gene was confirmed to map to the homologous region of human chromosome 15q13-q14. 26 refs., 3 figs.

  6. Electron microscopy of complexes of isolated acetylcholine receptor, biotinyl-toxin, and avidin

    SciTech Connect

    Holtzman, E.; Wise, D.; Wall, J.; Karlin, A.

    1982-01-01

    The principal curarimimetic toxin of Naja naja siamensis derivatized with biothinyl groups binds specifically both to acetylcholine receptor, isolated from Torpedo californica electric tissue, and to avidin. Isolated complexes of receptor monomer or dimer, biotinyl-toxin, and avidin were negatively stained and examined in the scanning transmission electron microscope. We measured the angle made by the radius to each avidin bound at the periphery of a monomeric unit in dimer to the axis connecting the centers of the monomers, starting at the crosslink between the monomers. We infer from the distribution of these angles that one toxin binding site is located in the range of 45/sup 0/ to 85/sup 0/ and another at about 100/sup 0/ further from the crosslink between the monomers. Because it is known that there are two toxin binding sites per monomer, associated with the two ..cap alpha.. chains, the bound avidins presumably point to portions of the ..cap alpha.. chains, indicating their positions relative to that portion of the delta chain located at the crosslink between monomers in dimer.

  7. Involvement of calpains in the destabilization of the acetylcholine receptor clusters in rat myotubes.

    PubMed

    Kim, S; Nelson, P G

    2000-01-01

    The effects of calpain inhibitors on the total number of acetylcholine receptors (AChRs) on cultured rat myotubes and on the stability of AChR clusters in these myotubes were investigated. The degradation rate of total AChRs labeled with (125)I-alpha-bungarotoxin was assessed from radioactivity remaining in the myotubes as a function of time. Treatment with calpain inhibitors resulted in a two- to three-fold increase in the half-life of total AChRs. Incubation with these inhibitors produced 40% increases in intracellular AChRs but no major changes in surface AChRs, indicating that the increased AChR half-life is due to intracellular accumulation. The rate loss of AChRs from the clusters was assessed by measuring the loss of fluorescence intensity in rhodaminated-alpha-bungarotoxin-labeled clusters with time. Treatment with calpain inhibitors resulted in twofold increases in cluster half-life. Thus, there was generally no change in total surface receptors with the calpain inhibitors, whereas cluster half-life was substantially increased. Furthermore, with a low dose of calpeptin there was no change in turnover of total cellular AChRs, whereas cluster half-life was doubled. Taken together, these results suggest that the increased half-life of clusters produced by the calpain inhibitors may be due to retardation of the lateral movement from AChRs in the clusters. PMID:10623898

  8. Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors

    PubMed Central

    Takahashi, Masazumi; Kubo, Tai; Mizoguchi, Akira; Carlson, C. George; Endo, Katsuaki; Ohnishi, Katsunori

    2002-01-01

    In mammals, two combinations of muscle nicotinic acetylcholine receptors (AChRs) are used: α2βγδ (γ-AChR) or α2βɛδ (ɛ-AChR). After birth, γ-AChRs are replaced by ɛ-AChRs (γ/ɛ-switch). The two receptors have different conductances and open times. During perinatal period, the long open time γ-AChRs generate random myofiber action potentials from uniquantal miniature end-plate potentials (mEPPs). ɛ-AChRs are suitable for strong adult muscle activities. Since the effect of the γ/ɛ-switch on neuromuscular development was unclear, despite the many differences in channel characteristics, we carried out this study to generate γ-subunit-deficient mice. Homozygotes born alive survived for 2 days in a stable condition, and were able to move their forelimbs. Endplate AChRs included ɛ-subunits, and muscle fibers had multiple neuromuscular junctions. Both pre- and postsynapses were abnormal and spontaneous action potentials generated from mEPPs were totally absent. Results suggest a requirement for γ-AChRs in mediating synaptically-induced action potential activity critical for neuromuscular development. PMID:12101101

  9. Recycling of Acetylcholine Receptors at Ectopic Postsynaptic Clusters Induced by Exogenous Agrin in Living Rats

    PubMed Central

    Brenner, Hans Rudolf; Akaaboune, Mohammed

    2014-01-01

    During the development of the neuromuscular junction, motor axons induce the clustering of acetylcholine receptors (AChRs) and increase their metabolic stability in the muscle membrane. Here, we asked whether the synaptic organizer agrin might regulate the metabolic stability and density of AChRs by promoting the recycling of internalized AChRs, which would otherwise be destined for degradation, into synaptic sites. We show that at nerve-free AChR clusters induced by agrin in extrasynaptic membrane, internalized AChRs are driven back into the ectopic synaptic clusters where they intermingle with pre-existing and new receptors. The extent of AChR recycling depended on the strength of the agrin stimulus, but not on the development of junctional folds, another hallmark of mature postsynaptic membranes. In chronically denervated muscles, in which both AChR stability and recycling are significantly decreased by muscle inactivity, agrin maintained the amount of recycled AChRs at agrin-induced clusters at a level similar to that at denervated original endplates. In contrast, AChRs did not recycle at agrin-induced clusters in C2C12 or primary myotubes. Thus, in muscles in vivo, but not in cultured myotubes, neural agrin promotes the recycling of AChRs and thereby increases their metabolic stability. PMID:25093969

  10. Autoradiographic localization of nicotinic acetylcholine receptors in the brain of the zebra finch (Poephila guttata)

    SciTech Connect

    Watson, J.T.; Adkins-Regan, E.; Whiting, P.; Lindstrom, J.M.; Podleski, T.R.

    1988-08-08

    We have localized nicotinic acetylcholine receptors in the zebra finch brain by using three 125I-labelled ligands: alpha bungarotoxin and two monoclonal antibodies to neuronal nicotinic receptors. Unfixed brains from intact adult male and female zebra finches were prepared for in vitro autoradiography. Low-resolution film autoradiograms and high-resolution emulsion autoradiograms were prepared for each of the three ligands. The major brain structures that bind all three of the ligands are hippocampus; hyperstriatum dorsalis; hyperstriatum ventralis; nucleus lentiformis mesencephali; nucleus pretectalis, some layers of the optic tectum; nucleus mesencephalicus lateralis; pars dorsalis; locus ceruleus; and all cranial motor nuclei except nucleus nervi hypoglossi. The major structures labelled only by (125I)-alpha bungarotoxin binding included hyperstriatum accessorium and the nuclei: preopticus medialis, medialis hypothalami posterioris, semilunaris, olivarius inferior, and the periventricular organ. Of the song control nuclei, nucleus magnocellularis of the anterior neostriatum; hyperstriatum ventralis, pars caudalis; nucleus intercollicularis; and nucleus hypoglossus were labelled. The binding patterns of the two antibodies were similar to one another but not identical. Both labelled nucleus spiriformis lateralis and nucleus geniculatus lateralis, pars ventralis especially heavily and also labelled the nucleus habenula medialis; nucleus subpretectalis; nucleus isthmi, pars magnocellularis; nucleus reticularis gigantocellularis; nucleus reticularis lateralis; nucleus tractus solitarii; nucleus vestibularis dorsolateralis; nucleus vestibularis lateralis; nucleus descendens nervi trigemini; and the deep cerebellar nuclei.

  11. The Actions of Piperidine Alkaloids at Fetal Muscle-Type and Autonomic-Type Nicotinic Acetylcholine Receptors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Piperidine alkaloids are found in many species of plants including Conium maculatum, Nicotiana spp., and Lupinus spp. A pharmacodynamic comparison was made of the alkaloids ammodendrine, anabasine, anabaseine, and coniine in; SH-SY5Y cells which express autonomic-type nicotinic acetylcholine recept...

  12. Evaluating the role of the alpha-7 nicotinic acetylcholine receptor in the pathophysiology and treatment of schizophrenia

    PubMed Central

    YOUNG, Jared W; GEYER, Mark A

    2013-01-01

    The group of schizophrenia disorders affects approximately 1% of the population and has both genetic and environmental etiologies. Sufferers report various behavioral abnormalities including hallucinations and delusions (positive symptoms), reduced joy and amotivation (negative symptoms), plus inattention and poor learning (cognitive deficits). Despite the heterogeneous symptoms experienced, most patients smoke. The self-medication hypothesis posits that patients smoke to alleviate symptoms, consistent with evidence for nicotine-induced enhancement of cognition. While nicotine acts on multiple nicotinic acetylcholine receptors (nAChRs), the primary target of research is often the homomeric α7 nAChR. Given genetic linkages between schizophrenia and this receptor, its association with P50 sensory gating deficits, and its reduced expression in post-mortem brains, many have attempted to develop α7 nAChR ligands for treating schizophrenia. Recent evidence that ligands can be orthosteric agonists or positive allosteric modulators (PAMs) has revitalized the hope for treatment discovery. Herein, we present evidence regarding: 1) Pathophysiological alterations of α7 nAChRs that might occur in patients; 2) Mechanistic evidence for the normal action of α7 nAChRs; 3) Preclinical studies using α7 nAChR orthosteric agonists and type I/II PAMs; and 4) Where successful translational testing has occurred for particular compounds, detailing what is still required. We report that the accumulating evidence is positive, but that greater work is required using positron emission tomography to understand current alterations in α7 nAChR expression and their relationship to symptoms. Finally, cross-species behavioral tasks should be used more regularly to determine the predictive efficacy of treatments. PMID:23856289

  13. L-theanine inhibits nicotine-induced dependence via regulation of the nicotine acetylcholine receptor-dopamine reward pathway.

    PubMed

    Di, Xiaojing; Yan, Jingqi; Zhao, Yan; Chang, Yanzhong; Zhao, Baolu

    2012-12-01

    In this study, the inhibitory effect of L-theanine, an amino acid derivative of tea, on the rewarding effects of nicotine and its underlying mechanisms of action were studied. We found that L-theanine inhibited the rewarding effects of nicotine in a conditioned place preference (CPP) model of the mouse and reduced the excitatory status induced by nicotine in SH-SY5Y cells to the same extent as the nicotine receptor inhibitor dihydro-beta-erythroidine (DHβE). Further studies using high performance liquid chromatography, western blotting and immunofluorescence staining analyses showed that L-theanine significantly inhibited nicotine-induced tyrosine hydroxylase (TH) expression and dopamine production in the midbrain of mice. L-theanine treatment also reduced the upregulation of the α(4), β(2) and α(7) nicotine acetylcholine receptor (nAChR) subunits induced by nicotine in mouse brain regions that related to the dopamine reward pathway, thus decreasing the number of cells that could react to nicotine. In addition, L-theanine treatment inhibited nicotine-induced c-Fos expression in the reward circuit related areas of the mouse brain. Knockdown of c-Fos by siRNA inhibited the excitatory status of cells but not the upregulation of TH induced by nicotine in SH-SY5Y cells. Overall, the present study showed that L-theanine reduced the nicotine-induced reward effects via inhibition of the nAChR-dopamine reward pathway. These results may offer new therapeutic strategies for treatment of tobacco addiction. PMID:23233221

  14. α6β2*-subtype nicotinic acetylcholine receptors are more sensitive than α4β2*-subtype receptors to regulation by chronic nicotine administration

    PubMed Central

    Marks, MJ; Grady, SR; Salminen, O; Paley, MA; Wageman, CR; McIntosh, JM; Whiteaker, P

    2014-01-01

    Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where * indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*-nAChR are downregulated following chronic nicotine exposure (unlike other subtypes that have been investigated – most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose-responses and quantitative ligand-binding autoradiography were used to define nicotine sensitivity of changes in α4β2*-nAChR and α6β2*-nAChR expression. α6β2*-nAChR downregulation by chronic nicotine exposure in dopaminergic and optic-tract nuclei was ≈three-fold more sensitive than upregulation of α4β2*-nAChR. In contrast, nAChR-mediated [3H]-dopamine release from dopamine-terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, while dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR-mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [3H]-DA release are primarily due to changes in nAChR, rather than in dopaminergic, function. PMID:24661093

  15. Structural characteristics of the recognition site for cholinergic ligands in the nicotinic acetylcholine receptor from squid optical ganglia

    SciTech Connect

    Plyashkevich, Yu.G.; Demushkin, V.P.

    1986-01-20

    The influence of chemical modification on the parameters of the binding of cholinergic ligands by the nicotinic acetylcholine receptor of squid optical ganglia was investigated. The presence of two subpopulations of recognition sites, differing in the composition of the groups contained in them, was detected. It was established with high probability that subpopulation I contains arginine and tyrosine residues and a carboxyl group while subpopulation II contains an amino group, a thyrosine residue, and a carboxyl group. Moreover, in both subpopulations there is an amino group important only for the binding of tubocurarin. On the basis of the results obtained, a model of the recognition sites for cholinergic ligands of the nicotinic acetylcholine receptor of squid optical ganglia is proposed.

  16. Polymerase chain reaction-based diagnosis of rhabdomyosarcomas: comparison of fetal type acetylcholine receptor subunits and myogenin.

    PubMed

    Gattenlöhner, S; Müller-Hermelink, H K; Marx, A

    1998-06-01

    The diagnosis of rhabdomyosarcoma (RMS) is usually straight-forward when light microscopy and immunohistochemistry are used. However, tumors that exhibit a low degree of differentiation and small biopsies can lead to confusion. In such patients and for the detection of minimal (residual) disease, a polymerase chain reaction (PCR)-based approach would be a valuable diagnostic adjunct. This type of approach would be highly sensitive and should be free from the risk for contamination of the tumor sample with normal tissue. Because myogenin and the alpha and gamma subunit of the fetal type acetylcholine receptor (AChR) are specific immunohistochemical markers for RMS, their expression on the mRNA level in RMS, other childhood and adult tumors, and normal tissues was studied. Although the sensitivity of both approaches was 100% in embryonal and alveolar RMS, detection of myogenin mRNA was not specific for RMS but occurred in normal muscle and the majority of the other normal tissues and childhood tumors. Conversely, detection of fetal AChR mRNA as defined by an alpha/tau ratio of < 1 was encountered only in RMS and denervated muscle. The authors conclude that mRNA of the fetal type AChR but not myogenin is a highly specific and sensitive target for the PCR-based diagnosis of RMS. PMID:9836066

  17. At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor.

    PubMed

    Otvos, Reka A; Mladic, Marija; Arias-Alpizar, Gabriela; Niessen, Wilfried M A; Somsen, Govert W; Smit, August B; Kool, Jeroen

    2016-06-01

    The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel expressed in different regions of the central nervous system (CNS). The α7-nAChR has been associated with Alzheimer's disease, epilepsy, and schizophrenia, and therefore is extensively studied as a drug target for the treatment of these diseases. Important sources for new compounds in drug discovery are natural extracts. Since natural extracts are complex mixtures, identification of the bioactives demands the use of analytical techniques to separate a bioactive from inactive compounds. This study describes screening methodology for identifying bioactive compounds in mixtures acting on the α7-nAChR. The methodology developed combines liquid chromatography (LC) coupled via a split with both an at-line calcium (Ca(2+))-flux assay and high-resolution mass spectrometry (MS). This allows evaluation of α7-nAChR responses after LC separation, while parallel MS enables compound identification. The methodology was optimized for analysis of agonists and positive allosteric modulators, and was successfully applied to screening of the hallucinogen mushroom Psilocybe Mckennaii The crude mushroom extract was analyzed using both reversed-phase and hydrophilic interaction liquid chromatography. Matching retention times and peak shapes of bioactives found with data from the parallel MS measurements allowed rapid pinpointing of accurate masses corresponding to the bioactives. PMID:26738519

  18. Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor.

    PubMed

    Kimura, Kumi; Tanida, Mamoru; Nagata, Naoto; Inaba, Yuka; Watanabe, Hitoshi; Nagashimada, Mayumi; Ota, Tsuguhito; Asahara, Shun-ichiro; Kido, Yoshiaki; Matsumoto, Michihiro; Toshinai, Koji; Nakazato, Masamitsu; Shibamoto, Toshishige; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2016-03-15

    Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity. PMID:26947072

  19. Arecoline inhibits and destabilizes agrin-induced acetylcholine receptor cluster formation in C2C12 myotubes.

    PubMed

    Chang, Yung-Fu; Liu, Ting-Yuan; Liu, Shao-Tung

    2013-10-01

    Areca nut (Areca catechu) is chewed as a medical and psychoactive food by roughly 10% of the world population. Areca nut chewing may lead to low birth weight, premature delivery and impaired muscle development. Our previous study showed that arecoline, a major alkaloid in the areca nut, inhibited the myogenic differentiation of C2C12 myoblastic cells. The clustering of acetylcholine receptors (AChRs) in the postsynaptic membrane at the neuromuscular junction (NMJ) by agrin, a signaling protein released by motor neurons, is critical for the development of functional muscles. Here, we further investigate whether arecoline affects the AChR clustering using cultured C2C12 myotubes. Rhodamine-conjugated α-bungarotoxin was used to detect the presence of AChR clusters. Our results showed that arecoline inhibited the formation of agrin-induced AChR clusters and destabilized agrin-induced or spontaneous AChR cluster formation. In addition, arecoline inhibited the expression of myogenin in C2C12 myotubes. These results shed light on the important role of arecoline on the detrimental effect of areca nut to muscle development. PMID:23933062

  20. Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn–plectin 1f complexes

    PubMed Central

    Mihailovska, Eva; Raith, Marianne; Valencia, Rocio G.; Fischer, Irmgard; Banchaabouchi, Mumna Al; Herbst, Ruth; Wiche, Gerhard

    2014-01-01

    Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuromuscular junctions (NMJs) in patients suffering from epidermolysis bullosa simplex (EBS)-muscular dystrophy (MS) with myasthenic syndrome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells. We found plectin isoform 1f (P1f) to bridge AChRs and IFs via direct interaction with the AChR-scaffolding protein rapsyn in an isoform-specific manner; forced expression of P1f in plectin-deficient cells rescued both compromised AChR clustering and IF network anchoring. In conditional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsynaptic membrane infoldings and disorganization of the NMJ microenvironment, including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion. PMID:25318670

  1. The Neuronal Nicotinic Acetylcholine Receptors α4* and α6* Differentially Modulate Dopamine Release in Mouse Striatal Slices

    PubMed Central

    Meyer, Erin L.; Yoshikami, Doju; McIntosh, J. Michael

    2008-01-01

    Striatal dopamine plays a major role in the regulation of motor coordination and in the processing of salient information. We used voltammetry to monitor dopamine-release evoked by electrical stimulation in striatal slices, where interneurons continuously release acetylcholine. Use of the α6-selective antagonist α-conotoxin MII[E11A] and α4 knockout mice enabled identification of two populations of dopaminergic fibers. The first population had a low action potential threshold, and action potential-evoked dopamine-release from these fibers was modulated by α6. The second population had a higher action potential threshold, and only α4(non-α6) modulated action potential-evoked dopamine-release. Striatal dopaminergic neurons fire in both tonic and phasic patterns. When stimuli were applied in a train to mimic phasic firing, more dopamine-released was observed in α4 knockout vs. wildtype mice. Furthermore, block of α4(non-α6), but not of α6, increased dopamine release evoked by a train. These results indicate that there are different classes of striatal dopaminergic fibers that express different subtypes of nicotinic receptors. PMID:18248619

  2. Roles of Amino Acids and Subunits in Determining the Inhibition of Nicotinic Acetylcholine Receptors by Competitive Antagonists

    PubMed Central

    Dilger, James P.; Vidal, Ana Maria; Liu, Man; Mettewie, Claire; Suzuki, Takahiro; Pham, Anh; Demazumder, Deeptankar

    2008-01-01

    Background Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the α–δ and α–ε subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the α–ε interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. Methods HEK293 cells were transfected with wild-type or mutant (αY198F, εD59A, εD59N, εD173A, εD173N, δD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcho-line in the absence and presence of antagonist. Concentration–response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. Results Effects of mutations were antagonist specific. αY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to εD173 mutations. εD59 mutations had large effects on metocurine and cisatracurium. δD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two δ subunits but no ε subunit. Differences in IC50 arose from differences in both dissociation and association rates. Conclusion Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the α–ε interface. Pancuronium, vecuronium, and cisatracurium were selective for the α–δ interface. This suggests the possibility of synergistic

  3. Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

    SciTech Connect

    Newsom-Davis, J.; Willcox, N.; Calder, L.

    1981-11-26

    We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases.

  4. Modal affinities of endplate acetylcholine receptors caused by loop C mutations

    PubMed Central

    Vij, Ridhima; Purohit, Prasad

    2015-01-01

    The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability (“modes”). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5–β5′ linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity. PMID:26503719

  5. Positive allosteric modulators as an approach to nicotinic acetylcholine receptor- targeted therapeutics: advantages and limitations

    PubMed Central

    Williams, Dustin K.; Wang, Jingyi; Papke, Roger L.

    2011-01-01

    Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues. PMID:21575610

  6. Multiple Pharmacophores for the Selective Activation of Nicotinic α7-Type Acetylcholine Receptors

    PubMed Central

    Horenstein, Nicole A.; Leonik, Fedra M.; Papke, Roger L.

    2010-01-01

    The activation of heteromeric and homomeric nicotinic acetylcholine receptors was studied in Xenopus laevis oocytes to identify key structures of putative agonist molecules associated with the selective activation of homomeric α7 receptors. We observed that selectivity between α7 and α4β2 was more readily obtained than selectivity between α7 and α3β4. Based on structural comparisons of previously characterized selective and nonselective agonists, we hypothesize at least three chemical motifs exist that, when present in molecules containing an appropriate cationic center, could be associated with the selective activation of α7 receptors. We identify the three distinct structural motifs based on prototypical drugs as the choline motif, the tropane motif, and the benzylidene motif. The choline motif involves the location of an oxygen-containing polar group such as a hydroxyl or carbonyl separated by two carbons from the charged nitrogen. The tropane motif provides α7-selectivity based on the addition of multiple small hydrophobic groups positioned away from the cationic center in specific orientations. We show that this motif can convert the nonselective agonists quinuclidine and ethyltrimethyl-ammonium to the α7-selective analogs methyl-quinuclidine and diethyldimethyl-ammonium, respectively. We have shown previously that the benzylidene group of 3–2,4, dimethoxy-benzylidene anabaseine (GTS-21) converts anabaseine into an α7-selective agonist. The benzylidene motif was also applied to quinuclidine to generate another distinct family of α7-selective agonists. Our results provide insight for the further development of nicotinic therapeutics and will be useful to direct future experiments with protein structure-based modeling and site-directed mutagenesis. PMID:18768388

  7. Nicotinic acetylcholine receptors contribute to learning-induced metaplasticity in the hippocampus.

    PubMed

    Becker, Benjamin; Klein, Eva M; Striepens, Nadine; Mihov, Yoan; Schlaepfer, Thomas E; Reul, Juergen; Goossens, Liesbet; Schruers, Koen; Kendrick, Keith M; Hurlemann, René

    2013-07-01

    Hippocampal learning is thought to induce metaplasticity, which can facilitate subsequent learning. Administered at single low doses, the N-methyl-d-aspartate-type glutamate receptor antagonist memantine predominantly blocks α7 nicotinic acetylcholine receptors (α7 nAChRs). Placebo-controlled administration of a single low dose of memantine in a pharmaco-fMRI experiment may thus help characterize the role of α7 nAChRs in hippocampal metaplasticity. We hypothesized that if α7 nAChRs contribute to learning-induced metaplasticity in the hippocampus, blockade of these receptors with low-dose memantine would selectively interfere with a facilitation of subsequent learning without impairing hippocampal learning per se. To specifically test this hypothesis, we devised a randomized controlled trial in which healthy volunteers were administered a 20-mg single oral dose of memantine or placebo and scanned on three subsequent runs of a hippocampal learning task. Our results indicate no discrepancies in behavioral learning between low-dose memantine- and placebo-treated participants in the first and second run of this task. In the third run, however, only the placebo-treated group showed facilitated behavioral learning, an effect paralleled by decreased neural responses in the hippocampal cornu ammonis region. Our findings suggest that blockade of α7 nAChRs selectively interfered with a learning-induced facilitation of subsequent learning while leaving unimpaired hippocampal learning per se. Taken together, our results provide support for a relevant contribution of α7 nAChRs to learning-associated metaplasticity in the hippocampus. PMID:23469888

  8. The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera

    PubMed Central

    Jones, Andrew K.; Raymond-Delpech, Valerie; Thany, Steeve H.; Gauthier, Monique; Sattelle, David B.

    2006-01-01

    Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission and play roles in many cognitive processes. They are under intense research as potential targets of drugs used to treat neurodegenerative diseases and neurological disorders such as Alzheimer's disease and schizophrenia. Invertebrate nAChRs are targets of anthelmintics as well as a major group of insecticides, the neonicotinoids. The honey bee, Apis mellifera, is one of the most beneficial insects worldwide, playing an important role in crop pollination, and is also a valuable model system for studies on social interaction, sensory processing, learning, and memory. We have used the A. mellifera genome information to characterize the complete honey bee nAChR gene family. Comparison with the fruit fly Drosophila melanogaster and the malaria mosquito Anopheles gambiae shows that the honey bee possesses the largest family of insect nAChR subunits to date (11 members). As with Drosophila and Anopheles, alternative splicing of conserved exons increases receptor diversity. Also, we show that in one honey bee nAChR subunit, six adenosine residues are targeted for RNA A-to-I editing, two of which are evolutionarily conserved in Drosophila melanogaster and Heliothis virescens orthologs, and that the extent of editing increases as the honey bee lifecycle progresses, serving to maximize receptor diversity at the adult stage. These findings on Apis mellifera enhance our understanding of nAChR functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species. PMID:17065616

  9. Delayed procedural learning in α7-nicotinic acetylcholine receptor knockout mice

    PubMed Central

    Young, J. W.; Meves, J. M.; Tarantino, I. S.; Caldwell, S.; Geyer, M. A.

    2014-01-01

    The α7-nicotinic acetylcholine receptor (nAChR) has long been a procognitive therapeutic target to treat schizophrenia. Evidence on the role of this receptor in cognition has been lacking, however, in part due to the limited availability of suitable ligands. The behavior of α7-nAChR knockout (KO) mice has been examined previously, but cognitive assessments using tests with cross-species translatability have been limited to date. Here, we assessed the cognitive performance of α7-nAChR KO and wild-type (WT) littermate mice in the attentional set-shifting task of executive functioning, the radial arm maze test of spatial working memory span capacity and the novel object recognition test of short-term memory. The reward motivation of these mutants was assessed using the progressive ratio breakpoint test. In addition, we assessed the exploratory behavior and sensorimotor gating using the behavioral pattern monitor and prepulse inhibition, respectively. α7-nAChR KO mice exhibited normal set-shifting, but impaired procedural learning (rule acquisition) in multiple paradigms. Spatial span capacity, short-term memory, motivation for food, exploration and sensorimotor gating were all comparable to WT littermates. The data presented here support the notion that this receptor is important for such procedural learning, when patterns in the environment become clear and a rule is learned. In combination with the impaired attention observed previously in these mice, this finding suggests that agonist treatments should be examined in clinical studies of attention and procedural learning, perhaps in combination with cognitive behavioral therapy. PMID:21679297

  10. Separate fractions of mRNA from Torpedo electric organ induce chloride channels and acetylcholine receptors in Xenopus oocytes.

    PubMed Central

    Sumikawa, K; Parker, I; Amano, T; Miledi, R

    1984-01-01

    Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor. Images Fig. 1. PMID:6094179

  11. Single-channel properties of α3β4, α3β4α5 and α3β4β2 nicotinic acetylcholine receptors in mice lacking specific nicotinic acetylcholine receptor subunits

    PubMed Central

    Ciuraszkiewicz, Anna; Schreibmayer, Wolfgang; Platzer, Dieter; Orr-Urtreger, Avi; Scholze, Petra; Huck, Sigismund

    2013-01-01

    Previous attempts to measure the functional properties of recombinant nicotinic acetylcholine receptors (nAChRs) composed of known receptor subunits have yielded conflicting results. The use of knockout mice that lack α5, β2, α5β2 or α5β2α7 nAChR subunits enabled us to measure the single-channel properties of distinct α3β4, α3β4α5 and α3β4β2 receptors in superior cervical ganglion (SCG) neurons. Using this approach, we found that α3β4 receptors had a principal conductance level of 32.6 ± 0.8 pS (mean ± SEM) and both higher and lower secondary conductance levels. α3β4α5 receptors had the same conductance as α3β4 receptors, but differed from α3β4 receptors by having an increased channel open time and increased burst duration. By contrast, α3β4β2 receptors differed from α3β4 and α3β4α5 receptors by having a significantly smaller conductance level (13.6 ± 0.5 pS). After dissecting the single-channel properties of these receptors using our knockout models, we then identified these properties – and hence the receptors themselves – in wild-type SCG neurons. This study is the first to identify the single-channel properties of distinct neuronal nicotinic receptors in their native environment. PMID:23613527

  12. Roles for N-terminal Extracellular Domains of Nicotinic Acetylcholine Receptor (nAChR) β3 Subunits in Enhanced Functional Expression of Mouse α6β2β3- and α6β4β3-nAChRs*

    PubMed Central

    Dash, Bhagirathi; Li, Ming D.; Lukas, Ronald J.

    2014-01-01

    Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where “*” indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5–8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′S subunits were substituted for mβ3V9′S subunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops “C” (Glu221 and Phe223), “E” (Ser144 and Ser148), and “β2-β3” (Gln94 and Glu101) increased function of mα6mβ2*- (∼2–3-fold) or mα6mβ4* (∼2–4-fold)-nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs. PMID:25028511

  13. The α6 nicotinic acetylcholine receptor subunit of Frankliniella occidentalis is not involved in resistance to spinosad.

    PubMed

    Hou, Wenjie; Liu, Qiulei; Tian, Lixia; Wu, Qingjun; Zhang, Youjun; Xie, Wen; Wang, Shaoli; Miguel, Keri San; Funderburk, Joe; Scott, Jeffrey G

    2014-05-01

    Insects evolve resistance which constrains the sustainable use of insecticides. Spinosyns, a class of environmentally-friendly macrolide insecticides, is not an exception. The mode of inheritance and the mechanisms of resistance to spinosad (the most common spinosyn insecticide) in Frankliniella occidentalis (Western flower thrips, WFT) were investigated in this study. Resistance (170,000-fold) was autosomal and completely recessive. Recent studies showed that deletion of the nicotinic acetylcholine receptor α6 subunit gene resulted in strains of Drosophila melanogaster, Plutella xylostella and Bactrocera dorsalis that are resistant to spinosad, indicating that nAChRα6 subunit maybe important for the toxic action of this insecticide. Conversely, a G275E mutation of this subunit in F. occidentalis was recently proposed as the mechanism of resistance to spinosad. We cloned and characterized nAChRα6 from three susceptible and two spinosad resistant strains from China and the USA. The Foα6 cDNA is 1873bp and the open reading frame is 1458bp which encodes 485 amino acid residues with a predicted molecular weight of 53.5-kDa, the 5' and 3' UTRs are 121 and 294bp, respectively. There was no difference in the cDNA sequence between the resistant and susceptible thrips, suggesting the G275E mutation does not confer resistance in these populations. Ten isoforms of Foα6, arising from alternative splicing, were isolated and did not differ between the spinosad-susceptible and resistant strains. Quantitative real time PCR analysis showed Foα6 was highly expressed in the first instar larva, pupa and adult, and the expression levels were 3.67, 2.47, 1.38 times that of the second instar larva. The expression level was not significantly different between the susceptible and resistant strains. These results indicate that Foα6 is not involved in resistance to spinosad in F. occidentalis from China and the USA. PMID:24861935

  14. Prior nicotine self-administration attenuates subsequent dopaminergic deficits of methamphetamine in rats: role of nicotinic acetylcholine receptors.

    PubMed

    Baladi, Michelle G; Nielsen, Shannon M; McIntosh, J Michael; Hanson, Glen R; Fleckenstein, Annette E

    2016-08-01

    Preclinical studies have demonstrated that oral nicotine exposure attenuates long-term dopaminergic damage induced by toxins, including repeated, high doses of methamphetamine. It is suggested that alterations in nicotinic acetylcholine receptor (nAChR) expression, including α4β2* and α6β2* subtypes, likely contribute to this protection. The current study extended these findings by investigating whether nicotine self-administration in male, Sprague-Dawley rats (a) attenuates short-term dopaminergic damage induced by methamphetamine and (b) causes alterations in levels of α4β2* and α6β2* nAChR subtypes. The findings indicate that nicotine self-administration (0.032 mg/kg/infusion for 14 days) per se did not alter α4β2* and α6β2* nAChR expression or dopamine transporter (DAT) expression and function. Interestingly, prior nicotine self-administration attenuated methamphetamine-induced decreases in DAT function when assessed 24 h, but not 1 h, after methamphetamine treatment (4×7.5 mg/kg/injection). The ability of nicotine to attenuate the effects of methamphetamine on DAT function corresponded with increases in α4β2*, but not α6β2*, nAChR binding density. Understanding the role of nAChRs in methamphetamine-induced damage has the potential to elucidate mechanisms underlying the etiology of disorders involving dopaminergic dysfunction, as well as to highlight potential new therapeutic strategies for prevention or reduction of dopaminergic neurodegeneration. PMID:26871405

  15. Isotopic rubidium ion efflux assay for the functional characterization of nicotinic acetylcholine receptors on clonal cell lines

    SciTech Connect

    Lukas, R.J.; Cullen, M.J.

    1988-11-15

    An isotopic rubidium ion efflux assay has been developed for the functional characterization of nicotinic acetylcholine receptors on cultured neurons. This assay first involves the intracellular sequestration of isotopic potassium ion analog by the ouabain-sensitive action of a sodium-potassium ATPase. Subsequently, the release of isotopic rubidium ion through nicotinic acetylcholine receptor-coupled monovalent cation channels is activated by application of nicotinic agonists. Specificity of receptor-mediated efflux is demonstrated by its sensitivity to blockade by nicotinic, but not muscarinic, antagonists. The time course of agonist-mediated efflux, within the temporal limitations of the assay, indicates a slow inactivation of receptor function on prolonged exposure to agonist. Dose-response profiles (i) have characteristic shapes for different nicotinic agonists, (ii) are described by three operationally defined parameters, and (iii) reflect different affinities of agonists for binding sites that control receptor activation and functional inhibition. The rubidium ion efflux assay provides fewer hazards but greater sensitivity and resolution than isotopic sodium or rubidium ion influx assays for functional nicotinic receptors.

  16. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees.

    PubMed

    Moffat, Christopher; Buckland, Stephen T; Samson, Andrew J; McArthur, Robin; Chamosa Pino, Victor; Bollan, Karen A; Huang, Jeffrey T-J; Connolly, Christopher N

    2016-01-01

    There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species. PMID:27124107

  17. Analysis and modulation of the immune response of mice to acetylcholine receptor by anti-idiotypes.

    PubMed

    Souroujon, M C; Barchan, D; Fuchs, S

    1985-01-01

    Anti-idiotypes were raised in mice against three well-characterized anti-acetylcholine receptor (AChR) monoclonal antibodies (mcAbs), as well as against polyclonal mouse anti-AChR antibodies. In binding experiments, the anti-idiotypic antibodies inhibited the binding of AChR only to the immunizing idiotype. However, a less restricted specificity was found in in vivo experiments. Mice producing anti-idiotypes were challenged with AChR and the idiotypic composition of their anti-AChR response was analysed using specific rabbit anti-idiotypic antibodies. It was found that preimmunization with a certain idiotype leads to the preferential suppression of this particular idiotype in the polyclonal response to AChR. However, preimmunization with either polyclonal or monoclonal anti-AChR antibodies resulted in a reduction of the overall anti-Torpedo AChR and anti-muscle AChR titers. This reduction was greater than would be expected from the representation of each of the respective idiotypes in the polyclonal anti-AChR serum, and may imply that in addition to the immunizing idiotype other anti-AChR idiotypes are also suppressed. Our results suggest that anti-idiotypes may have a potential for the modulation of the autoimmune response directed against AChR in myasthenia. PMID:3874156

  18. Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

    PubMed Central

    Papadouli, I; Potamianos, S; Hadjidakis, I; Bairaktari, E; Tsikaris, V; Sakarellos, C; Cung, M T; Marraud, M; Tzartos, S J

    1990-01-01

    The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible. PMID:1695844

  19. Suppression of ongoing experimental myasthenia by oral treatment with an acetylcholine receptor recombinant fragment

    PubMed Central

    Im, Sin-Hyeog; Barchan, Dora; Fuchs, Sara; Souroujon, Miriam C.

    1999-01-01

    Myasthenia gravis (MG) is an autoimmune disorder in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. In an attempt to develop an antigen-specific therapy for MG, we administered a nonmyasthenogenic recombinant fragment of AChR orally to rats. This fragment, corresponding to the extracellular domain of the human AChR α-subunit (Hα1-205), protected rats from subsequently induced experimental autoimmune myasthenia gravis (EAMG) and suppressed ongoing EAMG when treatment was initiated during either the acute or chronic phases of disease. Prevention and suppression of EAMG were accompanied by a significant decrease in AChR-specific humoral and cellular responses. The underlying mechanism for the Hα1-205–induced oral tolerance seems to be active suppression, mediated by a shift from a T-helper 1 (Th1) to a Th2/Th3 response. This shift was assessed by changes in the cytokine profile, a deviation of anti-AChR IgG isotypes from IgG2 to IgG1, and a suppressed AChR-specific delayed-type hypersensitivity response. Our results in experimental myasthenia suggest that oral administration of AChR-specific recombinant fragments may be considered for antigen-specific immunotherapy of myasthenia gravis. J. Clin. Invest. 104:1723–1730 (1999). PMID:10606626

  20. Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.

    PubMed

    Barchan, D; Balass, M; Souroujon, M C; Katchalski-Katzir, E; Fuchs, S

    1995-11-01

    We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR. PMID:7594584

  1. Suppression of ongoing experimental myasthenia by oral treatment with an acetylcholine receptor recombinant fragment.

    PubMed

    Im, S H; Barchan, D; Fuchs, S; Souroujon, M C

    1999-12-01

    Myasthenia gravis (MG) is an autoimmune disorder in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. In an attempt to develop an antigen-specific therapy for MG, we administered a nonmyasthenogenic recombinant fragment of AChR orally to rats. This fragment, corresponding to the extracellular domain of the human AChR alpha-subunit (Halpha1-205), protected rats from subsequently induced experimental autoimmune myasthenia gravis (EAMG) and suppressed ongoing EAMG when treatment was initiated during either the acute or chronic phases of disease. Prevention and suppression of EAMG were accompanied by a significant decrease in AChR-specific humoral and cellular responses. The underlying mechanism for the Halpha1-205-induced oral tolerance seems to be active suppression, mediated by a shift from a T-helper 1 (Th1) to a Th2/Th3 response. This shift was assessed by changes in the cytokine profile, a deviation of anti-AChR IgG isotypes from IgG2 to IgG1, and a suppressed AChR-specific delayed-type hypersensitivity response. Our results in experimental myasthenia suggest that oral administration of AChR-specific recombinant fragments may be considered for antigen-specific immunotherapy of myasthenia gravis. PMID:10606626

  2. A positive relationship between harm avoidance and brain nicotinic acetylcholine receptor availability.

    PubMed

    Storage, Steven; Mandelkern, Mark A; Phuong, Jonathan; Kozman, Maggie; Neary, Meaghan K; Brody, Arthur L

    2013-12-30

    Prior research indicates that disturbance of cholinergic neurotransmission reduces anxiety, leading to the hypothesis that people with heightened cholinergic function have a greater tendency toward anxiety-like and/or harm-avoidant behavior. We sought to determine if people with elevated levels of harm avoidance (HA), a dimension of temperament from the Temperament and Character Inventory (TCI), have high α4β2* nicotinic acetylcholine receptor (nAChR) availability. Healthy adults (n=105; 47 non-smokers and 58 smokers) underwent bolus-plus-continuous infusion positron emission tomography (PET) scanning using the radiotracer 2-[18F]fluoro-3-(2(S)azetidinylmethoxy) pyridine (abbreviated as 2-FA). During the uptake period of 2-FA, participants completed the TCI. The central study analysis revealed a significant association between total HA and mean nAChR availability, with higher total HA scores being linked with greater nAChR availability. In examining HA subscales, both 'Fear of Uncertainty' and 'Fatigability' were significant, based on higher levels of these characteristics being associated with greater nAChR availabilities. This study adds to a growing body of knowledge concerning the biological basis of personality and may prove useful in understanding the pathophysiology of psychiatric disorders (such as anxiety disorders) that have similar characteristics to HA. Study findings may indicate that heightened cholinergic neurotransmission is associated with increased anxiety-like traits. PMID:24148908

  3. Energy for Wild-Type Acetylcholine Receptor Channel Gating from Different Choline Derivatives

    PubMed Central

    Bruhova, Iva; Gregg, Timothy; Auerbach, Anthony

    2013-01-01

    Agonists, including the neurotransmitter acetylcholine (ACh), bind at two sites in the neuromuscular ACh receptor channel (AChR) to promote a reversible, global change in protein conformation that regulates the flow of ions across the muscle cell membrane. In the synaptic cleft, ACh is hydrolyzed to acetate and choline. Replacement of the transmitter’s ester acetyl group with a hydroxyl (ACh→choline) results in a +1.8 kcal/mol reduction in the energy for gating generated by each agonist molecule from a low- to high-affinity change of the transmitter binding site (ΔGB). To understand the distinct actions of structurally related agonist molecules, we measured ΔGB for 10 related choline derivatives. Replacing the hydroxyl group of choline with different substituents, such as hydrogen, chloride, methyl, or amine, increased the energy for gating (i.e., it made ΔGB more negative relative to choline). Extending the ethyl hydroxide tail of choline to propyl and butyl hydroxide also increased this energy. Our findings reveal the amount of energy that is available for the AChR conformational change provided by different, structurally related agonists. We speculate that a hydrogen bond between the choline hydroxyl and the backbone carbonyl of αW149 positions this agonist’s quaternary ammonium group so as to reduce the cation-π interaction between this moiety and the aromatic groups at the binding site. PMID:23442907

  4. Metabolic stabilization of acetylcholine receptors in vertebrate neuromuscular junction by muscle activity.

    PubMed

    Rotzler, S; Brenner, H R

    1990-08-01

    The effects of muscle activity on the growth of synaptic acetylcholine receptor (AChR) accumulations and on the metabolic AChR stability were investigated in rat skeletal muscle. Ectopic end plates induced surgically in adult soleus muscle were denervated early du