Jon Lindstrom

William Penn University, Filadelfia, Pennsylvania, United States

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Publications (323)1481.28 Total impact

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    ABSTRACT: Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries, and high vs. low agonist sensitivities, (HS & LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist binding sites in, and between, the isoforms have not been studied. We used a fully-linked subunit concatemeric nAChR approach to express uniform populations of HS- or LS-isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)face E-loop residues to their non-conserved α4 subunit counterparts, or vice-versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. ACh concentration-response curves and maximum function were measured using two-electrode voltage-clamp electrophysiology. Surface expression was measured with [(125)I]mAb 295 binding, and was used to define function/nAChR. If the α4(+)/(-)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly-differential outcomes within the HS-isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise-equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation, and that E-loop residues are an important contributor to this neighbor effect.
    Preview · Article · Dec 2015 · Journal of Biological Chemistry
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    ABSTRACT: Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in α2, α4 and β2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for β2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, α4(R336H) and β2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on α4β2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations' effects at alternate isoforms: high-sensitivity [HS; (α4)2(β2)3] or low-sensitivity [LS; (α4)3(β2)2] via 1:10 or 30:1 [α4:β2] cRNA injection ratios, respectively. An unbiased (1:1 [α4:β2] cRNA) injection ratio was also used to study potential shifts in isoform expression. α4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). β2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. α4β2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using β2-subunit-specific [(125)I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the β2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between TM- and C2- domain AD/NFLE-associated mutations. As we discuss, the shared features may be particularly salient to AD/NFLE etiology.
    No preview · Article · Nov 2015 · Neuropharmacology
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    ABSTRACT: Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs, but is without effect on α3* or α6* nAChRs ('*' indicates presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short-term desensitized nAChRs that have at least two α4 subunits, but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long-term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently, and overcomes short- and long-term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs.
    Preview · Article · Oct 2015 · Journal of Biological Chemistry
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    ABSTRACT: 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. Here we describe the development of α-Ctx LvIA(N9R,V10A) that is 3,000-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 PCR 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. The American Society for Pharmacology and Experimental Therapeutics.
    Preview · Article · Sep 2015 · Molecular pharmacology
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    Jie Luo · Jon Lindstrom
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    ABSTRACT: Myasthenia gravis (MG) is an organ-specific autoimmune disease characterized by muscle fatigability. In most cases, it is mediated by autoantibodies targeting muscle nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction. Experimental autoimmune myasthenia gravis (EAMG) is an animal model for MG, which is usually induced by immunization with AChR purified from fish electric organ. Pathological autoantibodies to AChRs are directed at the extracellular surface, especially the main immunogenic region (MIR). Current treatments for MG can help many but not all patients. Antigen-specific immunosuppressive therapy for MG that specifically suppresses the autoimmune response without affecting the entire immune system and avoids side effects of general immunosuppression is currently unavailable. Early attempts at antigen-specific immunosuppression for EAMG using AChR extracellular domain sequences that form epitopes for pathological autoantibodies risked provoking autoimmunity rather than suppressing it. We discovered a novel approach to specific immunosuppression of EAMG with a therapeutic vaccine consisting of bacterially-expressed human AChR cytoplasmic domains, which has the potential to specifically suppress MG without danger of causing exacerbation. This approach prevents development of chronic EAMG when initiated immediately after the acute phase of EAMG, and rapidly reverses established chronic EAMG when started during the chronic phase of EAMG. Successfully treated rats exhibited long-term resistance to re-induction of EAMG. In this review we also discuss the current understanding of the mechanisms by which the therapy works. Vaccination with AChR cytoplasmic domains in adjuvant is promising as a safe, antigen-specific, potent, effective, rapidly acting, and long lasting approach to therapy of MG. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Jul 2015 · Biochemical pharmacology
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    ABSTRACT: 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 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 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 is termed an accessory site-selective agonist (accessory SSAg). 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 thio-reactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Full-text · Article · Apr 2015 · Journal of Biological Chemistry
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    ABSTRACT: Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR) is characterized by a chronic, fatigable weakness of voluntary muscles. The production of autoantibodies involves the dysregulation of T cells which provide the environment for the development of autoreactive B cells. The symptoms are caused by destruction of the postsynaptic membrane and degradation of the AChR by IgG autoantibodies, predominantly of the G1 and G3 subclasses. Active immunization of animals with AChR from mammalian muscles, AChR form Torpedo or Electrophorus electric organs, and recombinant or synthetic AChR fragments generates a chronic model of MG, termed experimental autoimmune myasthenia gravis (EAMG). This model covers cellular mechanisms involved in the immune response against the AChR, e.g. antigen presentation, T cell-help and regulation, B cell selection and differentiation into plasma cells. Our aim is to define standard operation procedures and recommendations for the rat EAMG model using purified AChR from the Torpedo californica electric organ, in order to facilitate more rapid translation of preclinical proof of concept or efficacy studies into clinical trials and, ultimately, clinical practice. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Mar 2015 · Experimental Neurology
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    ABSTRACT: Antibodies against the muscle acetylcholine receptor (AChR) are the most common cause of myasthenia gravis (MG). Passive transfer of AChR antibodies from MG patients into animals reproduces key features of human disease, including antigenic modulation of the AChR, complement-mediated damage of the neuromuscular junction, and muscle weakness. Similarly, AChR antibodies generated by active immunization in experimental autoimmune MG models can subsequently be passively transferred to other animals and induce weakness. The passive transfer model is useful to test therapeutic strategies aimed at the effector mechanism of the autoantibodies. Here we summarize published and unpublished experience using the AChR passive transfer MG model in mice, rats and rhesus monkeys, and give recommendations for the design of preclinical studies in order to facilitate translation of positive and negative results to improve MG therapies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Mar 2015 · Experimental Neurology
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    ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) are highly conserved between humans and non-human primates. Conservation exists at the level of genomic structure, protein structure and epigenetics. Overall homology of nAChRs at the protein level is 98% in macaques versus 89% in mice, which is highly relevant for evaluating subtype-specific ligands that have different affinities in humans versus rodents. In addition to conservation at the protein level, there is high conservation of genomic structure in terms of intron and exon size and placement of CpG sites that play a key role in epigenetic regulation. Analysis of single nucleotide polymorphisms (SNPs) shows that while the majority of SNPs are not conserved between humans and macaques, some functional polymorphisms are. Most significantly, cynomolgus monkeys express a similar α5 nAChR Asp398Asn polymorphism to the human α5 Asp398Asn polymorphism that has been linked to greater nicotine addiction and smoking related disease. Monkeys can be trained to readily self-administer nicotine, and in an initial study we have demonstrated that cynomolgus monkeys bearing the α5 D398N polymorphism show a reduced behavioral sensitivity to oral nicotine and tend to consume it in a different pattern when compared to wild-type monkeys. Thus the combination of highly homologous nAChR, higher cortical functions and capacity for complex training makes non-human primates a unique model to study in vivo functions of nicotinic receptors. In particular, primate studies on nicotine addiction and evaluation of therapies to prevent or overcome nicotine addiction are likely to be highly predictive of treatment outcomes in humans. Copyright © 2015. Published by Elsevier Ltd.
    No preview · Article · Feb 2015 · Neuropharmacology
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    Jingyi Wang · Alexander Kuryatov · Jon Lindstrom
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    ABSTRACT: Nicotinic acetylcholine receptors (AChRs) are ACh-gated ion channels formed from five homologous subunits in subtypes defined by their subunit composition and stoichiometry. Some subtypes readily produce functional AChRs in Xenopus oocytes and transfected cell lines. α6β2β3* AChRs (subtypes formed from these subunits and perhaps others) are not easily expressed. This may be because the types of neurons in which they are expressed (typically dopaminergic neurons) have unique chaperones for assembling α6β2β3* AChRs, especially in the presence of the other AChR subtypes. Because these relatively minor brain AChR subtypes are of major importance in addiction to nicotine, it is important for drug development as well as investigation of their functional properties to be able to efficiently express human α6β2β3* AChRs. We review the issues and progress in expressing α6* AChRs. This article is part of a Special Issue entitled 'Nicotinic Acetylcholine Receptor'. Copyright © 2014. Published by Elsevier Ltd.
    Full-text · Article · Oct 2014 · Neuropharmacology
  • Jie Luo · Jon Lindstrom
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    ABSTRACT: Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are caused by Ab-mediated autoimmune responses to muscle nicotinic acetylcholine receptors (AChRs) that impair neuromuscular transmission, thereby causing muscle weakness. Previously, we discovered that i.p. injection of a therapeutic vaccine consisting of bacterially expressed cytoplasmic domains of human AChR subunits reduced the development of chronic EAMG in rats. In this article, we show that immunization with the therapeutic vaccine in adjuvants does not induce EAMG and, thus, is safe. The potency and efficacy of the therapeutic vaccine were greatly increased by s.c. administration of repeated low doses in IFA. Onset of chronic EAMG could be prevented. Established chronic EAMG could be rapidly reversed, modeling therapy of chronic MG. Therapy reduced pathological Abs assayed by immune precipitation of a main immunogenic region chimera. Successfully treated rats exhibited long-term resistance to reinduction of EAMG, suggesting a lasting cure of MG. A long-term effect of therapy was to change the isotype of the pathogenic Ab response from IgG2b, which fixes complement, to IgG1, which does not. Prevention and reversal of chronic EAMG was not caused by the isotype switch, but the isotype switch may contribute to resistance to reinduction of EAMG. Immunization with AChR cytoplasmic domains in adjuvant is promising as a safe, Ag-specific, potent, effective, rapidly acting, and long-lasting therapeutic approach to MG.
    No preview · Article · Oct 2014 · The Journal of Immunology
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    Carson Kai-Kwong Ley · Alexander Kuryatov · Jingyi Wang · Jon Martin Lindstrom
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    ABSTRACT: Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)2β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)2β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.
    Full-text · Article · Jul 2014 · PLoS ONE
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    Mi Shi · Zhifeng Yue · Alexandre Kuryatov · Jon M Lindstrom · Amita Sehgal
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    ABSTRACT: In this study, we report a new protein involved in the homeostatic regulation of sleep in Drosophila. We conducted a forward genetic screen of chemically mutagenized flies to identify short-sleeping mutants and found one, redeye (rye) that shows a severe reduction of sleep length. Cloning of rye reveals that it encodes a nicotinic acetylcholine receptor α subunit required for Drosophila sleep. Levels of RYE oscillate in light–dark cycles and peak at times of daily sleep. Cycling of RYE is independent of a functional circadian clock, but rather depends upon the sleep homeostat, as protein levels are up-regulated in short-sleeping mutants and also in wild type animals following sleep deprivation. We propose that the homeostatic drive to sleep increases levels of RYE, which responds to this drive by promoting sleep. DOI:
    Full-text · Article · Feb 2014 · eLife Sciences
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    ABSTRACT: Chronic nicotine administration increases the density of brain α4β2* nicotinic acetylcholine receptors (nAChRs), which may contribute to withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently we found that sazetidine-A, a potent partial agonist that desensitizes α4β2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here we investigated whether chronic sazetidine-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic sazetidine-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, sazetidine-A and varenicline each demonstrated anxiolytic effects in mice, but only sazetidine-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, sazetidine-A, or drugs like it, may be useful in achieving long-term abstinence from smoking. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jan 2014 · Journal of Neurochemistry
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    ABSTRACT: Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). α4β2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encodes the α4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (α4R336C, α4P451L and α4R487Q) to the common variant to determine their effects on α4β2 nAChR pharmacology. We examined [3H]-epibatidine binding, interacting proteins and phosphorylation of the α4 nAChR subunit with LC-MS/MS in HEK 293 cells, and voltage-clamp electrophysiology in Xenopus oocytes. We observed significant effects of the α4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified α4β2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in Xenopus oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these α4 rare variants, as well as shifts in receptor function following incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of α4β2 nAChRs resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common α4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.
    Full-text · Article · Jan 2014 · Journal of Pharmacology and Experimental Therapeutics
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    ABSTRACT: The design and synthesis of a series of substituted heteroaromatic α4β2α5 positive allosteric modulators is reported. The optimization and development of the heteroaromatic series was carried out from NS9283, and several potent analogues, such as 3-(5-(pyridin-3-yl)-2H-tetrazol-2-yl)benzonitrile (5k) and 3,3'-(2H-tetrazole-2,5-diyl)dipyridine (12h) with good in vitro efficacy were discovered.
    No preview · Article · Nov 2013 · Bioorganic & medicinal chemistry letters
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    ABSTRACT: The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained "smoldering activation" occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β2)2β2 and (α4β2)2α4. The (α4β2)2β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β2)2α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.
    Full-text · Article · Nov 2013 · PLoS ONE
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    ABSTRACT: Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS; (α4)2(β2)3) or low-sensitivity (LS; (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChR). Function was assessed using (86)Rb(+) efflux in a stably-transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously-studied agonists, desensitization bythe highly-selective agonists A-85380 and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase vs. LS-phase responses. The concatenated-nAChR experiments confirmed that ≈20% of LS-isoform ACh-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSP were generated, each targeting a conserved agonist-binding residue within the LS-isoform-only α4(+)/(-)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately 5x greater on a "per receptor" basis for unmodified-LSP vs. HSP α4β2-nAChR. Thus, recruitment of the α4(+)/(-)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(-)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS vs. LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChR are the predominant neuronal subtype these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.
    Preview · Article · Nov 2013 · Journal of Pharmacology and Experimental Therapeutics
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    ABSTRACT: Neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and β2 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affinity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between α4β2-containing (α 4 β2∗) nAChRs and other proteins remains limited. In this study, we identified proteins that interact with α 4 β2∗ nAChRs in a gene-dose dependent pattern by immunopurifying β2∗ nAChRs from mice that differ in α4 and β2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Reduced expression of either the α4 or the β2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-immunoprecipitated with these nAChRs. Furthermore, stratified linear regression analysis indicated that levels of 17 proteins was correlated significantly with expression of α4 β2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a β2∗ nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of α4 β2 nAChRs and their downstream signaling mechanisms.
    Full-text · Article · Jul 2013
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    ABSTRACT: Synthesis of acetylcholine (ACh) by non-neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na(+) -dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. By contrast, some non-neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non-neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter-like proteins, a five gene family (CTL1-5). Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na(+) -independent and CTL1-5 were expressed in all cells examined. CTL1,2,&5 were expressed at highest levels and knockdown of CTL1,2&5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1,2,3&5 had no effect on ACh synthesis in H82 cells. By contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non-neuronal cell lines and presents a mechanism to target non-neuronal ACh synthesis without affecting neuronal ACh synthesis. This article is protected by copyright. All rights reserved.
    No preview · Article · May 2013 · Journal of Neurochemistry

Publication Stats

17k Citations
1,481.28 Total Impact Points


  • 1993-2015
    • William Penn University
      Filadelfia, Pennsylvania, United States
  • 1992-2015
    • University of Pennsylvania
      • • Department of Neuroscience
      • • Department of Medicine
      • • The Mahoney Institute of Neurological Sciences
      Filadelfia, Pennsylvania, United States
  • 2011
    • University of Colorado at Boulder
      • Institute for Behavioral Genetics (IBG)
      Boulder, Colorado, United States
  • 1992-2008
    • Hospital of the University of Pennsylvania
      • Department of Neuroscience
      Philadelphia, Pennsylvania, United States
  • 1977-2007
    • Salk Institute
      La Jolla, California, United States
  • 2004
    • University of Utah
      • Department of Biology
      Salt Lake City, Utah, United States
  • 2003
    • University of the Sciences in Philadelphia
      Philadelphia, Pennsylvania, United States
    • University of Cologne
      Köln, North Rhine-Westphalia, Germany
  • 2000
    • University of Alabama at Birmingham
      • Vision Science Research Center
      Birmingham, Alabama, United States
    • The Children's Hospital of Philadelphia
      • Department of Neurology
      Philadelphia, Pennsylvania, United States
  • 1999
    • University of Florida
      • Department of Pharmacology and Therapeutics
      Gainesville, Florida, United States
  • 1994
    • Heidelberg University
      تيفين، أوهايو, Ohio, United States
    • University of Maryland, Baltimore
      • Department of Pharmacology
      Baltimore, Maryland, United States
    • University of Alicante
      • Department of Agrochemistry and Biochemistry
      Alicante, Valencia, Spain
  • 1987
    • Washington University in St. Louis
      • Department of Anatomy and Neurobiology
      San Luis, Missouri, United States
  • 1986
    • Stanford University
      Palo Alto, California, United States
    • University of Vermont
      • Department of Pathology
      Burlington, Vermont, United States
    • Cornell University
      Итак, New York, United States
  • 1983
    • The Rockefeller University
      • Laboratory of Cell Biology
      New York, New York, United States