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Identification and characterization of ML352: a novel, noncompetitive inhibitor of the presynaptic choline transporter.
Ennis EA, Wright J, Retzlaff CL, McManus OB, Lin Z, Huang X, Wu M, Li M, Daniels JS, Lindsley CW, Hopkins CR, Blakely RD
(2015) ACS Chem Neurosci 6: 417-27
MeSH Terms: Animals, Benzamides, Choline, Dose-Response Relationship, Drug, Enzyme Inhibitors, Gene Expression Regulation, HEK293 Cells, Hemicholinium 3, Humans, Isoxazoles, Male, Membrane Potentials, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Models, Biological, Mutation, Neural Inhibition, Prosencephalon, Protein Binding, Rats, Rats, Sprague-Dawley, Synaptosomes
Show Abstract · Added February 12, 2015
The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents.
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23 MeSH Terms
Transgenic overexpression of the presynaptic choline transporter elevates acetylcholine levels and augments motor endurance.
Holmstrand EC, Lund D, Cherian AK, Wright J, Martin RF, Ennis EA, Stanwood GD, Sarter M, Blakely RD
(2014) Neurochem Int 73: 217-28
MeSH Terms: Acetylcholine, Animals, Behavior, Animal, Choline, Cholinergic Agents, Chromosomes, Artificial, Bacterial, Gene Dosage, Hemicholinium 3, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Mice, Transgenic, Physical Endurance, Receptors, Presynaptic, Synaptosomes
Show Abstract · Added December 2, 2013
The hemicholinium-3 (HC-3) sensitive, high-affinity choline transporter (CHT) sustains cholinergic signaling via the presynaptic uptake of choline derived from dietary sources or from acetylcholinesterase (AChE)-mediated hydrolysis of acetylcholine (ACh). Loss of cholinergic signaling capacity is associated with cognitive and motor deficits in humans and in animal models. Whereas genetic elimination of CHT has revealed the critical nature of CHT in maintaining ACh stores and sustaining cholinergic signaling, the consequences of elevating CHT expression have yet to be studied. Using bacterial artificial chromosome (BAC)-mediated transgenic methods, we generated mice with integrated additional copies of the mouse Slc5a7 gene. BAC-CHT mice are viable, appear to develop normally, and breed at wild-type (WT) rates. Biochemical studies revealed a 2 to 3-fold elevation in CHT protein levels in the CNS and periphery, paralleled by significant increases in [(3)H]HC-3 binding and synaptosomal choline transport activity. Elevations of ACh in the BAC-CHT mice occurred without compensatory changes in the activity of either choline acetyltransferase (ChAT) or AChE. Immunohistochemistry for CHT in BAC-CHT brain sections revealed markedly elevated CHT expression in the cell bodies of cholinergic neurons and in axons projecting to regions known to receive cholinergic innervation. Behaviorally, BAC-CHT mice exhibited diminished fatigue and increased speeds on the treadmill test without evidence of increased strength. Finally, BAC-CHT mice displayed elevated horizontal activity in the open field test, diminished spontaneous alteration in the Y-maze, and reduced time in the open arms of the elevated plus maze. Together, these studies provide biochemical, pharmacological and behavioral evidence that CHT protein expression and activity can be elevated beyond that seen in wild-type animals. BAC-CHT mice thus represent a novel tool to examine both the positive and negative impact of constitutively elevated cholinergic signaling capacity.
Copyright © 2013 Elsevier Ltd. All rights reserved.
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Cognitive effects of dopamine depletion in the context of diminished acetylcholine signaling capacity in mice.
Zurkovsky L, Bychkov E, Tsakem EL, Siedlecki C, Blakely RD, Gurevich EV
(2013) Dis Model Mech 6: 171-83
MeSH Terms: Acetylcholine, Animals, Cholinergic Agents, Cognition Disorders, Dementia, Disease Models, Animal, Dopamine, Hemicholinium 3, Humans, Male, Membrane Transport Proteins, Memory, Mice, Mice, Inbred C57BL, Mice, Knockout, Parkinsonian Disorders, Signal Transduction
Show Abstract · Added July 10, 2013
A subset of patients with Parkinson's disease acquires a debilitating dementia characterized by severe cognitive impairments (i.e. Parkinson's disease dementia; PDD). Brains from PDD patients show extensive cholinergic loss as well as dopamine (DA) depletion. We used a mutant mouse model to directly test whether combined cholinergic and DA depletion leads to a cognitive profile resembling PDD. Mice carrying heterozygous deletion of the high-affinity, hemicholinium-3-sensitive choline transporter (CHT(HET)) show reduced levels of acetylcholine throughout the brain. We achieved bilateral DA depletion in CHT(HET) and wild-type (WT) littermates via intra-striatal infusion of 6-hydroxydopamine (6-OHDA), or used vehicle as control. Executive function and memory were evaluated using rodent versions of cognitive tasks commonly used with human subjects: the set-shifting task and spatial and novel-object recognition paradigms. Our studies revealed impaired acquisition of attentional set in the set-shifting paradigm in WT-6OHDA and CHT(HET)-vehicle mice that was exacerbated in the CHT(HET)-6OHDA mice. The object recognition test following a 24-hour delay was also impaired in CHT(HET)-6OHDA mice compared with all other groups. Treatment with acetylcholinesterase (AChE) inhibitors physostigmine (0.05 or 0.1 mg/kg) and donepezil (0.1 and 0.3 mg/kg) reversed the impaired object recognition of the CHT(HET)-6OHDA mice. Our data demonstrate an exacerbated cognitive phenotype with dual ACh and DA depletion as compared with either insult alone, with traits analogous to those observed in PDD patients. The results suggest that combined loss of DA and ACh could be sufficient for pathogenesis of specific cognitive deficits in PDD.
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17 MeSH Terms
Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice.
Bazalakova MH, Wright J, Schneble EJ, McDonald MP, Heilman CJ, Levey AI, Blakely RD
(2007) Genes Brain Behav 6: 411-24
MeSH Terms: Acetylcholine, Animals, Anxiety, Brain, Cholinergic Agents, Exploratory Behavior, Hemicholinium 3, Heterozygote, Maze Learning, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Mice, Knockout, Motor Activity, Receptors, Muscarinic, Rotarod Performance Test, Scopolamine, Spatial Behavior
Show Abstract · Added July 10, 2013
Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.
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Na+, Cl-, and pH dependence of the human choline transporter (hCHT) in Xenopus oocytes: the proton inactivation hypothesis of hCHT in synaptic vesicles.
Iwamoto H, Blakely RD, De Felice LJ
(2006) J Neurosci 26: 9851-9
MeSH Terms: Acetylcholine, Animals, Biological Transport, Chlorides, Choline, Female, Hemicholinium 3, Humans, Membrane Potentials, Membrane Transport Proteins, Models, Neurological, Oocytes, Patch-Clamp Techniques, Protons, Recombinant Fusion Proteins, Sodium, Symporters, Synaptic Vesicles, Xenopus laevis
Show Abstract · Added July 10, 2013
The recent cloning of the human choline transporter (hCHT) has allowed its expression in Xenopus laevis oocytes and the simultaneous measurement of choline transport and choline-induced current under voltage clamp. hCHT currents and choline transport are evident in cRNA-injected oocytes and significantly enhanced by the hCHT trafficking mutant L530A/V531A. The charge/choline ratio of hCHT varies from 10e/choline at -80 mV to 3e/choline at -20 mV, in contrast with the reported fixed stoichiometry of the Na+-coupled glucose transporter in the same gene family. Ion substitution shows that the choline uptake and choline-induced current are Na+ and Cl- dependent; however, the reversal potential of the induced current suggests a Na+-selective mechanism, consigning Cl- to a regulatory role rather than a coupled, cotransported-ion role. The hCHT-specific inhibitor hemicholinium-3 (HC-3) blocks choline uptake and choline-induced current; in addition, HC-3 alone reveals a constitutive, depolarizing leak current through hCHT. We show that external protons reduce hCHT current, transport, and binding with a similar pKa of 7.4, suggesting proton titration of residue(s) that support choline binding and transport. Given the localization of the choline transporter to synaptic vesicles, we propose that proton inactivation of hCHT prevents acetylcholine and proton leakage from the acidic interior of cholinergic synaptic vesicles. This mechanism would allow cholinergic, activity-triggered delivery of silent choline transporters to the plasma membrane, in which normal pH would reactivate the transporters for choline uptake and subsequent acetylcholine synthesis.
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19 MeSH Terms
The Caenorhabditis elegans choline transporter CHO-1 sustains acetylcholine synthesis and motor function in an activity-dependent manner.
Matthies DS, Fleming PA, Wilkes DM, Blakely RD
(2006) J Neurosci 26: 6200-12
MeSH Terms: Acetylcholine, Adaptation, Physiological, Animals, Animals, Genetically Modified, Binding, Competitive, Biological Transport, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Cells, Cultured, Choline, Cholinergic Agents, Electrophysiology, Gene Deletion, Green Fluorescent Proteins, Hemicholinium 3, Membrane Transport Proteins, Motor Activity, Nerve Tissue Proteins, Recombinant Fusion Proteins, Signal Transduction, Synapses, Tissue Distribution
Show Abstract · Added July 10, 2013
Cholinergic neurotransmission supports motor, autonomic, and cognitive function and is compromised in myasthenias, cardiovascular diseases, and neurodegenerative disorders. Presynaptic uptake of choline via the sodium-dependent, hemicholinium-3-sensitive choline transporter (CHT) is believed to sustain acetylcholine (ACh) synthesis and release. Analysis of this hypothesis in vivo is limited in mammals because of the toxicity of CHT antagonists and the early postnatal lethality of CHT-/- mice (Ferguson et al., 2004). In Caenorhabditis elegans, in which cholinergic signaling supports motor activity and mutant alleles impacting ACh secretion and response can be propagated, we investigated the contribution of CHT (CHO-1) to facets of cholinergic neurobiology. Using the cho-1 promoter to drive expression of a translational, green fluorescent protein-CHO-1 fusion (CHO-1:GFP) in wild-type and kinesin (unc-104) mutant backgrounds, we establish in the living nematode that the transporter localizes to cholinergic synapses, and likely traffics on synaptic vesicles. Using embryonic primary cultures, we demonstrate that CHO-1 mediates hemicholinium-3-sensitive, high-affinity choline uptake that can be enhanced with depolarization in a Ca(2+)-dependent manner supporting ACh synthesis. Although homozygous cho-1 null mutants are viable, they possess 40% less ACh than wild-type animals and display stress-dependent defects in motor activity. In a choline-free liquid environment, cho-1 mutants demonstrate premature paralysis relative to wild-type animals. Our findings establish a requirement for presynaptic choline transport activity in vivo in a model amenable to a genetic dissection of CHO-1 regulation.
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Lethal impairment of cholinergic neurotransmission in hemicholinium-3-sensitive choline transporter knockout mice.
Ferguson SM, Bazalakova M, Savchenko V, Tapia JC, Wright J, Blakely RD
(2004) Proc Natl Acad Sci U S A 101: 8762-7
MeSH Terms: Acetylcholine, Animals, Brain, Choline O-Acetyltransferase, Cholinergic Agents, Hemicholinium 3, In Vitro Techniques, Membrane Transport Proteins, Mice, Mice, Knockout, Motor Neurons, Neuromuscular Junction, Phenotype, Synaptic Transmission, Synaptosomes
Show Abstract · Added July 10, 2013
Presynaptic acetylcholine (ACh) synthesis and release is thought to be sustained by a hemicholinium-3-sensitive choline transporter (CHT). We disrupted the murine CHT gene and examined CHT-/- and +/- animals for evidence of impaired cholinergic neurotransmission. Although morphologically normal at birth, CHT-/- mice become immobile, breathe irregularly, appear cyanotic, and die within an hour. Hemicholinium-3-sensitive choline uptake and subsequent ACh synthesis are specifically lost in CHT-/- mouse brains. Moreover, we observe a time-dependent loss of spontaneous and evoked responses at CHT-/- neuromuscular junctions. Consistent with deficits in synaptic ACh availability, we also observe developmental alterations in neuromuscular junction morphology reminiscent of changes in mutants lacking ACh synthesis. Adult CHT+/- mice overcome reductions in CHT protein levels and sustain choline uptake activity at wild-type levels through posttranslational mechanisms. Our results demonstrate that CHT is an essential and regulated presynaptic component of cholinergic signaling and indicate that CHT warrants consideration as a candidate gene for disorders characterized by cholinergic hypofunction.
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The choline transporter resurfaces: new roles for synaptic vesicles?
Ferguson SM, Blakely RD
(2004) Mol Interv 4: 22-37
MeSH Terms: Acetylcholine, Animals, Cell Membrane, Choline, Cholinergic Agents, Hemicholinium 3, Humans, Membrane Transport Proteins, Neurons, Presynaptic Terminals, Synaptic Transmission, Synaptic Vesicles
Show Abstract · Added July 10, 2013
Presynaptic choline uptake is vital to sustained neuronal acetylcholine (ACh) release; however, only with the recent cloning of choline transporters (CHTs) (i.e., SLC5A7), has a picture emerged of the regulatory pathways supporting CHT modulation. Studies arising from the development of CHT-specific antibodies reveal a large, intracellular reserve of CHT proteins, localized to ACh-containing, synaptic vesicles. The intersection of mechanisms supporting vesicular ACh release and choline uptake demonstrates an elegant mechanism for linking regulation of CHT membrane density to rates of ACh release. Furthermore, these studies point to control of the CHT endocytic process as an important target for novel therapeutics that could offset functional deficits in disorders bearing diminished cholinergic tone, including myasthenias and dementias.
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Molecular cloning and characterization of a murine hemicholinium-3-sensitive choline transporter.
Apparsundaram S, Ferguson SM, Blakely RD
(2001) Biochem Soc Trans 29: 711-6
MeSH Terms: Animals, Blotting, Northern, CHO Cells, Cholinergic Agents, Cricetinae, DNA, Complementary, Dose-Response Relationship, Drug, Hemicholinium 3, Kinetics, Membrane Transport Proteins, Mice, Protein Binding, RNA, Messenger, Reverse Transcriptase Polymerase Chain Reaction
Show Abstract · Added July 10, 2013
In cholinergic neurons, a specific requirement for precursor choline in the biosynthesis of acetylcholine (ACh) is thought to be sustained by a presynaptic, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). This transporter exhibits micromolar affinity for choline and transport activity is Na(+)- and Cl(-)-dependent. Based on the sequence information available with the recent cloning of rat and human CHTs [Okuda, Haga, Kanai, Endou, Ishihara and Katsura (2000) Nat. Neurosci. 3, 120-125; Apparsundaram, Ferguson, George Jr and Blakely (2000) Biochem. Biophys. Res. Commun. 276, 862-867; Okuda and Haga (2000) FEBS Lett. 484, 92-97], we have identified a murine CHT orthologue (mCHT) by reverse transcriptase-PCR of spinal cord mRNA and confirmed this sequence using assembled mouse genomic DNA. Inferred splice junctions for mCHT exons are conserved with those of hCHT. The mCHT cDNA encodes a protein of 580 amino acids with 93% and 98% amino acid identity to human CHT and rat CHT1, respectively. Hydropathy analysis of the predicted amino acid sequence of mCHT indicates a protein containing 13 transmembrane domains (TMDs), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analysis of mouse tissues reveals the expression of mCHT as a single transcript of approximately 5 kb with highest expression in regions that are rich in cholinergic cell bodies, e.g. the spinal cord, brainstem, mid-brain and striatum, whereas hybridization signals are absent in regions lacking cholinergic soma, e.g. the cerebellum and kidney. Expression of mCHT in COS-7 cells results in high-affinity [(3)H]HC-3-binding sites (K(d)=5 nM), and Na(+)- and Cl(-)-dependent HC-3-sensitive choline uptake (K(m)=2 microM), assessed in resealed membrane vesicles. The availability of cloned, functional mCHT and its cognate genomic DNA should prove useful for studies of mCHT regulation and should open possibilities for evaluation of CHT dysfunction in murine models.
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14 MeSH Terms
Molecular cloning of a human, hemicholinium-3-sensitive choline transporter.
Apparsundaram S, Ferguson SM, George AL, Blakely RD
(2000) Biochem Biophys Res Commun 276: 862-7
MeSH Terms: Amino Acid Sequence, Animals, Binding Sites, Biological Transport, COS Cells, Carrier Proteins, Choline, Chromosomes, Human, Pair 2, Cloning, Molecular, Hemicholinium 3, Humans, Ligands, Membrane Transport Proteins, Molecular Sequence Data, Oocytes, Protein Binding, RNA, Messenger, Radiation Hybrid Mapping, Sequence Homology, Amino Acid, Transfection, Xenopus laevis
Show Abstract · Added July 10, 2013
Under many physiological circumstances, Na(+)- and Cl(-)-dependent, hemicholinium-3 (HC-3)-sensitive, high-affinity choline uptake (HACU) in cholinergic neurons is thought to be rate-limiting in the biosynthesis of acetylcholine (ACh). Based on sequence information provided by the Human Genome Project and the recently reported rat CHT1 (rCHT1), we cloned a human CHT cDNA from spinal cord. The hCHT cDNA encodes a protein of 580 amino acids having 93% identity to rCHT1 and 51% identity to the Caenorhabditis elegans homolog CHO-1, and is distantly related to members of the Na(+)-coupled glucose transporter (SGLT) gene family of Na(+)-coupled glucose (SGLT), nucleoside and iodide transporters. Northern blot analysis reveals the expression of a approximately 5 kb transcript in human brain regions rich in cholinergic neurons including the putamen, spinal cord, and medulla. Expression of hCHT cDNA in COS-7 cells results in saturable, Na(+)/Cl(-)-dependent choline uptake (K(m) = 1.2 microM) in membrane vesicles and [(3)H] HC-3 binding (K(d) = 4 nM) in membrane fractions, consistent with characteristics reported in mammalian cholinergic neurons. Using radiation hybrid mapping techniques, we localized the hCHT gene to human chromosome 2q12. These studies elucidate the primary structure and chromosomal localization of hCHT and provide a basis for mechanistic analysis of HACU regulation and an investigation of the role of hCHT in disease states.
Copyright 2000 Academic Press.
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