HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Summary Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ferguson, S. M. Articles by Blakely, R. D. Search for Related Content PubMed PubMed Citation Articles by Ferguson, S. M. Articles by Blakely, R. D. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CHOLINE BITARTRATE CHOLINE CHLORIDE

The Choline Transporter Resurfaces: New Roles for Synaptic Vesicles?

¹ Neuroscience Graduate Program, Vanderbilt Brain Institute and
² Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville TN 37232

	SUMMARY

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
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.

	INTRODUCTION

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
Acetylcholine (ACh) has widespread actions as a neurotransmitter in the central, peripheral, and autonomic nervous systems, leading to decades of research into strategies to enhance or diminish cholinergic tone for therapeutic ends. Within the mammalian central nervous system (CNS), cholinergic neurons sustain or modulate diverse cognitive and appetitive processes including arousal, attention, memory, and reward (1–3). The addictive properties of nicotine arise from its direct stimulatory effects on central ionotropic ACh receptors, whereas a deficit in cholinergic neurotransmission contributes to the cognitive impairment associated with Alzheimer Disease (AD) (4–6). As degeneration of the basal forebrain cholinergic neurons that project to the cerebral cortex is thought to contribute to dementia in AD (7), enhancement of cholinergic neurotransmission with acetylcholinesterase (AChE) inhibitors is an established, although limited, therapy for managing cognitive decline (6). ACh also serves as a neurotransmitter at the mammalian neuromuscular junction (NMJ) and is essential for neuronal control of muscular contraction (8). Thus, the lethal effects of clostridial neurotoxins arise from a blockade of ACh release at the NMJ located at the diaphragm (9). Here again, reversible AChE inhibitors are clinically beneficial in disorders associated with diminished cholinergic signaling, such as myasthenia gravis. Finally, ACh mediates chemical signaling by preganglionic neurons in both the sympathetic and parasympathetic branches of the autonomic nervous system (10) and is a neurotransmitter at parasympathetic postganglionic synapses, thereby controlling the output of many organs, including the heart, lungs, exocrine and endocrine glands, gut, and bladder (11). Muscarinic receptor agonists and antagonists are used in the treatment of bladder disorders and asthma, respectively; however, the therapeutic potential of current cholinergic drugs is limited by their lack of specificity and the multitude of physiological targets (11).

The presynaptic control of acetylcholine synthesis and release has received less attention as a therapeutic strategy for ACh cholinergic function, despite the fact that the cellular mechanisms controlling ACh synthesis and release at presynaptic terminals are generally well understood and conserved across targets and phyla. At the cellular level, neuronal activity triggers vesicular fusion and the release of ACh into the synaptic cleft. Here, ACh has only a brief opportunity to activate its receptors before being hydrolyzed by AChE to generate choline and acetate (12) (Figure 1). The hydrolysis of ACh terminates cholinergic signaling but the choline is efficiently recaptured by choline transporters (CHTs) to sustain new ACh synthesis. Indeed, pharmacological studies reveal that ACh release cannot be sustained without presynaptic transporter-mediated recapture of choline (10, 13). Once transported, ACh is very efficiently resynthesized by choline acetyltransferase (ChAT) (14, 15), loaded into synaptic vesicles by the vesicular ACh transporter (VAChT) (16–18), and the cycle continues. Acetate might also be recycled at the cholinergic presynaptic terminal (19), although if so, it is by an as yet undefined mechanism. Indeed, glucose metabolism and mitochondrial acetyl-CoA production can provide the needed acetyl moiety for presynaptic ACh homeostasis (20, 21). Regardless, the ChAT K_m for choline predicts that this enzyme is not saturated by presynaptic choline levels (22), and that the rate of ACh synthesis saturates in parallel with choline transport at choline concentrations above 5 µ M (23). To enhance ACh production, neurons enhance CHT activity in response to neuronal activity (24), pointing to a mechanism that, if harnessed with novel drugs, could offer an approach to control cholinergic signaling for therapeutic ends. Below, following a review of historical findings that shaped our view of the properties and role of CHT, we describe recently elucidated mechanisms of transporter regulation that depend on the vesicular release process and outline how these findings suggest possibilities for novel molecular interventions to mitigate cholinergic deficits.

View larger version (16K): [in this window] [in a new window]   Figure 1. CHT supports presynaptic ACh synthesis. (A) Molecular structures for choline, the substrate of CHT, the neurotransmitter acetylcholine (ACh), and hemicholinium-3 (HC-3), a selective, high-affinity CHT antagonist. (B) At cholinergic presynaptic terminals, CHT functions at the plasma membrane to transport choline into the cytoplasm where it is efficiently acetylated by choline acetyltransferase (ChAT) to generate acetylcholine (ACh). This ACh is packaged into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), a second cholinergic neuron specific transporter. Upon neuron stimulation, ACh-containing vesicles fuse with the plasma membrane in response to elevated cytoplasmic [Ca2+ ]. Within the synaptic cleft, ACh is inactivated by acetylcholinesterase- (AChE)-mediated hydrolysis, and the recapture of choline by CHT is the rate-limiting step in subsequent ACh synthesis.

	A DISTINCT CHOLINE TRANSPORTER SUPPORTS SYNTHESIS OF ACETYLCHOLINE

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

In retrospect, the observations that extracellular choline is required to sustain ACh release and that choline is efficiently recaptured in a saturable, HC-3–sensitive manner provided the essential evidence that cholinergic terminals possess a unique transporter critical for neurotransmitter release. However, the identification of CHT as a molecular entity was thwarted by the low density of mammalian cholinergic terminals in many preparations, relative to more numerous, lower-affinity (but higher-capacity) choline uptake mechanisms found in surrounding cells. Indeed, the relative contribution of cholinergic neuron–specific high-affinity choline uptake to total tissue choline accumulation is small at choline concentrations greater than 10 µM, where the neuronal specific transporter becomes saturated (33, 34). Nonetheless, many important features of the transporter were revealed in metabolic studies. For example, ACh synthesis from exogenous radiolabeled-choline requires extracellular Na⁺ (33), despite the fact that substantial synaptosomal choline uptake can be achieved at high concentrations in Na⁺-free buffers. This discrepancy led to the proposal that in cholinergic terminals a distinct high-affinity Na⁺-dependent choline transporter is dedicated to providing choline for ACh synthesis (23), whereas more widely available choline transport pathways are likely to be present in all cells to support other aspects of choline metabolism, such as phosphatidylcholine synthesis. This idea was confirmed with the identification of two kinetically distinct choline transport activities in rat brain presynaptic terminals: a Na+-dependent, high-affinity (K_m = 1–2 µM), low-capacity, HC-3–sensitive (HC-3 K_d = 4–30 nM) (23, 31, 35–39) process linked to cholinergic neurons and a second, low-affinity, higher capacity, Na⁺-independent uptake process with lower HC-3 sensitivity and ubiquitous distribution (35). As indicated, the majority of the choline transported by the high-affinity, Na⁺-dependent, HC-3–sensitive mechanism is efficiently converted to ACh (31, 40, 41), suggesting it is both metabolically linked and physically proximal to sites of ACh synthesis. Additionally, choline derived from the AChE-mediated hydrolysis of ACh is transported as efficiently as radiolabeled choline (42), suggesting an as yet unappreciated relationship between AChE and CHT. These findings also indicate that CHT must be highly localized near synaptic ACh release sites, compared to other sites on the presynaptic plasma membrane, to permit efficient recapture of choline.

Other defining characteristics of CHT activity include a requirement for extracellular Cl^- and a negative membrane potential, as revealed by inhibition of choline uptake into K⁺-depolarized synaptosomes (43). We now know that a CHT utilizing similar mechanisms is expressed by all cholinergic neurons (from vertebrates and invertebrates) that have been studied (10, 23, 24, 35, 44–48). Supporting this contention, studies involving the selective destruction (i.e., lesioning) of cholinergic axons––such as those of the septal–hippocampal pathway––eliminate the ability of the target region to support Na⁺-dependent, HC-3–sensitive, high-affinity choline uptake (49–52). The low-affinity choline uptake mechanism has not been clearly defined at a molecular level but might actually be supported multiple transporters. Among several candidates, we note the activity of the organic cation transporter type 2 (OCT2) that is expressed in neurons and which can transport choline with a K_m > 100 µM (53, 54).

	A CLOSE RELATIONSHIP BETWEEN NEUROTRANSMITTER RELEASE AND CHT REGULATION

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
The definition of a cholinergic neuron–specific choline transport process that supports ACh synthesis leads reasonably to questions concerning how the regulation of the CHT parallels changes in ACh synthesis. As noted, when the CHT is functional, ACh synthesis and release are sustained, even during persistent and prolonged stimulation (10, 55). Because ACh itself is not recycled and the transport of choline is rate limiting in the synthesis of ACh, the supply of choline must increase during periods of enhanced ACh release, otherwise the presynaptic vesicle stores of ACh will become depleted. Extracellular concentrations of choline (5–10 µ M) saturate CHT even in the absence of ACh release and hydrolysis; therefore, the only way to enhance CHT-mediated choline uptake is to increase the number of functional CHTs at the presynaptic plasma membrane. Indeed, the maximal rate of CHT-mediated choline uptake (V_max) is modulated in response to chemical or electrical manipulation of cholinergic firing rates (56–61) (Table 1). For example, in vivo treatments that decrease cholinergic firing and ACh release within the rat hippocampus decrease the V_max of the CHT, as measured using synaptosomal preparations (56, 57, 62). In contrast, stimuli that enhance in vivo cholinergic firing, ACh release, or both, support an increase in the CHT V_max (56, 62, 63). The specificity of these in vivo treatments is illustrated by the fact that the general anesthetic pentobarbital, which blunts activity in the septo-hippocampal projection, decreases both hippocampal ACh release and CHT function. Meanwhile, in the striatum where cholinergic activity and ACh release are unaffected by pentobarbital, the CHT V_max is unaltered. Conversely, scopolamine inhibits presynaptic muscarinic autoreceptors in the hippocampus and striatum and enhances both ACh release and the CHT V_max in both regions. Although the CHT V_max is regulated in parallel with the modulation of ACh release in vivo, it is important to note that regulation of CHT can also be demonstrated using nerve terminal preparations in vitro following depolarizing treatments that cause the opening of voltage-gated Ca²⁺ channels, synaptic vesicle fusion, and ACh release (64–66). These examples provide ample evidence that CHT capacity parallels electrically or pharmacologically induced changes in ACh release. That these changes are physiologically relevant is suggested by behavioral paradigms wherein changes in ACh release also elicit changes in CHT activity (67–71).

	TRACKING CHT WITH [3 H]-HC-3 BINDING

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

	MOLECULAR CLONING AND CHARACTERIZATION OF CHT

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
Although more than four decades of CHT characterization ensued from its original definition, only recently have the genes that encode CHT proteins been identified. Initial evidence supporting the existence of specific CHT-encoding genes was obtained by eliciting the expression of HC-3–sensitive choline uptake in Xenopus laevis oocytes following injection of rat spinal cord mRNAs (79). Elsewhere, purification and reconstitution efforts targeted CHT activity in cholinergic neuron–rich insect ganglia (47, 80). A false lead to the identification of CHT, perhaps triggered by the dual dependence of choline transport on both extracellular Na⁺ and Cl^-, placed CHT as a member of the Na⁺/Cl^- neurotransmitter-transporter gene family that also contains the transporters for -amino butyric acid (GABA), serotonin, and dopamine (81). The gene in question turned out, however, to encode a creatine transporter (82). Unfortunately, many databases still refer to the creatine transporter as a choline transporter, or CHOT1. An ingenious yeast expression cloning strategy also identified a choline transporter, now termed CTL1, in a search for the missing CHT (83). However, its ion-dependence, kinetic and pharmacological sensitivities, and regional distribution did not fit with expectations regarding presynaptic choline uptake. A major breakthrough in this saga was achieved with the discovery of a novel transporter-like protein exhibiting cholinergic neuron specific expression in the nematode Caenorhabditis elegans (84). When expressed in X. laevis oocytes, the corresponding C. elegans cRNA (now known as CHO-1), as well as a homologous rat CHT cRNA, were able to confer HC-3–sensitive, high-affinity, Na⁺ - and Cl^- -dependent choline transport activity. These observations paved the way for the identification of CHT cDNAs and genes in multiple species, including mouse and human (84–88). Initial cloning reports used the name CHT1 to describe this gene product in humans (87, 89), rats (84), and mice (86). Since that time, analysis of genome sequencing results predicts the presence of only a single CHT gene in multiple species. For example, in addition to the characterized human (NM_021815), rat (NM_053821), mouse (NM_022025), Torpedo marmorata (AJ420808) and C. elegans CHT (NM_070138) orthologs, a single CHT-encoding gene sequence is predicted in the Drosophila melanogaster (NM_142486) and Anopheles gambiae (XM_312589) genomes. Although initial caution concerning the possibility of multiple CHT genes and isoforms led to the term CHT1, in the absence of any additional predicted CHTs, we have adopted the simple "CHT" designation, leaving additional annotation as needed for possible mRNA processing variants.

CHT is a member of the SLC5 family of mammalian Na⁺ -dependent transporters (in humans, CHT is designated as SLC5A7) that currently consists of eight members (90) and that is part of a larger sodium-solute transporter superfamily, with several hundred members identified in species from humans to bacteria (91–93). Within the SLC5 family, the best-characterized members are the Na⁺ -dependent glucose transporters (SGLTs), which participate in the intestinal and renal absorption of water and glucose (94, 95), and the Na⁺/I^- symporter (NIS), responsible for iodide uptake in the thyroid gland (96). CHT is the only member of this family with a known presynaptic function related to neurotransmitter synthesis, although another family member (SLC5A4) has been proposed to serve as a glucose sensor in cholinergic motor neurons (97) and could contribute, in an as yet not understood role, to the control of cholinergic neurotransmission.

The CHT genes identified in mice, rats, and humans reside on chromosomes 17, 9, and 2, respectively (87). The structure of mammalian CHT genes is well conserved: each has nine exons and spans ~25 kb of genomic sequence (Figure 2A) (84, 86, 87, 89). Sequence similarity between CHT and other SLC5A family members, as well as hydrophilicity analyses and topology prediction algorithms, has led to the revision of initial predictions of twelve transmembrane segments to a new prediction comprising thirteen transmembrane domains with an extracellular N terminus and a cytoplasmic C terminus (87, 91) (Figure 2B and C). A consensus site for N -linked glycosylation is present at residue N³⁰¹ in the fourth extracellular loop of human, rat, and mouse CHTs, a prediction confirmed through immunoblots from COS-7 cells transfected with either wild-type human CHT or a CHT-N301Q mutant (Figure 2D). Using electron microscopy (EM) immunogold labeling techniques, we have demonstrated that the CHT C terminus resides in the cytoplasm (66) (see also Figure 3D and E). Although this initial model offers an important framework to guide structure-based investigations of CHT regulation, further analysis into the details of CHT transmembrane topology is warranted. With respect to predictions of the CHT quaternary structure, it is worth noting that freeze-fracture analysis of SGLT1 indicates that this SLC5A family member exists as a monomer on the plasma membrane (98).

View larger version (30K): [in this window] [in a new window]   Figure 2. CHT revealed. (A) The hCHT gene structure comprises 9 exons encoding a 5 kb transcript distributed over a span of ~25 kb on chromosome 2. (B) Hydrophilicity analysis of the 580 amino-acid protein encoded by the 1740 bp coding region of hCHT predicts 13 transmembrane-spanning domains (TMDs, marked by asterisks). (C) The predicted transmembrane topology for hCHT includes an extracellular N-terminus and an intracellular C terminus. Amino acids conserved from humans through mice and rats to C. elegans are depicted with filled circles. There is a single predicted extracellular N-linked glycosylation site (N301) and multiple cytoplasmic protein kinase A and protein kinase C consensus phosphorylation sites [Reproduced with permission from (87) ]. (D) The predicted N-linked glycosylation site was converted to glutamine (N301Q mutant) by site directed mutagenesis and the resulting construct was transfected into COS-7 cells. Compared to the wild-type hCHT, immunoblot analysis of this N301Q mutant reveals a loss of the major glycosylated form of hCHT and an increase in the abundance of nonglycosylated CHT. Similar shifts in mobility have previously been observed following Peptide N-Glycosidase F (PNGase F) treatment of hCHT (66).

View larger version (128K): [in this window] [in a new window]   Figure 3. CHT colocalizes with VAChT at presynaptic terminals of cholinergic neurons. (A) In the ventral horn of the mouse spinal cord, CHT immunoreactivity is present in motor neuron cell bodies as well in the large presynaptic C-boutons that surround them (Scale bar = 20 µ m). (B) VAChT is present in the same cell bodies and terminals as CHT, confirming their cholinergic identity. (C) The distributions of CHT and VAChT overlap completely demonstrating that CHT is a constant feature of cholinergic neurons and that it is enriched at presynaptic sites. (D) Immuno-electron microscopy with CHT-specific antibodies illustrates that within an individual cholinergic presynaptic C-bouton, CHT immunoreactivity revealed by silver enhanced gold particles is predominantly associated with presynaptic vesicles and is only occasionally detected at the plasma membrane (arrows). Note that the epitope is on the cytosolic face of the plasma membrane, consistent with the predicted topology of CHT (scale bar = 1 µ m). (E) In the cell body of a cholinergic neuron, a biosynthetic pool of CHT immunoreactivity is labeled with the C-terminal epitope found on the cytoplasmic face of the rough endoplasmic reticulum (scale bar = 1 µ m). [Data adapted from (66).]

	OBSERVING CHT AT THE SYNAPSE

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
As noted, the first direct visualization of CHT distribution was accomplished using [³ H]-HC-3 autoradiography (73, 74, 102–107). As with choline uptake, the specificity of [³ H]-HC-3 autoradiographic labeling of cholinergic terminals could be validated through the specific lesioning of cholinergic pathways (52). Although autoradiography is a powerful approach—for example, in offering the opportunity to discern the fine distribution of CHTs in distinct brain subregions (108) —no synaptic-level resolution is provided, and substantial evidence indicates that HC-3 binding to CHT is subject to regulation in response to changes in cholinergic activation (75). Because the abundance of HC-3 binding sites likely reflects a combination of CHT availability and the relative activity of cholinergic neurons, problems in interpretation of drug- or disease-induced changes arise. For example, such issues may confound the interpretation of [³ H]-HC-3 binding data to measure changes in the density of cholinergic terminals in AD as both decreases (105, 109) and increases (110, 111) in CHT density have been reported in postmortem samples. These discrepant findings could reflect an overall decrease in the number of cholinergic terminals (7) in AD, balanced in a region- or age-dependent manner by compensatory counter regulation of CHT in the remaining cholinergic axons. The limited resolution of autoradiographic studies also precludes an understanding of the fine localization of CHTs, such as might support the efficient recapture of synaptic choline released after AChE-catalyzed hydrolysis.

The development of CHT-specific antibodies has led to the direct, activity-independent detection of CHTs (66, 112–115). These studies, employing multiple CHT-specific antibodies raised against distinct epitopes, have confirmed a distribution of CHT protein in register with the localization and projections of cholinergic neurons. Consistent with its presumed primary role in support of ACh synthesis, CHT immunoreactivity is concentrated at presynaptic sites where it colocalizes with VAChT (Figure 3). There is no evidence at present to indicate the presence of CHT immunoreactivity in noncholinergic neurons or glia, although expression of CHT outside the nervous system has been reported (116, 117). With CHT-specific antibodies available that can reveal sites of transporter expression in the human CNS (66, 112), new opportunities are evident to evaluate CHT abundance relative to other cholinergic markers in AD and other disorders associated with cholinergic dysfunction.

	HIDDEN POOLS OF VESICULAR CHT

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
Although CHT immunoreactivity is present both in the cell bodies and presynaptic terminals of cholinergic neurons (66, 112, 113), choline uptake activity is restricted to the presynaptic terminals (118, 119). We used immuno-EM analysis to define CHT subcellular localization and identified several distinct pools of CHTs within cholinergic neurons (66). At the cell body level, abundant CHT immunoreactivity is evident in association with the rough endoplasmic reticulum membranes (Figure 3E). This biosynthetic pool likely accounts for the CHT signal associated with cholinergic soma, because CHT immunoreactivity is not abundant at the cell soma plasmalemma. In contrast, CHT immunoreactivity is evident at the plasma membrane of presynaptic terminals, although by far the greatest density of CHT labeling is surprisingly found to be associated with intracellular vesicles (Figure 3D). The possibility exists that epitope masking may limit the visualization of pools of plasma membrane–localized CHT, although support for a chiefly vesicular localization was also obtained using biochemical fractionation and vesicle immunoisolation techniques (66). Importantly, CHT-positive vesicles were found to harbor multiple synaptic vesicle proteins and to contain ACh. Because CHT requires a Na⁺ gradient to drive choline transport, it seems unlikely that CHT functions on the vesicle membrane as a choline importer. More likely, as we discuss below, a vesicular localization is one step in a multistep process that allows linkage of vesicular fusion, ACh release, and activity-dependent increases in choline transport, localized to the synapse.

The mechanisms supporting sorting and trafficking of CHT to nerve terminals are unknown but may involve directed axonal export of CHT vesicles or the retrieval of axonal proteins following constitutive sorting to dendrites and their subsequent retention at presynaptic sites (120, 121). Furthermore, distinct subtypes of vesicles and molecular motors serve to deliver scaffolding and synaptic vesicle components to presynaptic terminals (122–124). As CHT and VAChT both come to reside on common synaptic vesicles at the nerve terminal, it is possible that they share common targeting signals from an early stage to allow for their proper and coordinated trafficking. Additionally, we speculate that CHT and VAChT may share common structural motifs for endocytic vesicle targeting. For example, the internalization and sorting of VAChT to the synaptic-like microvesicles (SLMVs) in PC12 cells is dependent on an acidic residue–flanked dileucine motif in its cytoplasmic C terminus (125, 126). A similar motif is apparent in the cytoplasmic C terminus of mammalian CHTs (I⁵²⁹, L⁵³⁰) that warrants investigation with respect to a role in the endocytosis and vesicular sorting of CHT. The abundance of CHT at the plasma membrane and the uptake of choline into CHT-transfected cells are greatly enhanced (Figure 4) when clathrin-mediated endocytosis is blocked by cotransfection of a dominant-negative mutant of dynamin I (127). Enhanced choline transport has also been reported in CHT-transfected cells in response to disruption of endocytosis with a C-terminal fragment of AP180 (128). These studies illustrate how blocking the endocytosis of a constitutively recycling CHT protein results in its accumulation on the plasma membrane and indicate that efficient CHT endocytosis may serve both to target CHT to synaptic vesicles and negatively regulate choline uptake capacity. With an improved understanding of the mechanisms recruiting CHT for internalization, the development of selective pharmacological inhibitors of CHT endocytosis could provide a means to therapeutically enhance cholinergic function.

View larger version (28K): [in this window] [in a new window]   Figure 4. CHT accumulates on the plasma membrane when constitutive endocytosis is impaired. (A) We have observed that cotransfection of COS-7 cells with hCHT plus the dominant negative K44A mutant of dynamin I (127) elicits 15.6 +/– 1.1 fold more HC-3 sensitive [3 H]-choline uptake than cotransfection of hCHT with an empty vector control. (All assays performed in triplicate, n = 3, p < 0.0005.) (B) Cell surface biotinylation confirms that hCHT accumulates on the plasma membrane when clathrin-mediated endocytosis is blocked by expression of the K44A mutant of dynamin I.

	THE CHT RESURFACES

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

View larger version (21K): [in this window] [in a new window]   Figure 5. Localization of CHT to a subset of synaptic vesicles couples plasma membrane delivery of CHT to the rate of ACh release. Although CHT functions at the plasma membrane to capture choline for ACh synthesis, recent results indicate that it is also abundantly localized to a subset of VAChT-positive, ACh-containing synaptic vesicles (66). Thus, ACh release occurs by the fusion of two distinct but equally abundant subsets of synaptic vesicles, one that contains both CHT and VAChT and a second class of vesicles containing only VAChT. At steady state in presynaptic terminals, no major pools of CHT exist on VAChT-negative vesicles; therefore, ACh release and CHT insertion into the plasma membrane are very intimately linked.

	IS TRAFFICKING ALL THERE IS TO CHT REGULATION?

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
The large reserve of CHTs on cholinergic synaptic vesicles and the Ca²⁺ -dependent, botulinum neurotoxin C–sensitive regulation of choline uptake indicates a role for trafficking in coupling CHT regulation to ACh release (66). However, previous investigations of CHT regulation have arrived at models that account for CHT regulation based on the activation of previously silent plasma membrane CHTs (132). Therefore, it is important to critically reevaluate these earlier conclusions with the hindsight gained from the more recent investigations to integrate results into a coherent model that can guide future experiments. As noted, although at steady state most CHTs reside on synaptic vesicles, relative rates of CHT exocytosis and endocytosis and the half-life of CHT at the plasma membrane have not yet been carefully quantified. Depolarization of synaptosomes with high [K⁺ ] buffers evokes immediate increases in ACh release, whereas persistent changes in CHT function only become maximal after more than ten minutes of stimulation (64, 65, 76). However, once the rate of choline uptake has been modulated, the effect persists for many minutes following removal of the depolarizing stimulus. These temporal discrepancies between CHT regulation and ACh release suggest that in addition to trafficking, maximal CHT function may require further mechanisms to either activate or stabilize the transporter at the plasma membrane (Figure 6).

View larger version (20K): [in this window] [in a new window]   Figure 6. Potential mechanisms for the regulation of CHT function. Each of these mechanisms represents a candidate target for the pharmacological enhancement of CHT function. (A) A trafficking component is supported by the large cytoplasmic reserve of CHT on cholinergic synaptic vesicles that could serve to couple choline uptake capacity to the rate of ACh release (66). CHT abundance at the plasma membrane will reflect the balance between CHT exocytosis and endocytosis. (B) There are many possible ways to regulate CHT through interacting proteins. Different binding partners could either retain CHT at the plasma membrane or lead to CHT’s recruitment into clathrin-coated pits for endocytosis. Alternatively, the activity of CHTs within the plasma membrane could be regulated by interactions between CHT and other as yet unidentified proteins. (C) In conjunction with trafficking mechanisms and protein–protein interactions for controlling CHT function, posttranslational modification of the transporter could also serve control presynaptic choline uptake. The CHT phosphorylation state could directly regulate transport activity but might also determine the affinity of interactions between CHT and various regulatory or trafficking proteins.

An example of CHT regulation that is potentially trafficking-independent involves changes in HC-3 binding triggered by arachidonic acid. The treatment of rat striatal plasma membrane–enriched homogenates with phospholipase A₂ (PLA₂) or its product, arachidonic acid, results in a twofold increase in the B_max for [³ H]-HC-3 binding (132, 136, 137). If this membrane preparation is depleted of CHT-containing vesicles, then trafficking would be precluded and the reported appearance of new HC-3 binding sites must represent the activation of previously silent plasma membrane CHTs. However, the depletion of vesicular CHT from this preparation has not been established and will require more direct confirmation. Regardless, other evidence solidifies a role for PLA₂ activity in the regulation of CHT. For instance, the effects of depolarizing pretreatments on choline uptake into both mammalian and invertebrate cholinergic neurons can be blocked by quinacrine (132) or arachidonyltrifluoromethyl ketone (138), both PLA₂ inhibitors. Cytoplasmic PLA₂ activity is calmodulin (CaM)-dependent; thus, reports of the blockade by CaM inhibitors of depolarization-stimulated increases in CHT function are consistent with a role for PLA₂ in this process (139). Each of these examples supports an important role for PLA₂ activation in the regulation of CHT in response to neuronal activity, but the exact step regulated by arachidonic acid remains elusive. Although the concept of a post-insertion, arachidonic acid–dependent CHT activation is attractive, other explanations for these results are also plausible. Treating presynaptic plasma membranes with arachidonic acid can promote the fusion of synaptic vesicles (140, 141), which could also expose previously occluded HC-3 binding sites within the lumen of vesicles. Furthermore, because quinacrine can block depolarization-stimulated ACh release from synaptosomes (142), it is premature to conclude that quinacrine also blocks a subsequent trafficking-independent step in CHT modulation. Finally, with intact striatal slices or synaptosomes, the effects of arachidonic acid are not additive with the enhancement of HC-3 binding evoked by elevated K⁺ –induced depolarization (132). These observations suggest that a common mechanism is utilized for CHT enhancement linked to the mobilization of vesicular reserve pools or subsequent CHT membrane stabilization.

In most studies, there is a strong correlation between regulation of HC-3 binding and choline transport. However, observations that the affinity for HC-3 membrane binding can be shifted between two distinct affinity states in a Mg²⁺ - and ATP-dependent manner has further complicated the interpretation of HC-3 binding (38, 143). Only one of these two HC-3 affinity states is predicted to correspond to the active form of CHT that transports choline (38). Another example of divergence between regulation of choline uptake and HC-3 binding is the chelerythrine-induced stimulation of CHT-mediated choline uptake into horseshoe crab (Limulus polyphemus) brain slices that is not paralleled by an increase in HC-3 binding (144). Although the targets of chelerythrine in Limulus have not been identified—mammalian protein kinase C isoforms are sensitive to this drug (145) —the separable effects of this treatment on choline uptake and HC-3 binding are intriguing as they support the presence of a trafficking-independent form of CHT regulation. Interestingly, a coding SNP in the human CHT that results in an I89V substitution in transmembrane domain 3 has been reported to reduce the V_max for choline uptake without affecting whole-cell HC-3 binding (146), again suggesting that choline uptake and HC-3 binding by CHT are separable processes. Further studies taking advantage of specific antibodies, surface biotinylation protocols, and mutant CHT cDNAs are needed to advance our understanding of how CHT regulation is connected to changes in HC-3 binding.

	ADDING MUSTARD TO THE MIX

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
Hemicholinium mustard (HCM) is a derivative of HC-3 that can covalently modify and irreversibly inactive CHTs on the plasma membrane of living neurons (147). The rate of recovery of choline uptake in HCM-treated cholinergic neurons (in horseshoe crab brain-slice preparations) is acutely enhanced by treatments that cause depolarization (144). This finding supports the presence of a reserve pool of HCM-insensitive CHTs tucked within these neurons whose exposure is modulated by depolarization. However, because surface-expressed CHT possessing a very low affinity HC-3 binding site would not likely bind HCM either, these studies cannot unequivocally rule out the presence of silent transporters residing in the plasma membrane awaiting activation. Interestingly, choline mustard also selectively inactivates CHT in an irreversible manner but, in addition, is a substrate for the transporter (148). Unlike HCM, choline mustard pretreatment of synaptosomes effectively blocks subsequent depolarization-induced increases in choline uptake (149). As choline mustard is transported into the presynaptic cytoplasm, it can conceivably exert its effects by alkylating both plasma membrane as well as vesicular CHTs. Taken together, these studies are compatible with a trafficking explanation for CHT regulation. However, this interpretation is also limited by uncertainty concerning the nature of the HC-3 binding sites (and whether choline mustard targets the same sites), and it remains possible that interconversion of plasma membrane pools of CHT accompanies transporter activation.

	ANIMAL MODELS FOR STUDIES OF CHT REGULATION

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
Transgenic mice overexpressing AChE exhibit increased choline uptake and HC-3 binding in the hippocampus (150, 151). Furthermore, in vivo microdialysis revealed that hippocampal extracellular ACh levels are comparable between AChE transgenic mice and wild-type controls, suggesting that the increases in CHT function might have helped to normalize cholinergic function in these mice (151). Interestingly, CHT protein levels are also increased in the striatum of AChE knockout mice (152), whereas VAChT expression and ChAT activity remained unchanged. The apparent increases in CHT function in both the AChE knockout or transgenic mice suggest that multiple steps ranging from CHT transcription to CHT trafficking, posttranslational regulation, and degradation likely conspire via CHT to control presynaptic choline availability. Altered signaling through muscarinic ACh receptors is likely to be involved in some of the changes in CHT function in response to AChE manipulations. Knockout mice for the m2 and m4 muscarinic receptors have been generated and characterized with respect to the roles of these receptors in mediating negative feedback on presynaptic ACh release (153). In light of the potential role of synaptic vesicles in the delivery of CHT to the plasma membrane, diminished inhibition of ACh release in autoreceptor mutant mice could also increase CHT abundance at the plasma membrane. Of course, presynaptic muscarinic receptors are also logical candidates for communicating altered ACh release rates to plasmalemma CHTs via possible activation mechanisms independent of trafficking as well. One additional model that might provide information about CHT regulation is the ChAT hemizygous mouse. Whereas homozygous disruption of ChAT expression is lethal (8, 154), ChAT hemizygotes are viable and fertile. The loss of a single ChAT allele represents a cholinergic neuron-specific genetic alteration that could serve as a model for testing whether functional compensation targets CHT mobilization or activation to maintain stores of presynaptic ACh.

The mouse CHT gene maps to chromosome 17C, a region that has been associated with differences in high-affinity choline uptake by quantitative trait loci analysis (155). The mapping of variability in CHT function to the vicinity of the CHT gene indicates that these results could reflect functional polymorphisms in the CHT coding region or within the CHT promoter. However, until these loci are definitively mapped to the CHT gene, it remains possible that the effects on choline uptake have arisen from other alterations in cholinergic neuron function. A separate study indicates the presence of a nonsynonymous single nucleotide polymorphism (SNP) in the murine CHT gene (156) ; however, the impact of this or other polymorphisms on CHT function remains untested. We have recently developed CHT knockout mice (157), and our initial findings indicate that these mice die within an hour of birth, with symptoms consistent with paralysis and a loss of respiratory function, as expected for deficits in cholinergic neurotransmission at the NMJ and similar to the phenotype of the ChAT knockouts (8, 154). Interestingly, the CHT hemizygotes are viable and healthy and appear to have compensated for loss of a single CHT allele through posttranslational processes. Clearly, these mice offer many opportunities to illuminate endogenous mechanisms of CHT regulation and could well serve as a model to identify potential human phenotypes associated with carriers of CHT loss-of-function alleles.

	CHT AND HUMAN DISEASE

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
No diseases with obvious cholinergic implications have been associated with the hCHT locus at chromosome 2q12 (87). Nonetheless, CHT and ChAT function in a common pathway, and the physical characteristics of mice harboring knockouts for each gene singly appear similar; therefore, mutations of either of these genes in humans might have similar consequences. Mutations in ChAT elicit congenital myasthenic syndrome associated with episodic apnea (CMS-EA) (158, 159). Possibly, other cases of idiopathic myasthenic syndromes arise from a genetically determined loss of CHT expression or activity and a subsequent deficit in ACh synthesis. The presence of a relatively common nonsynonymous hCHT SNP (I89V) that yields diminished choline uptake activity in transfected cells (146) certainly raises intriguing questions as to whether CHT may be an important risk gene for disorders bearing deficits in cholinergic function. AD patients represent one candidate population that might be vulnerable to deficits in CHT expression or regulation: a loss of nucleus basalis cholinergic neurons occurs in the brains of AD patients (7), and the symptoms of dementia can be relieved with AChE inhibitors (6). Even moderately diminished CHT-mediated choline uptake might act synergistically on the sensitized background of a loss of cholinergic neurons to exacerbate functional cholinergic deficits. The reported increase of HC-3 binding in the cerebral cortex of AD brains suggests that CHT function may undergo compensatory upregulation in order to bolster the effectiveness of the surviving cholinergic neurons (110, 111, 160). Future efforts to target drugs that can enhance CHT function would thus appear to be desirable. Rather than developing CHT antagonists, small molecules that could bind to CHT or a CHT regulatory factor and block transporter endocytosis might be ideal. Although this seems highly ambitious, some progress has already been made in developing such choline uptake enhancers, (MKC-231, for example) (161). This agent reportedly enhances HC-3-sensitive choline uptake and reverses learning deficits in a rat model of hypocholinergic function (162). It is unknown whether MKC-231 targets CHT directly or whether it causes more global activation of cholinergic neurons. With the availability of CHT antibodies and additional insights arising from subcellular studies, an answer should now be readily attainable. Furthermore, the availability of CHT expressed in model cell systems should support high-throughput screens to identify agents eliciting enhanced choline uptake independent of changes in cholinergic neuronal activation. By targeting pathways likely to participate in posttranslational control of CHT trafficking, we have succeeded in enhancing choline uptake in model systems by as much as 15-fold (Figure 4). Studies are now planned to expand this effort to chemical libraries and to ascertain whether choline uptake enhancement exerts a meaningful impact on ACh synthesis and release.

	CONCLUSIONS

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 
CHT plays an important role in supporting and regulating presynaptic ACh synthesis. However, focused studies on the cell biology and pharmacology of the CHT protein have long been limited by the challenges surrounding the identification of the CHT gene. The recent cloning of CHTs from multiple species and advances in our understanding of CHT’s subcellular localization have revealed a large reserve of CHTs on synaptic vesicles, providing an elegant mechanism to link presynaptic choline uptake to antecedent levels of neuronal activity. Although, to date, the only drugs available to target CHT have been blockers whose effects are lethal, the stage is now set to identify positive modulators of CHT that could provide relief from cholinergic deficiency. Very likely, strategies to understand how CHT enhancement can be achieved will follow from an increasingly sophisticated understanding of how CHT localization and activity are coordinated in healthy cholinergic neurons.

Randy D. Blakely, PhD, is the Allan D. Bass Professor in the Department of Pharmacology and Director of the Center for Molecular Neuroscience at Vanderbilt University. Address correspondence to RDB. Email: randy.blakely{at}vanderbilt.edu; fax (615) 936-3040.

Shawn M. Ferguson, BS, is an advanced graduate student in the Neuroscience Graduate Program at Vanderbilt University and is performing thesis research in the Blakely laboratory.

	References

TOP Summary Introduction A Distinct Choline Transporter... A Close Relationship Between... Tracking CHT With [3... Molecular Cloning and... Observing CHT at the... Hidden Pools of Vesicular... The CHT Resurfaces Is Trafficking All There... Adding Mustard to the... Animal Models for Studies... CHT and Human Disease Conclusions References

 

1. Sarter, M. and Bruno, J.P. Cognitive functions of cortical acetylcholine: Toward a unifying hypothesis. Brain Res. Brain Res. Rev. 23, 28–46 (1997).[CrossRef][Medline]
2. Perry, E., Walker, M., Grace, J. and Perry, R. Acetylcholine in mind: A neurotransmitter correlate of consciousness? Trends Neurosci. 22, 273–280 (1999).[CrossRef][Medline]
3. Kitabatake, Y., Hikida, T., Watanabe, D., Pastan, I., and Nakanishi S. Impairment of reward-related learning by cholinergic cell ablation in the striatum. Proc. Natl. Acad. Sci. U.S.A. 100, 7965–7970 (2003).[Abstract/Free Full Text]
4. Zhou, F.M., Liang, Y., and Dani, J.A. Endogenous nicotinic cholinergic activity regulates dopamine release in the striatum. Nat. Neurosci. 4, 1224–1229 (2001).[CrossRef][Medline]
5. Gray, R., Rajan, A.S., Radcliffe, K.A., Yakehiro, M., and Dani, J.A. Hippocampal synaptic transmission enhanced by low concentrations of nicotine. Nature 383, 713–716 (1996).[CrossRef][Medline]
6. Doody, R.S. Current treatments for Alzheimer’s disease: Cholinesterase inhibitors. J. Clin. Psychiatry 64 Suppl 9, 11–17 (2003).[Medline]
7. Whitehouse P.J., Price, D.L., Struble, R.G., Clark, A.W., Coyle, J.T., and Delong, M.R. Alzheimer’s disease and senile dementia: Loss of neurons in the basal forebrain. Science 215, 1237–1239 (1982). This report revealed that the basal forebrain cholinergic neurons that project to the cerebral cortex are less abundant in Alzheimer Disease (AD) brains, findings that provided rationale, along with behavioral pharmacologic studies in rodents and humans, for cholinergic-based therapies as a symptomatic treatment for AD.[Abstract/Free Full Text]
8. Misgeld, T., Burgess, R.W., Lewis, R.M., Cunningham, J.M., Lichtman, J.W., and Sanes, J.R. Roles of neurotransmitter in synapse formation: Development of neuromuscular junctions lacking choline acetyltransferase. Neuron 36, 635–648 (2002).[CrossRef][Medline]
9. Davis, L.E. Botulism. Curr. Treat. Options Neurol. 5, 23–31 (2003).[Medline]
10. Birks, R.I. and MacIntosh, F.C. Acetylcholine Metabolism of a sympathetic ganglion. Can. J. Biochem. Physiol. 39, 787–827 (1961). This comprehensive early study established that sustained neuronal synthesis of ACh is dependent on the uptake of choline by what we now know to be a CHT-mediated mechanism.
11. Hardman, J.G. and Limbird, L.E. (eds) Goodman and Gilman’s: The Pharmacological Basis of Therapeutics. McGraw-Hill, New York (2001).
12. Soreq, H. and Seidman, S. Acetylcholinesterase—new roles for an old actor. Nat. Rev. Neurosci. 2, 294–302 (2001).[CrossRef][Medline]
13. Van der Kloot, W., Molgo, J., Cameron, R., and Colasante, C. Vesicle size and transmitter release at the frog neuromuscular junction when quantal acetylcholine content is increased or decreased. J. Physiol. 541, 385–393 (2002).[Abstract/Free Full Text]
14. Hersh, L.B. Kinetic studies of the choline acetyltransferase reaction using isotope exchange at equilibrium. J. Biol. Chem. 257, 12820–12825 (1982).[Abstract/Free Full Text]
15. Engel, A.G., Ohno, K., and Sine, S.M. Sleuthing molecular targets for neurological diseases at the neuromuscular junction. Nat. Rev. Neurosci. 4, 339–352 (2003).[CrossRef][Medline]
16. Eiden, L.E. The cholinergic gene locus. J. Neurochem. 70, 2227–2240 (1998).[Medline]
17. Gilmor, M.L., Nash, N.R., Roghani, A., Edwards, R.H., Yi, H., Hersch, S.M., and Levey, A.I. Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles. J. Neurosci. 16, 2179–2190 (1996).[Abstract/Free Full Text]
18. Erickson, J.D., Varoqui, H., Schafer, M.K. et al. Functional identification of a vesicular acetylcholine transporter and its expression from a "cholinergic" gene locus. J. Biol. Chem. 269, 21929–21932 (1994).[Abstract/Free Full Text]
19. Corthay, J., Dunant, Y., Eder, L., and Loctin, F. Incorporation of acetate into acetylcholine, acetylcarnitine, and amino acids in the Torpedo electric organ. J. Neurochem. 45, 1809–1819 (1985).[CrossRef][Medline]
20. Jope, R.S. High-affinity choline transport and acetylCoA production in brain and their roles in the regulation of acetylcholine synthesis. Brain Res. 180, 313–344 (1979).[CrossRef][Medline]
21. Tucek, S. Regulation of acetylcholine synthesis in the brain. J. Neurochem. 44, 11–24 (1985).[Medline]
22. Wu, D. and Hersh, L.B. Choline acetyltransferase: Celebrating its fiftieth year. J. Neurochem. 62, 1653–1663 (1994).[Medline]
23. Haga, T. Synthesis and release of [¹⁴ C]-acetylcholine in synaptosomes. J. Neurochem. 18, 781–798 (1971). The authors characterized a link between high-affinity, Na⁺ -dependent choline uptake and ACh synthesis in synaptosomes.[Medline]
24. Kuhar, M.J. and Murrin, L.C. Sodium-dependent, high-affinity choline uptake. J. Neurochem. 30, 15–21 (1978). A review of early studies concerning the identification and characterization of presynaptic choline transport mechanisms.[Medline]
25. Jones, S.F. and Kwanbunbumpen, S. Some effects of nerve stimulation and hemicholinium on quantal transmitter release at the mammalian neuromuscular junction. J. Physiol. 207, 51–61 (1970).[Abstract/Free Full Text]
26. Potter, L.T. Synthesis, storage and release of [¹⁴ C]-acetylcholine in isolated rat diaphragm muscles. J. Physiol. 206, 145–166 (1970).[Abstract/Free Full Text]
27. Schueler, F.W. A new group of respiratory paralyzants. I. The "hemicholiniums". J. Pharmacol. Exp. Ther. 115, 127–143 (1955). The original report on the synthesis of HC-3 and the in vivo demonstration that it has lethal effects consistent with a depletion of presynaptic ACh synthesis.[Abstract/Free Full Text]
28. Birks, R.I., Macintosh, F.C., and Sastry, P.B. Pharmacological inhibition of acetylcholine synthesis. Nature 178, 1181 (1956).[Medline]
29. Freeman, J.J., Macri, J.R., Choi, R.L., and Jenden, D.J. Studies on the behavioral and biochemical effects of hemicholinium in vivo. J. Pharmacol. Exp. Ther. 210, 91–97 (1979).[Abstract/Free Full Text]
30. Yamamura, H.I. and Snyder, S.H. High-affinity transport of choline into synaptosomes of rat brain. J. Neurochem. 21, 1355–1374 (1973).[CrossRef][Medline]
31. Guyenet, P., Lefresne, P., Rossier, J., Beaujouan, J.C., and Glowinski, J. Inhibition by hemicholinium-3 of [¹⁴ C]-acetylcholine synthesis and [³ H]-choline high-affinity uptake in rat striatal synaptosomes. Mol. Pharmacol. 9, 630–639 (1973).[Abstract/Free Full Text]
32. Okuda, T. and Haga, T. High-affinity choline transporter. Neurochem. Res. 28, 483–488 (2003).[CrossRef][Medline]
33. Marchbanks, R.M. The conversion of [¹⁴ C]-choline to [¹⁴ C]-acetylcholine in synaptosomes in vitro. Biochem. Pharmaco.l 18, 1763–1766 (1969).[CrossRef][Medline]
34. Diamond, I. and Kennedy, E. Carrier-mediated transport of choline into synaptic nerve endings. J. Biol. Chem. 244, 3258–3263 (1969).[Abstract/Free Full Text]
35. Yamamura, H.I. and Snyder, S.H. Choline: High-affinity uptake by rat brain synaptosomes. Science 178, 626–628 (1972). This paper presents the results of kinetic analyses of synaptosomal choline uptake that distinguish between a Na⁺ -dependent, high-affinity choline transport mechanism supporting ACh synthesis and a lower-affinity, Na⁺ -independent choline transport mechanism.[Abstract/Free Full Text]
36. Sandberg, K. and Coyle, J.T. Characterization of [³ H]-hemicholinium-3 binding associated with neuronal choline uptake sites in rat brain membranes. Brain Res. 348, 321–330 (1985). The first demonstration that [³ H]-HC-3 membrane binding could serve as a measure of CHT abundance and/or activity.[CrossRef][Medline]
37. Kuhar, M.J. and Zarbin, M.A. Synaptosomal transport: A chloride dependence for choline, GABA, glycine and several other compounds. J. Neurochem. 31, 251–256 (1978).[Medline]
38. Ferguson, S.S. and Collier, B. Stereoselectivity of the inhibition of [3H]-hemicholinium-3 binding to the sodium-dependent high-affinity choline transporter by the enantiomers of - and ß -methylcholine. J. Neurochem. 62, 1449–1457 (1994).[Medline]
39. Chatterjee T.K., Cannon, J.G., and Bhatnagar, R.K. Characteristics of [³ H]-hemicholinium-3 binding to rat striatal membranes: Evidence for negative cooperative site-site interactions. J. Neurochem. 49, 1191–1201 (1987).[CrossRef][Medline]
40. Haga, T. and Noda, H. Choline uptake systems of rat brain synaptosomes. Biochim. Biophys. Acta 291, 564–575 (1973).[Medline]
41. Mulder, A.H., Yamamura, H.I., Kuhar, M.J., and Snyder, S.H. Release of acetylcholine from hippocampal slices by potassium depolarization: Dependence on high affinity choline uptake. Brain Res. 70, 372–376 (1974).[CrossRef][Medline]
42. Raiteri, M., Marchi, M., and Caviglia, A.M. Studies on a possible functional coupling between presynaptic acetylcholinesterase and high-affinity choline uptake in the rat brain. J. Neurochem. 47, 1696–1699 (1986).[Medline]
43. Simon, J.R. and Kuhar, M.J. High-affinity choline uptake: Ionic and energy requirements. J. Neurochem. 27, 93–99 (1976).[Medline]
44. Wetzel, G.T. and Brown, J.H. Relationships between choline uptake, acetylcholine synthesis and acetylcholine release in isolated rat atria. J. Pharmacol. Exp. Ther. 226, 343–348 (1983).[Abstract/Free Full Text]
45. Pert, C.B. and Snyder, S.H. High-affinity transport of choline into the myenteric plexus of guinea pig intestine. J. Pharmacol. Exp. Ther. 191, 102–108 (1974).[Abstract/Free Full Text]
46. Ducis, I. and Whittaker, V.P. High-affinity, sodium-gradient-dependent transport of choline into vesiculated presynaptic plasma membrane fragments from the electric organ of Torpedo marmorata and reconstitution of the solubilized transporter into liposomes. Biochim. Biophys. Acta 815, 109–127 (1985).[Medline]
47. Knipper, M., Boekhoff, I., and Breer, H. Isolation and reconstitution of the high-affinity choline carrier. FEBS Lett. 245, 235–237 (1989).[CrossRef][Medline]
48. Hoover, D.B., Ganote, C.E., Ferguson, S.M., Blakely, R.D., and Parsons, R.L. Localization of cholinergic innervation in guinea pig heart by immunohistochemistry for high affinity choline transporters. Cardiovas. Res. (in press).
49. Sethy, V.H., Kuhar, M.J., Roth, R.H., Van Woert, M.H., and Aghajanian, G.K. Cholinergic neurons: Effect of acute septal lesion on acetylcholine and choline content of rat hippocampus. Brain Res. 55, 481–484 (1973).
    The loss of choline transport after lesioning cholinergic projections to the hippocampus provided support for the selective presence of CHT on cholinergic presynaptic terminals.[CrossRef][Medline]
50. Kuhar, M.J. Neurotransmitter uptake: A tool in identifying neurotransmitter- specific pathways. Life Sci. 13, 1623–1634 (1973).[CrossRef][Medline]
51. Kuhar, M.J., DeHaven, R.N., Yamamura, H.I., Rommel-Spacher, H., and Simon, J.R. Further evidence for cholinergic habenulo-interpeduncular neurons: Pharmacologic and functional characteristics. Brain Res. 97, 265–275 (1975).[CrossRef][Medline]
52. Rossner, S., Schliebs, R., Perez-Polo, J.R., Wiley, R.G., and Bigl, V. Differential changes in cholinergic markers from selected brain regions after specific immunolesion of the rat cholinergic basal forebrain system. J. Neurosci. Res. 40, 31–43 (1995).[CrossRef][Medline]
53. Busch, A.E., Karbach, U., Miska, D. et al. Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine. Mol. Pharmacol. 54, 342–352 (1998).[Abstract/Free Full Text]
54. Sweet, D.H., Miller, D.S., and Pritchard, J.B. Ventricular choline transport: A role for organic cation transporter 2 expressed in choroid plexus. J. Biol. Chem. 276, 41611–41619 (2001).[Abstract/Free Full Text]
55. Maire, J.C. and Wurtman, R.J. Effects of electrical stimulation and choline availability on the release and contents of acetylcholine and choline in superfused slices from rat striatum. J. Physiol. 80, 189–195 (1985).
56. Simon, J.R. and Kuhar, M.G. Impulse-flow regulation of high affinity choline uptake in brain cholinergic nerve terminals. Nature 255, 162–163 (1975). An important study showing that in vivo changes in ACh release rates elicited parallel increases or decreases in rates of choline uptake.[CrossRef][Medline]
57. Atweh, S., Simon, J.R., and Kuhar, M.J. Utilization of sodium-dependent high-affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo. Life Sci. 17, 1535–1544 (1975).[CrossRef][Medline]
58. Collier, B. and Ilson, D. The effect of preganglionic nerve stimulation on the accumulation of certain analogues of choline by a sympathetic ganglion. J. Physiol. 264, 489–509 (1977).[Abstract/Free Full Text]
59. Collier, B., Kwok, Y.N., and Welner, S.A. Increased acetylcholine synthesis and release following presynaptic activity in a sympathetic ganglion. J. Neurochem. 40, 91–98 (1983). The concomitant electrical stimulation of cholinergic neurons and measurement of [³ H]-choline uptake demonstrated that CHT can be rapidly regulated during increases in neuronal activity.[Medline]
60. Lindmar, R., Loffelholz, K., Weide, W., and Witzke, J. Neuronal uptake of choline following release of acetylcholine in the perfused heart. J. Pharmacol. Exp. Ther. 215, 710–715 (1980).[Free Full Text]
61. Vaca, K. and Pilar, G. Mechanisms controlling choline transport and acetylcholine synthesis in motor nerve terminals during electrical stimulation. J. Gen. Physiol. 73, 605–628 (1979).[Abstract/Free Full Text]
62. Atweh, S.F. and. Kuhar, M.J. Effects of anesthetics and septal lesions and stimulation on [³ H]-acetylcholine formation in rat hippocampus. Eur. J. Pharmacol. 37, 311–319 (1976).[CrossRef][Medline]
63. Simon, J.R., Atweh, S., and Kuhar, M.J. Sodium-dependent high-affinity choline uptake: A regulatory step in the synthesis of acetylcholine. J. Neurochem. 26, 909–922 (1976).[CrossRef][Medline]
64. Murrin, L.C. and Kuhar, M.J. Activation of high-affinity choline uptake in vitro by depolarizing agents. Mol. Pharmacol. 12, 1082–1090 (1976).
    Stimuli that evoke ACh release from synaptosomes also regulate choline uptake, establishing a capacity for the acute regulation of CHT at presynaptic terminals.[Abstract/Free Full Text]
65. Murrin, L.C., DeHaven, R.N., and Kuhar, M.J. On the relationship between [³ H]-choline uptake activation and [³ H]-acetylcholine release. J. Neurochem. 29, 681–687 (1977).[CrossRef][Medline]
66. Ferguson, S.M., Savchenko, V., Apparsundaram, S., Zwick, M., Wright, J., Heilman, C.J., Yi, H., Levey, A.I., and Blakely, R.D. Vesicular localization and activity-dependent trafficking of presynaptic choline transporters. J. Neurosci. 23, 9697–9709 (2003). In addition to establishing that CHT is exclusively detected in cholinergic neurons, this paper characterizes a largely intracellular localization of CHT on cholinergic synaptic vesicles and demonstrates a role for this vesicular CHT reserve in the activity dependent regulation of choline uptake.[Abstract/Free Full Text]
67. Burgel, P. and Rommelspacher, H. Changes in high-affinity choline uptake in behavioral experiments. Life Sci. 23, 2423–2427 (1978).[CrossRef][Medline]
68. Wenk, G., Hepler, D., and Olton, D. Behavior alters the uptake of [³ H]-choline into acetylcholinergic neurons of the nucleus basalis magnocellularis and medial septal area. Behav. Brain Res. 13, 129–138 (1984). In this study, both rapid and long lasting changes in choline uptake were observed in the hippocampus and neocortex in response to a variety of behavioral tasks.[CrossRef][Medline]
69. Lai, H. and Carino, M.A. Effects of noise on high-affinity choline uptake in the frontal cortex and hippocampus of the rat are blocked by intracerebroventricular injection of corticotropin-releasing factor antagonist. Brain Res. 527, 354–358 (1990).[CrossRef][Medline]
70. Toumane, A., Durkin, T., Marighetto, A., Galey, D., and Jaffard, R. Differential hippocampal and cortical cholinergic activation during the acquisition, retention, reversal and extinction of a spatial discrimination in an 8-arm radial maze by mice. Behav. Brain Res. 30, 225–234. (1988).[CrossRef][Medline]
71. Lebrun, C., Durkin, T.P., Marighetto, A., and Jaffard, R.A comparison of the working memory performances of young and aged mice combined with parallel measures of testing and drug-induced activations of septo-hippocampal and nbm-cortical cholinergic neurones. Neurobiol. Aging 11, 515–521 (1990).[CrossRef][Medline]
72. O’Regan, S. Binding of [³ H]-hemicholinium-3 to the high-affinity choline transporter in electric organ synaptosomal membranes. J. Neurochem. 51, 1682–1688 (1988).[CrossRef][Medline]
73. Rainbow, T.C., Parsons, B., and Wieczorek, C.M. Quantitative autoradiography of [³ H]-hemicholinium-3 binding sites in rat brain. Eur. J. Pharmacol. 102, 195–196 (1984).[CrossRef][Medline]
74. Quirion, R. Characterization and autoradiographic distribution of hemicholinium-3 high-affinity choline uptake sites in mammalian brain. Synapse 1, 293–303 (1987).[CrossRef][Medline]
75. Lowenstein, P.R. and Coyle, J.T. Rapid regulation of [³ H]-hemicholinium-3 binding sites in the rat brain. Brain Res. 381, 191–194 (1986). The observation is made that the maximum capacity for HC-3 binding can be rapidly regulated and that this binding measurement likely reflects the previous in vivo activity of cholinergic neurons rather than simply CHT abundance.[CrossRef][Medline]
76. Saltarelli, M.D., Lowenstein, P.R., and Coyle, J.T. Rapid in vitro modulation of [³ H]-hemicholinium-3 binding sites in rat striatal slices. Eur. J. Pharmacol. 135, 35–40 (1987).[CrossRef][Medline]
77. Yamada, K., Saltarelli, M.D., and Coyle, J.T. [³ H]-hemicholinium-3 binding in rats with status epilepticus induced by lithium chloride and pilocarpine. Eur. J. Pharmacol. 195, 395–397 (1991).[CrossRef][Medline]
78. Swann, A.C. and Hewitt, L.O. Hemicholinium-3 binding: Correlation with high-affinity choline uptake during changes in cholinergic activity. Neuropharmacology 27, 611–615 (1988).[CrossRef][Medline]
79. Blakely, R.D., Robinson, M.B., and Amara, S.G. Expression of neurotransmitter transport from rat brain mRNA in Xenopus laevis oocytes. Proc. Natl. Acad. Sci. 85, 9846–9850 (1988).[Abstract/Free Full Text]
80. Knipper, M., Kahle, C., and Breer, H. Purification and reconstitution of the high affinity choline transporter. Biochim. Biophys. Acta 1065, 107–113 (1991).[Medline]
81. Mayser, W., Schloss, P., and Betz, H. Primary structure and functional expression of a choline transporter expressed in the rat nervous system. FEBS Lett. 305, 31–36 (1992).[CrossRef][Medline]
82. Schloss, P., Mayser, W., and Betz, H. The putative rat choline transporter CHOT1 transports creatine and is highly expressed in neural and muscle-rich tissues. Biochem. Biophys. Res. Commun. 198, 637–645 (1994).[CrossRef][Medline]
83. O’Regan, S., Traiffort, E., Ruat, M., Cha, N., Compaore, D., and Meunier, F.M. An electric lobe suppressor for a yeast choline transport mutation belongs to a new family of transporter-like proteins. Proc. Natl. Acad. Sci. U.S.A. 97, 1835–1840 (2000).[Abstract/Free Full Text]
84. Okuda, T., Haga, T., Kanai, Y., Endou, H., Ishihara, T., and Katsura, I. Identification and characterization of the high-affinity choline transporter. Nat. Neurosci. 3, 120–125 (2000). This landmark study represents the first molecular identification and cloning of presynaptic, HC-3-sensitive choline transporter cDNAs.[CrossRef][Medline]
85. Wang, Y., Cao, Z., Newkirk, R.F., Ivy, M.T., and Townsel, J.G. Molecular cloning of a cDNA for a putative choline co-transporter from Limulus CNS. Gene 268, 123–131 (2001).[CrossRef][Medline]
86. Apparsundaram, S., Ferguson, S.M., and Blakely, R.D. Molecular cloning and characterization of a murine hemicholinium-3- sensitive choline transporter. Biochem. Soc. Trans. 29, 711–716 (2001).[CrossRef][Medline]
87. Apparsundaram, S., Ferguson, S.M., George, A.L., Jr., and Blakely, R.D. Molecular cloning of a human, hemicholinium-3-sensitive choline transporter. Biochem. Biophys. Res. Commun. 276, 862–867 (2000). The first report of a human CHT cDNA, in addition to providing a novel assay of CHT function, the human CHT gene was localized to chromosome 2q12.[CrossRef][Medline]
88. Guermonprez, L., O’Regan, S., Meunier, F.M., and Morot-Gaudry-Talarmain, Y. The neuronal choline transporter CHT1 is regulated by immunosuppressor-sensitive pathways. J. Neurochem. 82, 874–884 (2002).[CrossRef][Medline]
89. Okuda, T. and Haga, T. Functional characterization of the human high-affinity choline transporter. FEBS Lett. 484, 92–97 (2000).[CrossRef][Medline]
90. Wright, E.M. and Turk, E. The sodium/glucose cotransport family SLC5. Pflugers Arch. (2003).
91. Turk, E. and Wright, E.M. Membrane topology motifs in the SGLT cotransporter family. J. Membr. Biol. 159, 1–20 (1997).[CrossRef][Medline]
92. Jung, H. Towards the molecular mechanism of Na⁺/solute symport in prokaryotes. Biochim. Biophys. Acta 1505, 131–143 (2001).[Medline]
93. Jung, H. The sodium/substrate symporter family: Structural and functional features. FEBS Lett. 529, 73–77 (2002).[CrossRef][Medline]
94. Wright, E.M., Turk, E., and Martin, M.G. Molecular basis for glucose-galactose malabsorption. Cell Biochem. Biophys. 36, 115–121 (2002).[CrossRef][Medline]
95. Wright, E.M. Renal Na⁺ -glucose cotransporters. Am. J. Physiol. Renal Physiol. 280, F10–F18 (2001).[Abstract/Free Full Text]
96. Dohan, O., De la Vieja, A., Paroder, V., Riedel, C., Artani, M., Reed, M., Ginter, C.S. and Carrasco, N. The sodium/iodide Symporter (NIS): Characterization, regulation, and medical significance. Endocr. Rev. 24, 48–77 (2003).[Abstract/Free Full Text]
97. Diez-Sampedro, A., Hirayama, B.A., Osswald, C., Gorboulev, V., Baumgarten, K., Volk, C., Wright, E.M., and Koepsell, H. A glucose sensor hiding in a family of transporters. Proc. Natl. Acad. Sci. U.S.A. 100, 11753–11758 (2003).[Abstract/Free Full Text]
98. Eskandari, S., Wright, E.M., Kreman, M., Starace, D.M., and Zampighi, G.A. Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy. Proc. Natl. Acad. Sci. U.S.A. 95, 11235–11240 (1998).[Abstract/Free Full Text]
99. Gates, J., Ferguson, S.M., Blakely, R.D., and Apparsundaram, S. Phosphorylation and trafficking of the hemicholinum-3 sensitive choline transporter. Soc. Neurosci. Abstr. 28, 144.122 (2002).
100. Bauman, A.L., Apparsundaram, S., Ramamoorthy, S., Wadzinski, B.E., Vaughan, R.A., and Blakely, R.D. Cocaine- and antidepressant-sensitive biogenic amine transporters exist in regulated complexes with protein phosphatase 2A. J. Neurosci. 20, 7571–7578 (2000).[Abstract/Free Full Text]
101. Issa, A.M., Gauthier, S., and Collier, B. Effects of the phosphatase inhibitors calyculin A and okadaic acid on acetylcholine synthesis and content of rat hippocampal formation. J. Neurochem. 66, 1924–1932 (1996).[Medline]
102. Manaker, S., Wieczorek, C.M., and Rainbow, T.C. Identification of sodium-dependent, high-affinity choline uptake sites in rat brain with [³ H]-hemicholinium-3. J. Neurochem. 46, 483–488 (1986).[Medline]
103. Vickroy, T.W., Roeske, W.R., Gehlert, D.R., Wamsley, J.K., and Yamamura, H.I. Quantitative light microscopic autoradiography of [³ H]-hemicholinium-3 binding sites in the rat central nervous system: A novel biochemical marker for mapping the distribution of cholinergic nerve terminals. Brain Res. 329, 368–373 (1985).[CrossRef][Medline]
104. Pascual, J., Gonzalez, A.M., and Pazos, A. Autoradiographic distribution of [³ H]-hemicholinium-3 binding sites in human brain. Brain Res. 505, 306–310 (1989).[CrossRef][Medline]
105. Rodriguez-Puertas, R., Pazos, A., Zarranz, J.J., and Pascual, J. Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer’s disease: An autoradiographic study using [³ H]-hemicholinium-3. J. Neural. Transm. Park. Dis. Dement. Sect. 8, 161–169 (1994).[CrossRef][Medline]
106. Lowenstein, P.R., Slesinger, P.A., Singer, H.S., Walker, L.C., Casanova, M.F., Raskin, L.S., Price, D.L., and Coyle, J.T. Compartment-specific changes in the density of choline and dopamine uptake sites and muscarinic and dopaminergic receptors during the development of the baboon striatum: A quantitative receptor autoradiographic study. J. Comp. Neurol. 288, 428–446 (1989).[CrossRef][Medline]
107. Bekenstein, J.W., and Wooten, G.F. Hemicholinium-3 binding sites in rat brain: A quantitative autoradiographic study. Brain Res. 481, 97–105 (1989).[CrossRef][Medline]
108. Lowenstein P.R., Joyce, J.N., Coyle, J.T., and Marshall, J.F. Striosomal organization of cholinergic and dopaminergic uptake sites and cholinergic M1 receptors in the adult human striatum: A quantitative receptor autoradiographic study. Brain Res. 510, 122–126 (1990).[CrossRef][Medline]
109. Pascual, J., Fontan, A., Zarranz, J.J., Berciano, J., Florez, J., and Pazos, A. High-affinity choline uptake carrier in Alzheimer’s disease: Implications for the cholinergic hypothesis of dementia. Brain Res. 552, 170–174 (1991).[CrossRef][Medline]
110. Bissette, G., Seidler, F.J., Nemeroff, C.B., and Slotkin, T.A. High-affinity choline transporter status in Alzheimer’s disease tissue from rapid autopsy. Ann. N.Y. Acad. Sci. 777, 197–204 (1996).[Medline]
111. Slotkin, T.A., Nemeroff, C.B., Bissette, G., and Seidler, F.J. Overexpression of the high-affinity choline transporter in cortical regions affected by Alzheimer’s disease. Evidence from rapid autopsy studies. J. Clin. Invest. 94, 696–702 (1994).
112. Kus, L., Borys, E., Ping Chu, Y., Ferguson, S.M., Blakely, R.D., Emborg, M.E., Kordower, J.H., Levey, A.I. and Mufson, E.J. Distribution of high-affinity choline transporter immunoreactivity in the primate central nervous system. J. Comp. Neurol. 463, 341–357 (2003).[CrossRef][Medline]
113. Lips, K.S., Pfeil, U., Haberberger, R.V., and Kummer, W. Localisation of the high-affinity choline transporter-1 in the rat skeletal motor unit. Cell Tissue Res. 307, 275–280 (2002).[CrossRef][Medline]
114. Misawa, H., Nakata, K., Matsuura, J., Nagao, M., Okuda, T., and Haga, T. Distribution of the high-affinity choline transporter in the central nervous system of the rat. Neuroscience 105, 87–98 (2001). This study found that the distribution of CHT immunoreactivity was consistent with the selective expression of CHT in all major classes of cholinergic neurons.[CrossRef][Medline]
115. Kobayashi, Y., Okuda, T., Fujioka, Y., Matsumura, G., Nishimura, Y., and Haga, T. Distribution of the high-affinity choline transporter in the human and macaque monkey spinal cord. Neurosci. Lett. 317, 25–28 (2002).[CrossRef][Medline]
116. Haberberger, R.V., Pfeil, U., Lips, K.S., and Kummer, W. Expression of the high-affinity choline transporter, CHT1, in the neuronal and non-neuronal cholinergic system of human and rat skin. J. Invest. Dermatol. 119, 943–948 (2002).[CrossRef][Medline]
117. Song, P., Sekhon, H.S., Jia Y., Keller, J.A., Blusztajn, J.K., Mark, G.P., and Spindel, E.R. Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma. Cancer Res. 63, 214–221 (2003).[Abstract/Free Full Text]
118. Bussiere, M., Vance, J.E., Campenot, R.B., and Vance, D.E. Compartmentalization of choline and acetylcholine metabolism in cultured sympathetic neurons. J. Biochem. 130, 561–568 (2001).[Abstract/Free Full Text]
119. Suszkiw, J.B. and Pilar, G. Selective localization of a high-affinity choline uptake system and its role in ACh formation in cholinergic nerve terminals. J. Neurochem. 26, 1133–1138 (1976).[CrossRef][Medline]
120. Gu, C., Jan, Y.N., and Jan, L.Y. A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels. Science 301, 646–649 (2003).[Abstract/Free Full Text]
121. Sampo, B., Kaech, S., Kunz, S., and Banker, G. Two distinct mechanisms target membrane proteins to the axonal surface. Neuron 37, 611–624 (2003).[CrossRef][Medline]
122. J. M. Scholey. Rafting along the axon on Unc104 motors. Dev. Cell 2, 515–516 (2002).[CrossRef][Medline]
123. Zhai, R.G., Vardinon-Friedman, H., Cases-Langhoff, C., Becker, B., Gundelfinger, E.D., Ziv, N.E., and Garner, C.C. Assembling the presynaptic active zone: A characterization of an active one-precursor vesicle. Neuron 29, 131–143 (2001).[CrossRef][Medline]
124. Yonekawa, Y., Harada, A., Okada, Y., Funakoshi, T., Kanai, Y., Takei, Y., Terada, S., Noda, T., and Hirokawa, N. Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice. J. Cell Biol. 141, 431–441 (1998).[Abstract/Free Full Text]
125. Tan, P.K., Waites, C., Liu, Y., Krantz, D.E., and Edwards, R.H. A leucine-based motif mediates the endocytosis of vesicular monoamine and acetylcholine transporters. J. Biol. Chem. 273, 17351–17360 (1998).[Abstract/Free Full Text]
126. Krantz, D.E., Waites, C., Oorschot, V., Liu, Y., Wilson, R.I., Tan, P.K., Klumperman, J., and Edwards, R.H. A phosphorylation site regulates sorting of the vesicular acetylcholine transporter to dense core vesicles. J. Cell Biol. 149, 379–396 (2000).[Abstract/Free Full Text]
127. van der Bliek, A.M., Redelmeier, T.E., Damke, H., Tisdale, E.J., Meyerowitz, E.M., and Schmid, S.L. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J. Cell Biol. 122, 553–563 (1993).[Abstract/Free Full Text]
128. Ribeiro, F.M., Alves-Silva, J., Volknandt, W. et al. The hemicholinium-3 sensitive high-affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles. J. Neurochem. 87, 136–146 (2003).[CrossRef][Medline]
129. Blasi, J., Chapman, E.R., Yamasaki, S., Binz, T., Niemann, H., and Jahn, R. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin. EMBO J. 12, 4821–4828 (1993).[Medline]
130. O’Leary, M.E. and Suszkiw, J.B. Effect of colchicine on 45Ca and choline uptake, and acetylcholine release in rat brain synaptosomes. J. Neurochem. 40, 1192–1195 (1983).[CrossRef][Medline]
131. Burgoyne, R.D. and Cumming, R. Taxol stabilizes synaptosomal microtubules without inhibiting acetylcholine release. Brain Res. 280, 190–193 (1983).[CrossRef][Medline]
132. Saltarelli, M.D., Yamada, K., and Coyle, J.T. Phospholipase A2 and ³ H-hemicholinium-3 binding sites in rat brain: A potential second-messenger role for fatty acids in the regulation of high-affinity choline uptake. J. Neurosci. 10, 62–72 (1990).[Abstract]
133. Somwar, R., Niu, W., Kim, D.Y., Sweeney, G., Randhawa, V.K., Huang, C., Ramlal, T., and Klip, A. Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport. J. Biol. Chem. 276, 46079–46087 (2001).[Abstract/Free Full Text]
134. Dowd, B.F. and Forbush, B. PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1). J. Biol. Chem. 278, 27347–27353 (2003).[Abstract/Free Full Text]
135. Apparsundaram, S., Sung, U., Price, R.D., and Blakely, R.D. Trafficking-dependent and -independent pathways of neurotransmitter transporter regulation differentially involving p38 mitogen-activated protein kinase revealed in studies of insulin modulation of norepinephrine transport in SK-N-SH cells. J. Pharmacol. Exp. Ther. 299, 666–677 (2001).[Abstract/Free Full Text]
136. Yamada, K., Saltarelli, M.D., and Coyle, J.T. Involvement of phospholipase A2 in the regulation of [³ H]-hemicholinium-3 binding. Biochem. Pharmacol. 37, 4367–4373 (1988).[CrossRef][Medline]
137. Yamada, K., Saltarelli, M.D., and Coyle, J.T. Specificity of the activation of [³ H]-hemicholinium-3 binding by phospholipase A2. J. Pharmacol. Exp. Ther. 249, 836–842 (1989).[Abstract/Free Full Text]
138. Ford, B.D., Ivy, M.T., Mtshali, C.P., and Townsel, J.G. The involvement of protein kinase C in the regulation of choline cotransport in Limulus. Comp. Biochem. Physiol. Mol. Integr. Physiol. 123, 255–261 (1999).
139. Yamada, K., Saltarelli, M.D. and Coyle, J.T. Effects of calmodulin antagonists on sodium-dependent high-affinity choline uptake. Brain Res. 542, 132–134. (1991).[CrossRef][Medline]
140. Nishio, H., Takeuchi, T., Hata, F., and Yagasaki, O. Ca²⁺ -independent fusion of synaptic vesicles with phospholipase A2–treated presynaptic membranes in vitro. Biochem. J. 318, 981–987 (1996).
141. Schiavo, G., Matteoli, M., and Montecucco, C. Neurotoxins affecting neuroexocytosis. Physiol. Rev. 80, 717–766 (2000).[Abstract/Free Full Text]
142. Baba, A., Ohta, A., and Iwata, H. Inhibition by quinacrine of depolarization-induced acetylcholine release and calcium influx in rat brain cortical synaptosomes. J. Neurochem. 40, 1758–1761 (1983).[CrossRef][Medline]
143. Chatterjee, T.K. and Bhatnagar, R.K. Ca²⁺ -dependent, ATP-induced conversion of the [³ H]-hemicholinium-3 binding sites from high- to low-affinity states in rat striatum: Effect of protein kinase inhibitors on this affinity conversion and synaptosomal choline transport. J. Neurochem. 54, 1500–1508 (1990). This is the first report of multiple, regulated affinity states for the HC-3 binding site on CHT.[CrossRef][Medline]
144. Ivy, M.T., Newkirk, R.F., Karim, M.R., Mtshali, C.M., and Townsel, J.G. Hemicholinium-3 mustard reveals two populations of cycling choline cotransporters in Limulus. Neuroscience 102, 969–978 (2001).[CrossRef][Medline]
145. Herbert, J.M., Augereau, J.M., Gleye, J., and Maffrand, J.P. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 172, 993–999 (1990).[CrossRef][Medline]
146. Okuda, T., Okamura, M., Kaitsuka, C., Haga, T., and Gurwitz, D. Single nucleotide polymorphism of the human high-affinity choline transporter alters transport rate. J. Biol. Chem. 277, 45315–45322 (2002). In this paper, the authors characterize the functional consequences of a coding SNP in CHT that may be quite common in some human populations.[Abstract/Free Full Text]
147. Gylys, K.H., Abdalah, I., and Jenden, D.J. Selectivity of hemicholinium mustard, an affinity ligand, for the high-affinity choline transport system. Neuropharmacology 36, 1741–1746 (1997).[CrossRef][Medline]
148. Rylett, R.J. and Walters, S.A. Uptake and metabolism of [³ H]-choline mustard by cholinergic nerve terminals from rat brain. Neuroscience 36, 483–489 (1990).[CrossRef][Medline]
149. Ferguson, S.S., Rylett, R.J., and Collier, B. Regulation of rat brain synaptosomal [³ H]-hemicholinium-3 binding and [³ H]-choline transport sites following exposure to choline mustard aziridinium ion. J. Neurochem. 63, 1328–1337 (1994).[Medline]
150. Beeri, R., Le Novere, N., Mervis, R., Huberman, T., Grauer, E., Changeux, J.P., and Soreq, H. Enhanced hemicholinium binding and attenuated dendrite branching in cognitively impaired acetylcholinesterase-transgenic mice. J. Neurochem. 69, 2441–2451 (1997).
     The first report of altered CHT regulation as a major compensatory response to a genetic manipulation of cholinergic function.[Medline]
151. Erb, C., Troost, J., Kopf, S., Schmitt, U., Loffelholz, K., Soreq, H., and Klein, J. Compensatory mechanisms enhance hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase. J. Neurochem. 77, 638–646 (2001).[CrossRef][Medline]
152. Volpicelli-Daley, L.A., Hrabovska, A., Duysen, E.G., Ferguson, S.M., Blakely, R.D., Lockridge, O., and Levey, A.I. Altered striatal function and muscarinic cholinergic receptors in acetylcholinesterase knockout mice. Mol. Pharmacol. 64, 1309–1316 (2003).[Abstract/Free Full Text]
153. Zhang, W., Basile, A.S., Gomeza, J., Volpicelli, L.A., Levey, A.I., and Wess, J. Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice. J. Neurosci. 22, 1709–1717 (2002).[Abstract/Free Full Text]
154. Brandon, E.P., Lin, W., D’Amour, K.A. et al. Aberrant patterning of neuromuscular synapses in choline acetyltransferase–deficient mice. J. Neurosci. 23, 539–549 (2003).[Abstract/Free Full Text]
155. Tarricone, B.J., Hwang, W.G., Hingtgen, J.N., Mitchell, S.R., Belknap, J.K., and Nurnberger, J.I., Jr. Identification of a locus on mouse chromosome 17 associated with high-affinity choline uptake using BXD recombinant inbred mice and quantitative trait loci analysis. Genomics 27, 161–164 (1995).[CrossRef][Medline]
156. Marshall, K.E., Godden, E.L., Yang, F., Burgers, S., Buck, K.J., and Sikela, J.M. In silico discovery of gene-coding variants in murine quantitative trait loci using strain-specific genome sequence databases. Genome Biol. 3, 78 (2002).
157. Bazalakova, M., Ferguson, S.M., Savchenko, V., Wright, J., and Blakely, R.D. Characterization of the choline transporter knockout mouse. Soc. Neurosci. Abstr. 29, 580–586 (2003).
158. Ohno, K., Tsujino, A., Brengman, J.M. et al. Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans. Proc. Natl. Acad. Sci. U.S.A. 98, 2017–2022 (2001). This is the first paper to show that human mutations that impair ACh synthesis lead to a potentially lethal myasthenic syndrome.[Abstract/Free Full Text]
159. Maselli, R.A., Chen, D., Mo, D., Bowe, C., Fenton, G., and Wollmann, R.L. Choline acetyltransferase mutations in myasthenic syndrome due to deficient acetylcholine resynthesis. Muscle Nerve 27, 180–187 (2003).[CrossRef][Medline]
160. Slotkin, T.A., Seidler, F.J., Crain, B.J., Bell, J.M., Bissette, G., and Nemeroff, C.B. Regulatory changes in presynaptic cholinergic function assessed in rapid autopsy material from patients with Alzheimer disease: Implications for etiology and therapy. Proc. Natl. Acad. Sci. U.S.A. 87, 2452–2455 (1990).[Abstract/Free Full Text]
161. Murai, S., Saito, H., Abe, E., Masuda, Y., Odashima, J., and Itoh, T. MKC–231, a choline uptake enhancer, ameliorates working memory deficits and decreased hippocampal acetylcholine induced by ethylcholine aziridinium ion in mice. J. Neural. Transm. Gen. Sect. 98, 1–13 (1994).[CrossRef][Medline]
162. Bessho, T., Takashina, K., Tabata, R., Ohshima, C., Chaki, H., Yamabe, H., Egawa, M., Tobe, A., and Saito, K. Effect of the novel high-affinity choline uptake enhancer 2-(2-oxopyrrolidin-1-yl)-N -(2,3-dimethyl-5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-yl) acetoamide on deficits of water maze learning in rats. Arzneimittelforschung 46, 369–373 (1996).[Medline]
163. Collier, B. and Katz, H.S. Acetylcholine synthesis from recaptured choline by a sympathetic ganglion. J. Physiol. 238, 639–655 (1974).[Abstract/Free Full Text]
164. Saltarelli, M.D., Lopez, J., Lowenstein, P.R., and Coyle, J.T. The role of calcium in the regulation of [³ H]-hemicholinium-3 binding sites in rat brain. Neuropharmacology 27, 1301–1308 (1988).[CrossRef][Medline]
165. Knipper, M., Kahle, C., and Breer, H. Regulation of hemicholinium binding sites in isolated nerve terminals. J. Neurobiol. 23, 163–172 (1992).[CrossRef][Medline]

This article has been cited by other articles:

				M. Pinthong, S. A. G. Black, F. M. Ribeiro, C. Pholpramool, S. S. G. Ferguson, and R. J. Rylett Activity and Subcellular Trafficking of the Sodium-Coupled Choline Transporter CHT Is Regulated Acutely by Peroxynitrite Mol. Pharmacol., March 1, 2008; 73(3): 801 - 812. [Abstract] [Full Text] [PDF]

				D. B. Goodenowe, L. L. Cook, J. Liu, Y. Lu, D. A. Jayasinghe, P. W. K. Ahiahonu, D. Heath, Y. Yamazaki, J. Flax, K. F. Krenitsky, et al. Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer's disease and dementia J. Lipid Res., November 1, 2007; 48(11): 2485 - 2498. [Abstract] [Full Text] [PDF]

				G. P. Mullen, E. A. Mathews, M. H. Vu, J. W. Hunter, D. L. Frisby, A. Duke, K. Grundahl, J. D. Osborne, J. A. Crowell, and J. B. Rand Choline Transport and de novo Choline Synthesis Support Acetylcholine Biosynthesis in Caenorhabditis elegans Cholinergic Neurons Genetics, September 1, 2007; 177(1): 195 - 204. [Abstract] [Full Text] [PDF]

				H. Iwamoto, R. D. Blakely, and L. J. De Felice Na+, Cl-, and pH Dependence of the Human Choline Transporter (hCHT) in Xenopus Oocytes: The Proton Inactivation Hypothesis of hCHT in Synaptic Vesicles J. Neurosci., September 27, 2006; 26(39): 9851 - 9859. [Abstract] [Full Text] [PDF]

				D. S. Matthies, P. A. Fleming, D. M. Wilkes, and R. D. Blakely The Caenorhabditis elegans choline transporter CHO-1 sustains acetylcholine synthesis and motor function in an activity-dependent manner. J. Neurosci., June 7, 2006; 26(23): 6200 - 6212. [Abstract] [Full Text] [PDF]

				V. Michel, Z. Yuan, S. Ramsubir, and M. Bakovic Choline Transport for Phospholipid Synthesis. Experimental Biology and Medicine, May 1, 2006; 231(5): 490 - 504. [Abstract] [Full Text] [PDF]

				S. Apparsundaram, V. Martinez, V. Parikh, R. Kozak, and M. Sarter Increased Capacity and Density of Choline Transporters Situated in Synaptic Membranes of the Right Medial Prefrontal Cortex of Attentional Task-Performing Rats J. Neurosci., April 13, 2005; 25(15): 3851 - 3856. [Abstract] [Full Text] [PDF]

				J. Gates Jr., S. M. Ferguson, R. D. Blakely, and S. Apparsundaram Regulation of Choline Transporter Surface Expression and Phosphorylation by Protein Kinase C and Protein Phosphatase 1/2A J. Pharmacol. Exp. Ther., August 1, 2004; 310(2): 536 - 545. [Abstract] [Full Text] [PDF]

				S. M. Ferguson, M. Bazalakova, V. Savchenko, J. C. Tapia, J. Wright, and R. D. Blakely Lethal impairment of cholinergic neurotransmission in hemicholinium-3-sensitive choline transporter knockout mice PNAS, June 8, 2004; 101(23): 8762 - 8767. [Abstract] [Full Text] [PDF]

This Article Summary Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ferguson, S. M. Articles by Blakely, R. D. Search for Related Content PubMed PubMed Citation Articles by Ferguson, S. M. Articles by Blakely, R. D. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CHOLINE BITARTRATE CHOLINE CHLORIDE