Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

CHT1 (High-Affinity Choline Transporter)

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101645



Historical Background

The neurotransmitter acetylcholine (ACh) interacts with the nicotinic and muscarinic ACh receptors and is involved in a variety of physiological, behavioral, and cognitive functions in the central and peripheral nervous system. ACh is synthesized from choline and acetyl coenzyme A by choline acetyltransferase in the cholinergic presynaptic terminals. Although it has long been recognized from classic studies on ACh metabolism that exogenous choline is critical for ACh synthesis, it was not until the 1970s that the existence of high-affinity choline transporters in cholinergic neurons was postulated based on studies using isolated nerve ending particles (synaptosomes). Extracellular choline is actively transported into the presynaptic terminals by the Na+-dependent, high-affinity choline transporter in the plasma membrane and subsequently used for ACh synthesis. The high-affinity choline uptake is the rate-limiting step in ACh synthesis. Choline acetyltransferase is present in the cholinergic terminals in kinetic excess and therefore is not thought to be the rate-limiting step in ACh synthesis. The high-affinity choline transporter was first cloned in 2000 and designated as CHT1 (Okuda et al. 2000).

Structure and Functional Properties of CHT1

CHT1 is a member of the solute carrier family 5 (SLC5, sodium/glucose cotransporter family) and is also known as SLC5A7. It shares 20–25% amino acid sequence homology with other family members. Almost all SLC5 family members are Na+-coupled transporters for various solutes such as glucose, myoinositol, and iodide. CHT1 is a 13-transmembrane-spanning domain protein with an extracellular N-terminus and an intracellular C-terminus (Fig. 1). CHT1 may form homo-oligomers as determined by chemical cross-linking and immunoprecipitation assays, although the role of homo-oligomerization has been unclear (Okuda et al. 2012). CHT1 is N-linked glycosylated at asparagine residue 301, and glycosylation is necessary for substrate transport function and/or trafficking to the cell surface. CHT1 contains potential serine/threonine phosphorylation sites within the intracellular regions. CHT1 is shown to be indeed a phosphoprotein and its trafficking is regulated by phosphorylation (Gates et al. 2004). The exact phosphorylation site of CHT1 has yet to be determined. The three-dimensional structure of CHT1 is unknown; not even a single structure within the SLC5 family has been determined. However, the crystal structure of the Vibrio parahaemolyticus sodium/galactose cotransporter (vSGLT), a bacterial homolog of the sodium/glucose cotransporter, has already been reported (Faham et al. 2008). A three-dimensional homology model of CHT1 can be generated based on the vSGLT crystal structure (Okuda et al. 2012).
CHT1 (High-Affinity Choline Transporter), Fig. 1

Model of transmembrane topology of CHT1. CHT1 contains 13 transmembrane-spanning domains (marked in gray). The N-linked glycosylation site at position 301 is highlighted by a Y-shaped symbol

CHT1 has been functionally characterized by its capacity to transport choline with high affinity when expressed in cultured cells or Xenopus oocytes. CHT1 transports choline with a Km value of 1–5 μM in a Na+- and Cl-dependent manner (Okuda et al. 2000). The only known specific inhibitor of CHT1 is hemicholinium-3 (HC-3), which inhibits the choline transport in a competitive manner with a Ki value of 1–10 nM. HC-3 is hydrophilic, and a radiolabeled ligand ([3H]HC-3) is useful to assess the cell surface expression level of CHT1. Recently, the compound called ML352 was reported to inhibit CHT1-mediated choline uptake in a noncompetitive manner (Ennis et al. 2015). Iwamoto et al. performed a detailed biophysical analysis of choline transport and choline-induced current under voltage clamp conditions in Xenopus oocytes expressing human CHT1 (Iwamoto et al. 2006). They reported that Na+ was cotransported with substrate by CHT1, but Cl was not transported (Fig. 2). CHT1-mediated choline uptake is dependent on the membrane voltage and is an electrogenic process, but unlike other members of the SLC5 family, CHT1 shows variable charge/substrate stoichiometry from ~10/1 at −80 mV to 3/1 at −20 mV. CHT1-mediated choline uptake is also dependent on extracellular pH. External protons reduce the choline uptake with a pKa value of 7.4, suggesting proton titration of a functional group on the CHT1 protein (Fig. 2).
CHT1 (High-Affinity Choline Transporter), Fig. 2

Transport properties of CHT1. CHT1 mediates coupled transport of several Na+ ions and one choline molecule. The number of cotransported Na+ ions varies depending on the membrane potential. Cl is necessary for the choline transport, but is not cotransported. External protons reduce the CHT1-mediated choline transport, possibly by titration of a functional group on the protein

Regulation of CHT1 Expression/Trafficking

Immunohistochemical studies have established that CHT1 is specifically expressed in cholinergic neurons (Misawa et al. 2001). CHT1 is highly enriched in their presynaptic terminals as well as cell bodies. In the central nervous system, CHT1 is specifically expressed in regions known to contain cholinergic neurons, such as the basal forebrain, striatum, brainstem, and spinal cord. Since the cellular distribution of CHT1 correlates well with that of choline acetyltransferase or the vesicular ACh transporter, CHT1 can be considered as a cholinergic neuronal marker. In the nematode Caenorhabditis elegans, the expression of cholinergic marker genes is co-regulated by the COE (Collier, Olf, EBF)-type transcription factor UNC-3 (Kratsios et al. 2012). Several members of the COE transcription factor family are expressed in mammalian cholinergic neurons, and a conserved COE motif is found in the 5′ upstream regulatory region of the CHT1 gene. It is therefore highly likely that this transcriptional regulation is critical for cholinergic neuron-specific expression of CHT1. The expression of CHT1 gene is regulated at the transcriptional level by several factors, including nerve growth factor (NGF) and bone morphogenetic proteins (BMPs) (Berse et al. 2005; Madziar et al. 2008). In superior cervical ganglion neurons, it has been reported that an activity-dependent retrograde signal regulates the CHT1 expression (Krishnaswamy and Cooper 2009). This retrograde signal has not been identified and it is also unknown whether this regulation can be generalized to other cholinergic neurons.

Studies on CHT1 localization at the subcellular level have established that CHT1, despite functioning at the plasma membrane, is predominantly localized intracellularly at synaptic vesicles (Ferguson et al. 2003). In the rat neuromuscular junctions, more than 90% of CHT1 proteins are observed on synaptic vesicles (Nakata et al. 2004). CHT1 is constitutively internalized primarily via the clathrin-mediated endocytosis pathway (Ribeiro et al. 2003). A dileucine-like motif (Leu-531 and Val-532) located within the C-terminal cytoplasmic region of CHT1 is important for its constitutive endocytosis as demonstrated by heterologous expression systems (Ribeiro et al. 2005). Extracellular choline stimulates a dynamin-dependent internalization of CHT1, and the specific inhibitor HC-3 blocks it (Okuda et al. 2011). It has long been known that high-affinity choline uptake is acutely regulated by neuronal activity in the cholinergic nerve terminals (Simon and Kuhar 1975). In brain synaptosomes, depolarization induces a significant increase in the maximal uptake rate of choline or in the maximal binding capacity of HC-3, suggesting the increase in cell surface expression of CHT1. It is now understood that these observations are due to the fact that intracellular CHT1 residing in synaptic vesicles is translocated to the plasma membrane by exocytosis. It is considered that this neuronal activity-dependent trafficking of CHT1 is critical for sustaining ACh synthesis in cholinergic neurons.

Physiological Roles of CHT1

It has long been known that administration of HC-3 to animals causes lethal respiratory failure (Schueler 1955). CHT1 knockout mice were generated and characterized by Blakely and coworkers (Ferguson et al. 2004). The mice fail to survive the first hours of life as a result of hypoxia from a respiratory failure, and their phenotype suggests an impaired ability to sustain ACh synthesis. These results suggest that CHT1 is an essential protein to sustain cholinergic neurotransmission by transporting extracellular choline. In contrast, CHT1 heterozygous knockout mice appear normal and maintain normal levels of the high-affinity choline uptake, even though the expression levels of CHT1 protein are reduced by approximately half. However, they display deficits in endurance performance during a treadmill test (Bazalakova et al. 2007) and in attention-demanding cognitive tasks (Parikh et al. 2013). These deficits may be due to the limited intracellular pool of CHT1 available for neuronal activity-dependent vesicular trafficking that is required to sustain ACh synthesis in cholinergic neurons.

To date, there have been two reports regarding the relationship of CHT1 with human diseases, such as a distal hereditary motor neuropathy type VII and a congenital myasthenic syndrome with episodic apnea. In the former case, a dominantly acting mutation identified was a single-base deletion (c.1497delG), resulting in a near-complete deletion of the cytoplasmic C-terminal region of CHT1 (Barwick et al. 2012), and in the latter case, 11 recessive missense mutations were identified (Bauche et al. 2016). The high-affinity choline uptake activity in patients with these diseases was predicted to be diminished by mutations that impaired CHT1 function and/or trafficking.

There is a functionally relevant, nonsynonymous single nucleotide polymorphism in the coding region of the human CHT1 gene (c.265A>G, rs1013940) (Okuda et al. 2002). This polymorphism results in an isoleucine to valine substitution (p.Ile89Val, I89V) within transmembrane domain 3 of the protein. The I89V variant transporter shows a 40–50% decrease in the choline uptake rate with no change in the affinity for choline compared with the wild-type transporter, when expressed in cultured cells. This variant is considered to have a deficit in substrate translocation or reorientation of the protein and consequently has a lower transport rate. This polymorphism is reported to be associated with pediatric attention-deficit hyperactivity disorder (ADHD) (English et al. 2009) and the severity in unipolar major depressive disorder (Hahn et al. 2008).


Cholinergic neurons are endowed with the high-affinity choline transporter for ACh synthesis at the presynaptic terminals. CHT1 mediates the Na+-dependent, high-affinity choline uptake, which is the rate-limiting step in ACh synthesis. It is specifically expressed in cholinergic neurons. An important regulatory mechanism of CHT1 is neuronal activity-dependent trafficking, which is critical for sustaining ACh synthesis. A lethal impairment of ACh synthesis in CHT1 knockout mice underscores the physiological significance of CHT1 function in cholinergic neurotransmission.


  1. Barwick KE, Wright J, Al-Turki S, McEntagart MM, Nair A, Chioza B, et al. Defective presynaptic choline transport underlies hereditary motor neuropathy. Am J Hum Genet. 2012;91:1103–7. doi:10.1016/j.ajhg.2012.09.019.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bauche S, O’Regan S, Azuma Y, Laffargue F, McMacken G, Sternberg D, et al. Impaired presynaptic high-affinity choline transporter causes a congenital myasthenic syndrome with episodic apnea. Am J Hum Genet. 2016;99:753–61. doi:10.1016/j.ajhg.2016.06.033.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bazalakova MH, Wright J, Schneble EJ, McDonald MP, Heilman CJ, Levey AI, et al. Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice. Genes Brain Behav. 2007;6:411–24. doi:10.1111/j.1601-183X.2006.00269.x.CrossRefPubMedGoogle Scholar
  4. Berse B, Szczecinska W, Lopez-Coviella I, Madziar B, Zemelko V, Kaminski R, et al. Expression of high affinity choline transporter during mouse development in vivo and its upregulation by NGF and BMP-4 in vitro. Brain Res Dev Brain Res. 2005;157:132–40. doi:10.1016/j.devbrainres.2005.03.013.CrossRefPubMedGoogle Scholar
  5. English BA, Hahn MK, Gizer IR, Mazei-Robison M, Steele A, Kurnik DM, et al. Choline transporter gene variation is associated with attention-deficit hyperactivity disorder. J Neurodev Disord. 2009;1:252–63. doi:10.1007/s11689-009-9033-8.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Ennis EA, Wright J, Retzlaff CL, McManus OB, Lin Z, Huang X, et al. Identification and characterization of ML352: a novel, noncompetitive inhibitor of the presynaptic choline transporter. ACS Chem Neurosci. 2015;6:417–27. doi:10.1021/cn5001809.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Faham S, Watanabe A, Besserer GM, Cascio D, Specht A, Hirayama BA, et al. The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport. Science. 2008;321:810–4. doi:10.1126/science.1160406.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Ferguson SM, Savchenko V, Apparsundaram S, Zwick M, Wright J, Heilman CJ, et al. Vesicular localization and activity-dependent trafficking of presynaptic choline transporters. J Neurosci. 2003;23:9697–709.PubMedGoogle Scholar
  9. Ferguson SM, Bazalakova M, Savchenko V, Tapia JC, Wright J, Blakely RD. Lethal impairment of cholinergic neurotransmission in hemicholinium-3-sensitive choline transporter knockout mice. Proc Natl Acad Sci U S A. 2004;101:8762–7.CrossRefPubMedPubMedCentralGoogle Scholar
  10. Gates Jr J, Ferguson SM, Blakely RD, Apparsundaram S. Regulation of choline transporter surface expression and phosphorylation by protein kinase C and protein phosphatase 1/2A. J Pharmacol Exp Ther. 2004;310:536–45.CrossRefPubMedGoogle Scholar
  11. Hahn MK, Blackford JU, Haman K, Mazei-Robison M, English BA, Prasad HC, et al. Multivariate permutation analysis associates multiple polymorphisms with subphenotypes of major depression. Genes Brain Behav. 2008;7:487–95. doi:10.1111/j.1601-183X.2007.00384.x.CrossRefPubMedGoogle Scholar
  12. Iwamoto H, Blakely RD, De Felice LJ. 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. 2006;26:9851–9.CrossRefPubMedGoogle Scholar
  13. Kratsios P, Stolfi A, Levine M, Hobert O. Coordinated regulation of cholinergic motor neuron traits through a conserved terminal selector gene. Nat Neurosci. 2012;15:205–14. doi:10.1038/nn.2989.CrossRefGoogle Scholar
  14. Krishnaswamy A, Cooper E. An activity-dependent retrograde signal induces the expression of the high-affinity choline transporter in cholinergic neurons. Neuron. 2009;61:272–86. doi:10.1016/j.neuron.2008.11.025.CrossRefPubMedGoogle Scholar
  15. Madziar B, Shah S, Brock M, Burke R, Lopez-Coviella I, Nickel AC, et al. Nerve growth factor regulates the expression of the cholinergic locus and the high-affinity choline transporter via the Akt/PKB signaling pathway. J Neurochem. 2008;107:1284–93. doi:10.1111/j.1471-4159.2008.05681.x.CrossRefPubMedGoogle Scholar
  16. Misawa H, Nakata K, Matsuura J, Nagao M, Okuda T, Haga T. Distribution of the high-affinity choline transporter in the central nervous system of the rat. Neuroscience. 2001;105:87–98.CrossRefPubMedGoogle Scholar
  17. Nakata K, Okuda T, Misawa H. Ultrastructural localization of high-affinity choline transporter in the rat neuromuscular junction: enrichment on synaptic vesicles. Synapse. 2004;53:53–6.CrossRefPubMedGoogle Scholar
  18. Okuda T, Haga T, Kanai Y, Endou H, Ishihara T, Katsura I. Identification and characterization of the high-affinity choline transporter. Nat Neurosci. 2000;3:120–5.CrossRefPubMedGoogle Scholar
  19. Okuda T, Okamura M, Kaitsuka C, Haga T, Gurwitz D. Single nucleotide polymorphism of the human high affinity choline transporter alters transport rate. J Biol Chem. 2002;277:45315–22.CrossRefPubMedGoogle Scholar
  20. Okuda T, Konishi A, Misawa H, Haga T. Substrate-induced internalization of the high-affinity choline transporter. J Neurosci. 2011;31:14989–97. doi:10.1523/JNEUROSCI.2983-11.2011.CrossRefPubMedGoogle Scholar
  21. Okuda T, Osawa C, Yamada H, Hayashi K, Nishikawa S, Ushio T, et al. Transmembrane topology and oligomeric structure of the high-affinity choline transporter. J Biol Chem. 2012;287:42826–34. doi:10.1074/jbc.M112.405027.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Parikh V, St Peters M, Blakely RD, Sarter M. The presynaptic choline transporter imposes limits on sustained cortical acetylcholine release and attention. J Neurosci. 2013;33:2326–37. doi:10.1523/jneurosci.4993-12.2013.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Ribeiro FM, Alves-Silva J, Volknandt W, Martins-Silva C, Mahmud H, Wilhelm A, 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. 2003;87:136–46.CrossRefPubMedGoogle Scholar
  24. Ribeiro FM, Black SA, Cregan SP, Prado VF, Prado MA, Rylett RJ, et al. Constitutive high-affinity choline transporter endocytosis is determined by a carboxyl-terminal tail dileucine motif. J Neurochem. 2005;94:86–96.CrossRefPubMedGoogle Scholar
  25. Schueler FW. A new group of respiratory paralyzants. I. The “hemicholiniums”. J Pharmacol Exp Ther. 1955;115:127–43.PubMedGoogle Scholar
  26. Simon JR, Kuhar MG. Impulse-flow regulation of high affinity choline uptake in brain cholinergic nerve terminals. Nature. 1975;255:162–3.CrossRefPubMedGoogle Scholar

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Pharmacology, Faculty of PharmacyKeio UniversityTokyoJapan