Single Nucleotide Polymorphism of the Human High Affinity Choline Transporter Alters Transport Rate

Abstract

High affinity choline uptake plays a critical role in the regulation of acetylcholine synthesis in cholinergic neurons. Recently, we succeeded in molecular cloning of the high affinity choline transporter (CHT1), which is specifically expressed in cholinergic neurons. Here we demonstrate the presence of functionally relevant, nonsynonymous single nucleotide polymorphism in the human CHT1 gene by comprehensive sequence analysis of the exons and the intron/exon boundaries including the transcription start site. The deduced amino acid change for the polymorphism is isoleucine to valine at amino acid 89 (I89V) located within the third transmembrane domain of the protein. The allele frequency of I89V was 6% for Ashkenazi Jews. Functional assessment of the I89V transporter in mammalian cell lines revealed a 40 -50% decrease in V max for choline uptake rate compared with the wild type, whereas there was no alteration in the apparent affinities for choline, sodium, chloride, and the specific inhibitor hemicholinum-3. There also was no change in the specific hemicholinum-3 binding activity. The decreased choline uptake was not associated with the surface expression level of the protein as assessed by biotinylation assay. These results suggest an impaired substrate translocation in the I89V transporter. The Caenorhabditis elegans ortholog of CHT1 has a valine residue at the corresponding position and a single replacement from valine to isoleucine caused a decrease in the choline uptake rate by 40%, suggesting that this hydrophobic residue is generally critical in the choline transport rate in CHT1. This polymorphism in the allelic CHT1 gene may represent a predisposing factor for cholinergic dysfunction.

A Single Nucleotide Polymorphism Alters the Activity of the Renal Na+:Cl- Cotransporter and Reveals a Role for Transmembrane Segment 4 in Chloride and Thiazide Affinity

“…due to a decrease in the intrinsic activity of the cotransporter. A similar negative effect on the intrinsic activity or on the receptor signaling capacity has been documented for the SNP I89V and the SNP S268P that occur in the human high affinity choline transporter (37) and in the human -opioid receptor (38), respectively.…”

Lethal impairment of cholinergic neurotransmission in hemicholinium-3-sensitive choline transporter knockout mice

“…The generation and initial characterization of CHTknockout mice reported here demonstrates that choline transported by CHT contributes to ACh synthesis and is essential for sustaining cholinergic neurotransmission at levels required to support life. The recent report of a relatively common, nonsynonymous, single nucleotide polymorphism (SNP) in the coding region of human CHT that results in diminished choline transport (44), as well as the identification of nonsynonymous ChAT SNPs with clinical phenotypes (45,46), predicts that variations in absolute levels of cholinergic capacity might be relatively common across human populations and set risk thresholds for a variety of disorders or their onset͞severity. In addition to the well recognized deficits in cholinergic function in myasthenic syndromes, defects in the capacity for ACh synthesis in the CNS may contribute to cognitive dysfunction and dementia and may place demands on the regulatory mechanisms revealed by our studies that control CHT function.…”

The high-affinity choline transporter CHT1 is regulated by the ubiquitin ligase Nedd4-2

“…cDNA for the human CHT1 (15,17) and anti-CHT1 antibody (10, 17) were prepared as described previously.…”

“…The expression of CHT1 on the cell surface was directly identified by the cell surface biotinylation assay, which was performed as described previously (17). Briefly, cells grown in 6-well culture plates were transiently transfected using Lipofectamine 2000.…”

“…CHT1-mediated choline uptake is essential for postnatal viability (6). CHT1 functions at the cell surface but is predominantly localized in synaptic vesicles in cholinergic neurons and in endosomal compartments in cultured cells, while many other neurotransmitter transporters are predominantly localized at the cell surface in neurons or cultured cells (7,14,17,19). CHT1 has been reported to be internalized in a clathrin-dependent manner in which a dileucine-like motif present in the carboxyl terminus interacts with adaptor proteins (19).…”