A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation.
The human serotonin transporter (hSERT) is responsible for reuptake of serotonin (5-HT) from the synaptic cleft and is target for antidepressant medicine. Differential hSERT activity caused by genetic polymorphisms is believed to affect the risk of developing depression and, moreover, to affect the response to antidepressant therapy. The hSERT contains in the second extracellular loop (EL2) two sites for N-linked glycosylation that are critical for functional transporter expression. Here we examine a non-synonymous single nucleotide polymorphism (SNP) in EL2 that gives rise to a potential third glycosylation site due to substitution of a lysine at position 201 with an asparagine (K201N). In agreement with introduction of an additional glycosylation site, western blot analysis showed migration of hSERT K201N corresponding to a higher molecular weight than wild type hSERT upon expression in both HEK293 cells and primary cultures of cortical neurons. An increase in molecular weight was not observed after removal of glycans with peptide N-glycosidase F (PNGase F). Quantitative analysis of western blots indicated significantly increased total transporter expression ( approximately 30%) for hSERT K201N as compared to hSERT in both cell systems. The increase in expression was accompanied by corresponding significant increases in the number of [(3)H]citalopram binding sites and in the V(max) for [(3)H]5-HT uptake. Characterization of mutants carrying all possible combinations of glycosylation sites demonstrated clear correlation between the number of glycosylation sites and the level of transporter activity, and showed that K201N could substitute for either one of the two original glycosylation sites.
Rasmussen
Trine N
TN
Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Plenge
Per
P
Bay
Tina
T
Egebjerg
Jan
J
Gether
Ulrik
U
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D015283:Citalopram / Q000494:pharmacology; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D051381:Rats; D017208:Rats, Wistar; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology
D015283:Citalopram; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; C493230:SLC6A4 protein, human