A mutation in the human norepinephrine transporter gene (SLC6A2) associated with orthostatic intolerance disrupts surface expression of mutant and wild-type transporters.

Hahn MK, Robertson D, Blakely RD
J Neurosci. 2003 23 (11): 4470-8

PMID: 12805287 · PMCID: PMC6740799

The norepinephrine transporter (NET) mediates reuptake of norepinephrine released from neurons, and, as such, it is an important regulator of noradrenergic neurotransmission. Recently, our laboratory reported a polymorphism in the human NET (hNET) gene A457P in an individual with the autonomic disorder orthostatic intolerance (OI). The presence of the hNET-A457P allele tracked with elevated heart rates and plasma NE levels in family members. hNET-A457P lacks >98% transport activity in several heterologous expression systems. In the present work, Western blot and biotinylation analyses performed in transiently transfected COS-7 cells revealed impairment in processing of hNET-A457P to the fully glycosylated form and a decrease in surface expression to approximately 30% of hNET-wild type (hNET-wt). Because the hNET-A457P mutation is carried on a single allele in OI subjects, we examined the influence of cotransfection of hNET-wt and hNET-A457P and found that hNET-A457P exerts a dominant-negative effect on hNET-wt uptake activity. Experiments to determine oligomerization as a potential mechanism of the dominant-negative effect demonstrated that hNET-A457P coimmunoprecipitates with, and diminishes surface expression of, hNET-wt. These results reveal that hNET-A457P causes a conformational disruption that interferes with transporter biosynthetic progression and trafficking of both the mutant transporter and hNET-wt. These results elucidate a molecular mechanism for the disrupted NE homeostasis and cardiovascular function evident in OI patients with the hNET-A457P mutation.

MeSH Terms (20)

Amino Acid Substitution Animals Autonomic Nervous System Diseases Binding, Competitive Binding Sites Blotting, Western Cell Membrane COS Cells Genes, Dominant Glycosylation Humans Mutation Norepinephrine Plasma Membrane Transport Proteins Posture Precipitin Tests Protein Binding Protein Processing, Post-Translational Protein Transport Symporters Transfection

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