A mutation in the human norepinephrine transporter gene (SLC6A2) associated with orthostatic intolerance disrupts surface expression of mutant and wild-type transporters.
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.
Hahn
Maureen K
MK
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8548, USA.
Robertson
David
D
Blakely
Randy D
RD
D019943:Amino Acid Substitution; D000818:Animals; D001342:Autonomic Nervous System Diseases / Q000235:genetics* / Q000503:physiopathology; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D005799:Genes, Dominant; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011187:Posture; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D027981:Symporters / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; D027981:Symporters