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The Journal of Neuroscience, June 1, 2003, 23(11):4470-4478
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A Mutation in the Human Norepinephrine Transporter Gene (SLC6A2) Associated with Orthostatic Intolerance Disrupts Surface Expression of Mutant and Wild-Type Transporters
Maureen K. Hahn,1,2 David Robertson,2,3,4 andRandy D. Blakely1,2,4 1 Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8548, 2 Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8548, 3 Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8548, and 4 Autonomic Dysfunction Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8548
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
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.
Key words: norepinephrine; transporter; SLC6A2; orthostatic intolerance; trafficking; antidepressant
Received Sep. 6, 2002; revised Mar. 13, 2003; accepted Mar. 17, 2003.
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