4470 • The Journal of Neuroscience, June 1, 2003 • 23(11):4470–4478 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 and Randy D. Blakely1,2,4 1Department of Pharmacology, 2Center for Molecular Neuroscience, 3Department of Medicine, and 4Autonomic 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 regulatorofnoradrenergicneurotransmission.Recently,ourlaboratoryreportedapolymorphisminthehumanNET(hNET)geneA457P 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 Ͼ98% transport activity in several heterologous expression systems. In the present work, Western blot and biotinylation analyses performed in transiently transfected COS-7 cells revealed impair- ment in processing of hNET-A457P to the fully glycosylated form and a decrease in surface expression to ϳ30% 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 coimmunopre- cipitates 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 Introduction lants, including amphetamine and cocaine (Ritz et al., 1990; Tat- sumi et al., 1997; Sacchetti et al., 1999). Noradrenergic neurotransmission in the brain mediates atten- ϩ Ϫ tion, learning and memory, and emotion and pain perception The human NET (hNET) is a member of the Na /Cl - (Foote et al., 1983). Norepinephrine (NE) is also involved in dependent GAT (GABA transporter)/NET transporter family autonomic control via its actions in the brainstem and as the (Nelson, 1998; Hahn and Blakely, 2002a) and is a single-copy primary neurotransmitter used at postganglionic sympathetic gene (SLC6A2) located on chromosome 16 (Bru¨ss et al., 1993). The hNET cDNA encodes a 617 amino acid protein sufficient to nerve terminals. NE released at central and peripheral synapses is ϩ inactivated through active transport into terminals by the presyn- confer saturable, Na -dependent NE uptake in transfected cells aptically localized norepinephrine transporter (NET) (Iversen, (Pacholczyk et al., 1991). hNET contains three canonical 1961). Localized expression of NET to NE neurons is demon- N-glycosylation sites in the second extracellular loop, and immu- strated by [ 3H]nisoxetine autoradiography (Tejani-Butt, 1992), nohistochemical studies confirm a progression of hNET from gene expression (Lorang et al., 1994), NE uptake (Mitchell et al., light to more heavily glycosylated forms during synthesis of the 1994), and selective antibodies (Schroeter et al., 2000). NET re- mature protein (Pacholczyk et al., 1991; Melikian et al., 1994, captures as much as 90% of released NE, making it a critical 1996). mediator of NE inactivation and presynaptic catecholamine ho- Although a role for compromised function of NE systems and meostasis (Schomig et al., 1989). Indeed, NET knock-out mice NET in mood disorders has long been suspected (Schildkraut, (Xu et al., 2000) exhibit both a diminished NE clearance rate and 1965; Leonard, 1997), we recently demonstrated that NET dys- elevated extracellular NE concentrations, despite lowered tissue function may also manifest as diseases of the cardiovascular sys- content. Finally, NET is a target for tricyclic antidepressants, tem (Blakely, 2001; Hahn and Blakely, 2002b). Moreover, de- NET-selective reuptake inhibitors and multiple psychostimu- creases in NE uptake sites and activity are observed in hypertension, diabetes, cardiomyopathy, and heart failure (Esler et al., 1981; Merlet et al., 1992; Bohm et al., 1995; Schnell et al., 1996; Backs et al., 2001), and insufficient NE clearance may con- Received Sept., 6, 2002; revised March 13, 2003; accepted March 17, 2003. tribute to disease progression (Bohm et al., 1998). NETs mediate This work was supported by National Institutes of Health Grants MH58921 (R.D.B.) and F32 MH12896 (M.K.H.). a nonvesicular form of catecholamine release (Schomig et al., Correspondence should be addressed to Dr. Randy D. Blakely, Center for Molecular Neuroscience, 6133 Medical 1984). Efflux of cytoplasmic NE through NET during ischemia Research Building III, Suite 7140, Vanderbilt School of Medicine, Nashville, TN 37232-8548. E-mail: [email protected]. may contribute to fatal arrhythmias (Wilkerson and Sanders, Copyright © 2003 Society for Neuroscience 0270-6474/03/234470-09$15.00/0 1978; Schomig et al., 1991). Hahn et al. • Human Norepinephrine Transporter Mutation A457P J. Neurosci., June 1, 2003 • 23(11):4470–4478 • 4471 Our laboratory identified hNET-A457P, a mutant highly de- counter. Binding isotherms, Scatchard analyses, and competition curves ficient in NE transport, in a subject with orthostatic intolerance were analyzed using Kaleidagraph curve-fitting software (Synergy Soft- (OI), a disorder characterized by elevated heart rate and accom- ware, Reading, PA). 125 panied by indices of a hyperadrenergic state (Shannon et al., [ I]RTI-55 radioligand whole-cell binding. To ascertain the density of hNET binding sites on the cell surface, [ 125I]RTI-55 whole-cell binding 2000). The proband and family members carrying the heterozy- ϫ gous mutation also exhibited evidence of NET dysfunction (Ja- was performed. Cells were washed three times with 1 PBS before incu- bation with radiolabel and competitors. To assess nonspecific binding, cob et al., 1999; Shannon et al., 2000). Although hNET-A457P cells were preincubated with or without 100 ␮M dopamine for 10 min at has been shown to be deficient in transport, the molecular and 125 4°C in binding buffer. [ I]RTI-55 (10 nM) was added for 1 hr at 4°C. cellular basis of its loss of its activity has not been defined. In the Cells were washed three times with binding buffer at 4°C, solubilized in present study, we show that the defect of hNET-A457P lies in scintillation fluid (National Diagnostics, Atlanta, GA), and counted in a both disrupted biosynthetic processing and negligible function of gamma counter. residual surface transporters. Furthermore, coexpression of the [3H]NE uptake assays. NE transport was assayed in Krebs’–Ringer’s– mutant with hNET-wild type (hNET-wt) decreases the surface HEPES (KRH) buffer as described previously (Melikian et al., 1994; Ap- expression of hNET-wt and diminishes transport activity. Fi- parsundaram et al., 1998a). Briefly, cells were preincubated for 10 min at nally, we show that hNET-A457P coimmunoprecipitates with 37°C, with or without 1 ␮M desipramine to assess nonspecific accumu- hNET-wt, providing evidence of oligomeric complexes that lation, followed by the addition of 50 nM (single-point) or varying con- 3 ϳ could underlie the presentation of severe hNET dysfunction in centrations of (kinetic analysis) [ H]NE ( 30 Ci/mmol; Amersham Bio- heterozygous subjects. sciences, Uppsala, Sweden). After 10 min, cells were washed three times in KRH and incubated for 2 hr in scintillation fluid (National Diagnos- tics), and accumulated [ 3H]NE was determined by scintillation count- Materials and Methods ing. Student’s t test (two-tailed) was used to compare means of transport Plasmids constructs. The expression vector pcDNA3 (Invitrogen, Carls- and binding assays, and p values Ͻ0.05 were considered significant. bad, CA) containing the coding sequence for hNET, or hNET engineered Immunoblots. For preparation of detergent extracts of transfected cells, with tags or substitutions, was used in all transfection experiments. Con- cells were solubilized in radioimmunoprecipitation assay (RIPA) buffer struction of the plasmids pcDNA3-hNET-wt bearing an introduced AflII (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton site and pcDNA3-hNET-A457P has been described previously (Galli et X-100, 1% sodium deoxycholate, 250 ␮M PMSF, 1 ␮g/ml aprotinin, 1 al., 1995; Shannon et al., 2000; Bauman and Blakely, 2002). The plasmid ␮g/ml leupeptin, and 1 ␮M pepstatin) for 1 hr at 4°C and centrifuged at pcDNA3-hNET-A457P contains a single nucleotide substitution of a C 20,000 ϫ g for 30 min, and supernatants were separated on 8% SDS- foraGatposition 237 (GenBank accession number 91127) to create the PAGE gels. Proteins were transferred electrophoretically to polyvinyli- alanine to proline substitution found in OI subjects (Shannon et al., dene fluoride membrane (Millipore, Bedford, MA). Membranes were 2000). His-hNET-wt and HA-hNET-wt, containing a [His]6-Gly or a incubated with a monoclonal antibody directed against hNET at a dilu- hemagluttinin (HA) epitope sequence