title: Pharmacological properties of the naturally occurring Ala(457)Pro variant of the human norepinephrine transporter.
abstract: Recently, another research group has reported an almost complete loss of function of the human norepinephrine transporter (hNET) in patients who had orthostatic intolerance and who were heterozygous for a guanine to cytosine exchange, resulting in a hNET Ala(457)Pro variant. To explore the reason for the deficiency in NET function, we compared in detail the pharmacology of the Ala(457)Pro variant with that of the wild-type hNET in COS-7 cells transiently transfected with hNET or Ala(457)Pro cDNA. Compared to the wild-type hNET, the Ala(457)Pro variant exhibited a five-fold higher affinity for cocaine, but a two-fold lower affinity for the NET inhibitor nisoxetine, and an unchanged affinity for the antidepressant desipramine. Plasma membrane expression (measured as Bmax of [3H]nisoxetine binding) of the Ala(457)Pro variant was only 40% of that of the wild-type hNET. The Ala(457)Pro variant showed a six- to 10-fold decrease in affinity for the substrates dopamine and 1-methyl-4-phenylpyridinium (MPP(+)). Compared with the wild-type hNET, the maximum rate (V(max)) of norepinephrine uptake by the Ala(457)Pro variant was slightly reduced, whereas the turnover number (calculated from V(max)/B(max)) was approximately two-fold higher. However, the Ala(457)Pro variant exhibited a 50-fold higher K(m) (i.e. lower apparent affinity) for norepinephrine than the wild-type hNET. Thus, the previously reported loss of function of the Ala(457)Pro variant associated with orthostatic intolerance is only partly due to a reduction in plasma membrane expression of the transporter, and is mainly caused by the pronounced reduction in the apparent affinity of norepinephrine.
authors.affiliation: Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Filip A
authors.initials: FA
authors.lastname: Paczkowski
authors.forename: Heinz
authors.initials: H
authors.lastname: Bönisch
authors.forename: Lesley J
authors.initials: LJ
authors.lastname: Bryan-Lluka
mesh_terms: D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003042:Cocaine / Q000494:pharmacology / Q000652:urine; D003891:Desipramine / Q000494:pharmacology; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D005473:Fluoxetine / Q000031:analogs & derivatives* / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009638:Norepinephrine / Q000037:antagonists & inhibitors / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D027981:Symporters / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
chemical_list: D018759:Adrenergic Uptake Inhibitors; D018765:Dopamine Uptake Inhibitors; D008164:Luminescent Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D020033:Protein Isoforms; C493227:SLC6A2 protein, human; D027981:Symporters; D005473:Fluoxetine; D049452:Green Fluorescent Proteins; C011386:nisoxetine; D003042:Cocaine; D003891:Desipramine; D009638:Norepinephrine