title: chi-Conotoxin and tricyclic antidepressant interactions at the norepinephrine transporter define a new transporter model.
abstract: Monoamine neurotransmitter transporters for norepinephrine (NE), dopamine and serotonin are important targets for antidepressants and analgesics. The conopeptide chi-MrIA is a noncompetitive and highly selective inhibitor of the NE transporter (NET) and is being developed as a novel intrathecal analgesic. We used site-directed mutagenesis to generate a suite of mutated transporters to identify two amino acids (Leu(469) and Glu(382)) that affected the affinity of chi-MrIA to inhibit [(3)H]NE uptake through human NET. Residues that increased the K(d) of a tricyclic antidepressant (nisoxetine) were also identified (Phe(207), Ser(225), His(296), Thr(381), and Asp(473)). Phe(207), Ser(225), His(296), and Thr(381) also affected the rate of NE transport without affecting NE K(m). In a new model of NET constructed from the bLeuT crystal structure, chi-MrIA-interacting residues were located at the mouth of the transporter near residues affecting the binding of small molecule inhibitors.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Filip A
authors.initials: FA
authors.lastname: Paczkowski
authors.forename: Iain A
authors.initials: IA
authors.lastname: Sharpe
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dutertre
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lewis
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000929:Antidepressive Agents, Tricyclic / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D020916:Conotoxins / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004298:Dopamine / Q000378:metabolism; D005473:Fluoxetine / Q000031:analogs & derivatives / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000235:genetics / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D000316:Adrenergic alpha-Agonists; D000929:Antidepressive Agents, Tricyclic; D020916:Conotoxins; D009498:Neurotoxins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D020033:Protein Isoforms; C493227:SLC6A2 protein, human; C507563:chi-conopeptide MrIA, Conus; D005473:Fluoxetine; C011386:nisoxetine; D018698:Glutamic Acid; D007930:Leucine; D004298:Dopamine; D009638:Norepinephrine