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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adrenergic alpha-Agonists / metabolism
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Amino Acid Sequence
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Animals
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Antidepressive Agents, Tricyclic / metabolism*
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Biological Transport / physiology
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Conotoxins / genetics
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Conotoxins / metabolism*
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Crystallography, X-Ray
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Dopamine / metabolism
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Fluoxetine / analogs & derivatives
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Fluoxetine / metabolism
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Glutamic Acid / metabolism
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Humans
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Leucine / metabolism
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Models, Molecular
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Neurotoxins / genetics
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Neurotoxins / metabolism*
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Norepinephrine / metabolism
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Norepinephrine Plasma Membrane Transport Proteins / chemistry
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Norepinephrine Plasma Membrane Transport Proteins / genetics
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Norepinephrine Plasma Membrane Transport Proteins / metabolism*
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Protein Isoforms / genetics
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Protein Isoforms / metabolism*
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Protein Structure, Tertiary
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Sequence Alignment
Substances
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Adrenergic alpha-Agonists
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Antidepressive Agents, Tricyclic
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Conotoxins
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Neurotoxins
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Norepinephrine Plasma Membrane Transport Proteins
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Protein Isoforms
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SLC6A2 protein, human
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chi-conopeptide MrIA, Conus
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Fluoxetine
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nisoxetine
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Glutamic Acid
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Leucine
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Dopamine
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Norepinephrine