Functional significance of a highly conserved glutamate residue of the human noradrenaline transporter

• Published in: Journal of neurochemistry Vol. 81; no. 2; pp. 344 - 354
• Main Authors: Sucic, Sonja, Paczkowski, Filip A., Runkel, Fabian, Bönisch, Heinz, Bryan‐Lluka, Lesley J.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-04-2002; Blackwell
• Subjects: Amino Acid Substitution; Animals; Binding, Competitive - drug effects; Biological and medical sciences; Cell Line; Cell physiology; Cocaine - pharmacokinetics; Conserved Sequence; COS Cells; Desipramine - pharmacokinetics; Dopamine Agents - pharmacokinetics; expression; Female; Fluoxetine - analogs & derivatives; Fluoxetine - pharmacokinetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Glutamic Acid - physiology; Humans; ion dependence; Kidney - drug effects; Kidney - metabolism; Membrane and intracellular transports; Molecular and cellular biology; Molecular Sequence Data; Mutagenesis, Site-Directed; nisoxetine binding; noradrenaline (norepinephrine) transporter; Norepinephrine - pharmacokinetics; Norepinephrine Plasma Membrane Transport Proteins; Ovary - drug effects; Ovary - metabolism; Sequence Homology, Amino Acid; site‐directed mutagenesis; Sodium - metabolism; Structure-Activity Relationship; Symporters - antagonists & inhibitors; Symporters - genetics; Symporters - metabolism; Transfection; uptake; Human; Site directed mutagenesis; Catecholamine; Gene expression; Glutamate; In vitro; Cell surface; Conserved sequence; Norepinephrine; Mutation; Noradrenaline transporter; Carrier protein; Aminoacid sequence
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2002.00826.x

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+‐ and Cl–‐dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site‐directed mutagenesis and the mutants were expressed in transfected COS‐7 or HEK‐293 cells. Cell surface expression of hE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in Vmax values for noradrenaline and dopamine uptake of 2–3‐fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82–260‐fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.