Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis

• Published in: Journal of neurochemistry Vol. 94; no. 6; pp. 1620 - 1630
• Main Authors: Sucic, Sonja, Bryan‐Lluka, Lesley J.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-09-2005; Blackwell; Blackwell Publishing Ltd
• Subjects: Adrenergic Agonists - metabolism; Amino Acid Motifs - physiology; Analytical, structural and metabolic biochemistry; Animals; Binding and carrier proteins; Binding, Competitive - genetics; Biological and medical sciences; Brain - metabolism; Brain Chemistry - physiology; Cell Membrane - chemistry; Cell Membrane - metabolism; Cell physiology; Cells; Cercopithecus aethiops; COS Cells; cysteine; Cysteine - chemistry; Ethyl Methanesulfonate - analogs & derivatives; Ethyl Methanesulfonate - chemistry; Fundamental and applied biological sciences. Psychology; Humans; Membrane and intracellular transports; Membranes; Mesylates - chemistry; Molecular and cellular biology; Mutagenesis, Site-Directed; Mutation; Neurochemistry - methods; Neurons - metabolism; Neuropharmacology - methods; nisoxetine binding; noradrenaline transporter; Norepinephrine - metabolism; Norepinephrine Plasma Membrane Transport Proteins; Pharmacology; Protein Structure, Tertiary - physiology; Proteins; Radioligand Assay; site‐directed mutagenesis; substituted‐cysteine accessibility method; Symporters - chemistry; Symporters - genetics; Symporters - metabolism; uptake; Human; site-directed mutagenesis; Alanine; Cysteine; Interaction; Site directed mutagenesis; noradrenaline transporter; nisoxetine binding; Cell surface; Uptake; substituted-cysteine accessibility method; Substrate; Sensitivity; Endogenous; Analysis method; Intracellular; Mutation
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/j.1471-4159.2005.03316.x

The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted‐cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild‐type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell‐surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7‐fold for hP97C and 10‐fold for hF101C and increased 12‐fold for hY98C. [3H]Nisoxetine binding affinities were decreased 13‐fold for hP97C and 5‐fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mm MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.