Abstract
The human norepinephrine transporter (hNET) is a member of the neurotransmitter/sodium symporter family, which also includes the neuronal monoamine transporters for serotonin (SERT) and dopamine (DAT). Its involvement in chronic pain and many neurological disorders underlies its pharmaceutical importance. Using the X-ray crystal structures of the human serotonin transporter (hSERT) (PDB 5I6X) and Drosophila melanogaster dopamine transporter (dDAT) (PDB 4M48 and PDB 4XPA) as templates, we developed molecular models for norepinephrine (NE) bound to its high affinity binding site (S1) in the hNET. Our model suggests that the S1 site for NE is deeply buried between transmembrane helices (TMHs) 1, 3, 6, and 8 and overlaps the binding site for leucine in the bacterial leucine transporter (LeuT) and dopamine (DA) in dDAT. Mutational studies identified the functional binding pocket for NE comprised residues A73, A77, N78, V148, N153, I156, G320, F329, N350, S420, G423, and M424, which all influenced NE affinity and/or transport. These effects support a NE-hNET docking model where A73, A77, G320, S420, G423, and M424 form H-bond interactions with NE, V148, I156, and F329 form hydrophobic interactions with NE, whereas N78 affects NE transport and N350 affects NE affinity and transport via an influence on the octahedral co-ordination of the Na1+ ion. Consistent with a conserved structure-function amongst sodium-dependent neurotransmitter transporters, S1 residues A73, A77 (G100 in hSERT), N78, V148 (I150 in hSERT), N153, G320, F329 (Y331 in d DAT), N350, and G423 are conserved in DAT and SERT, indicating they likely play conserved functional roles.
Highlights
Spatio-temporal regulation of NE neurotransmission, presynaptic homeostasis and maintenance of extra synaptic monoamine levels is maintained by the NET (Torres et al, 2003)
Western blot analysis revealed that the A73G, A73V, A73L, A73R, A77L, A77R, N78A, V148A, N153A, G320A, V325A, N350A, S420A, G423A, and M424A mutants of human NET (hNET) had similar expression levels compared to wild type (WT) hNET when expressed in COS-7 cells
The Y152A mutant did not express and the F72A, I156A, S318A, F323A, and F329A mutants showed reduced expression levels compared with WT hNET (Figure 6)
Summary
Spatio-temporal regulation of NE neurotransmission, presynaptic homeostasis and maintenance of extra synaptic monoamine levels is maintained by the NET (Torres et al, 2003). The SLC6 gene family includes the monoamine [DA, NE, and 5-HT], amino acid [GABA, glycine, and proline] and osmolyte (betaine, creatine, and taurine) transporters (Dougherty et al, 1999; Jayanthi and Ramamoorthy, 2005; Klimek et al, 1997). The human NET (hNET) is of particular clinical relevance because dysregulated NE neurotransmission plays a key role in diseases such as chronic pain (Torebjörk et al, 1995), depression (Klimek et al, 1997), OCD (Hollander et al, 1988), anxiety, ADHD (Dougherty et al, 1999), cardiac diseases (Jayanthi and Ramamoorthy, 2005), obesity, and orthostatic intolerance (Hahn et al, 2003; Zhou, 2004). Several studies have identified hNET single nucleotide polymorphisms (SNPs) and their associated pathologies These studies shed light on how alterations in the protein’s structure, expression and/or function lead to disease (Supplementary Table S1). Alterations in the structure, function and expression of this transporter can produce significant anatomical and functional sequelae
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