Effects of serine mutations in transmembrane domain 7 of the human norepinephrine transporter on substrate binding and transport.
Two serine residues in the beta-adrenergic receptor (beta-AR) have been proposed to form hydrogen bonds with the catechol moiety of the ligand and contribute to the activation of the receptor. These conserved serine residues in the dopamine (DA) and norepinephrine transporters (DAT and NET, respectively) have also been shown to affect substrate transport in the rat DAT. In the present work, hydrogen bonding interactions between the corresponding serine residues in the human NET (hNET), 354 and 357, and the hydroxyl groups on the substrate were systematically evaluated by examining the transport and binding properties of DA and several single hydroxyl analogues of DA at wild-type and serine-to-alanine-substituted transporters. A comparison of [3H]nisoxetine binding at the serine 354 mutant, in which K(D) increased 70-fold from the wild-type value, with the binding of DA, m-tyramine (m-TYR), and p-tyramine (p-TYR) at mutant 354, where the increase in Ki was less dramatic, revealed that serine 354 is more influential in inhibitor than substrate binding. The binding of m-TYR and p-TYR at the serine 354 and serine 357 mutants did not show a direct interaction between one serine and one substrate catechol hydroxyl group. DA, m-TYR, and p-TYR binding affinity did not deviate from the wild-type value at the serine 357 and double mutant transporters. At these two transporters, however, the Km of DA uptake increased, suggesting that the roles of serine 357 and serine 354 in substrate transport are different from their roles in binding. The K'm for induced efflux of DA decreased at the serine 357 mutant compared with the wild-type, whereas the K'm at the serine 354 mutant was the same as that of the wild-type. Further investigation of the role of substrate hydroxyls in the transport process revealed no difference between the transport of m-TYR or p-TYR, as measured indirectly through their induced efflux of DA, at any of the mutants. Although these serines are influential in inhibitor and substrate binding to the transporter and substrate uptake and efflux, they do not appear to be involved in a direct hydrogen bond interaction with substrate, suggesting that the pattern of distinct hydrogen bonding interactions at the beta-AR does not exist at the hNET.
Danek Burgess
K S
KS
Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA.
Justice
J B
JB
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004298:Dopamine / Q000493:pharmacokinetics; D004566:Electrodes; D005473:Fluoxetine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D012694:Serine; D027981:Symporters; D014316:Tritium; D014439:Tyramine / Q000493:pharmacokinetics
D002352:Carrier Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; D027981:Symporters; D005473:Fluoxetine; D014316:Tritium; C011386:nisoxetine; D012694:Serine; C028169:3-tyramine; D004298:Dopamine; D014439:Tyramine