Helix XI contributes to the entrance of the serotonin transporter permeation pathway.
The sodium-dependent transporters for dopamine, norepinephrine, and serotonin that regulate neurotransmission, also translocate the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Previous studies implicated residues in transmembrane helix (TMH) XI of DAT as important sites for MPP(+) transport. We examined the importance of TMH XI residues F551 and F556 for MPP(+) translocation by human SERT. Mutations at hSERT F556, but not F551, reduced both 5-HT and MPP(+) transport compared to wild type. However, F556S/hSERT showed a reduction in surface expression explaining the decrease of transport activity for 5-HT, but did not account for the decrease in MPP(+) transport observed. Cysteine mutants at those positions confirmed the accessibility of hSERT/F556 to different methanethiosulfonate (MTS) reagents, suggesting its presence in a hydrophilic environment of the protein. In the presence of MTSET, current induced by 5-HT and MPP(+) was inhibited at the F556C mutant. In agreement with our homology model of SERT, based on the leucine transporter (LeuT(Aa)) from Aquifex aeolicus structure, these results are consistent with the hypothesis that a portion of TMH XI lines the entrance into the substrate permeation pathway.
Torres-Altoro
Melissa I
MI
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy and Pharmaceutical Sciences, West Lafayette, Indiana 47907-2091, USA.
White
Kellie J
KJ
Rodríguez
Gustavo J
GJ
Nichols
David E
DE
Barker
Eric L
EL
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D006801:Humans; D009154:Mutation; D009498:Neurotoxins / Q000378:metabolism*; D009865:Oocytes; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D009498:Neurotoxins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D010649:Phenylalanine; D015655:1-Methyl-4-phenylpyridinium