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Engineered zinc‐binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter
Author(s) -
White Kellie J.,
Kiser Philip D.,
Nichols David E.,
Barker Eric L.
Publication year - 2006
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.062386106
Subject(s) - aquifex aeolicus , transmembrane domain , transporter , histidine , homology modeling , serotonin transporter , binding site , mutant , chemistry , biochemistry , biophysics , biology , amino acid , stereochemistry , serotonin , receptor , enzyme , escherichia coli , gene
The human serotonin transporter (hSERT) regulates neurotransmission by removing released serotonin (5‐HT) from the synapse. Previous studies identified residues in SERT transmembrane helices (TMHs) I and III as interaction sites for substrates and antagonists. Despite an abundance of data supporting a 12‐TMH topology, the arrangement of the TMHs in SERT and other biogenic amine transporters remains undetermined. A high‐resolution structure of a bacterial leucine transporter that demonstrates homology with SERT has been reported, thus providing the basis for the development of a SERT model. Zn 2+ ‐binding sites have been utilized in transporters and receptors to define experimentally TMH proximity. Focusing on residues near the extracellular ends of hSERT TMHs I and III, we engineered potential Zn 2+ ‐binding sites between V102 or W103 (TMH I) and I179–L184 (TMH III). Residues were mutated to either histidine or cysteine. TMH I/III double mutants were constructed from functional TMH I mutants, and Zn 2+ sensitivity was assessed. Dose‐response assays suggest an approximately twofold increase in sensitivity to Zn 2+ inhibition at the hSERT V102C/M180C and approximately fourfold at the V102C/I179C mutant compared to the hSERT V102C single mutant. We propose that the increased sensitivity to Zn 2+ confirms the proximity and the orientation of TMHs I and III in the membrane. Homology modeling of the proposed Zn 2+ ‐binding sites using the coordinates of the Aquifex aeolicus leucine transporter structure provided a structural basis for interpreting the results and developing conclusions.