Engineered zinc‐binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter

Abstract

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.