A critical site in the human serotonin transporter defines Na+ and Cl‐ dependence

The Na+ and Cl− coupled serotonin (5HT) transporter (SERT) is the principal site of antidepressant action. Mutation of a single residue in trans-membrane segment 1 (TM 1) of hSERT, N101, uncouples Na+ and Cl−-dependent 5HT translocation. Our previous studies (Henry et al., 2003) suggested that TM 1 forms part of the permeation pathway for 5HT, and residues there likely interact with Na+ during transport. The recent high-resolution structure for LeuTAa, (Yamashita et al., 2005) a Na+-dependent bacterial homolog of SERT suggests an interaction between our substituted residue (conserved in LeuTAa) and Na+. Electrophysiological analysis of this mutant reveals a 5HT-induced current in the absence of Na+ and/or Cl−. Remarkably, the N101 mutant also transports 5HT in the absence of Na+ and Cl− although Na+ further stimulates uptake. Furthermore, pH specifically modulates the contribution of this residue in voltage- and Na-dependent 5HT transport. A complete loss of Cl− dependence (in the presence of Na+) for the N101 mutation suggests that Cl− is tightly coordinated with Na+ and 5HT in a multi-ion/substrate pocket. This hSERT mutant demonstrates the first example in the SLC6 family of substrate transport uncoupled from normally co-transported ions. Further analysis including structural modeling may help in uncovering the mechanisms of ion coupling and substrate movement through neurotransmitter transporters. Supported by NIH NS34075 grant to LJD.